Target gene disruption method and immunogenic composition

Abstract

Disruption of specific gene function in obligate intracellular bacteria and subsequent restoration of its activity remains extremely challenging due to their absolute necessity within host cells for replication. In this study, targeted allelic exchange mutants were generated to inactivate two genes in the rickettsial pathogen Ehrlichia chaffeensis and subsequently restore one of the two genes. This method was also successfully used in Ehrlichia canis and Anaplasma phagocytophilum. The generated mutant pathogens are useful in immunogenic compositions to reduce the incidence or severity of infection with rickettsial pathogens. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] This invention was made with United States government support under Grant No. AI070908 awarded by the National Institutes of Health (NIH). The United States government has certain rights in this invention. [Background technology]

[0002] Disruption of specific gene functions in obligate intracellular bacteria and subsequent restoration of their activity remains extremely challenging due to their absolute requirement within host cells for replication. Here, we generated targeted mutants by allelic exchange in two genes and one genetically complemented gene in the rickettsial pathogen Ehrlichia chaffeensis (E. chaffeensis). In theory, this approach could be applied to other obligate intracellular bacteria, enabling structure-function analyses routinely performed in intracellular bacteria. This method could also be applied to generate attenuated strains of obligate intracellular bacteria, which could be useful as live vaccine candidates.

[0003] Obligate intracellular bacteria cause disease in millions of people worldwide. These bacteria include many pathogenic Gram-negative bacteria in the orders Rickettsiales and Chlamydiales. The lack of an efficient system for targeted mutagenesis in Rickettsia and Chlamydiae (Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, Orientia, and Chlamydiae) remains a major obstacle to understanding microbial pathogenesis and defining the functional significance of many bacterial genes. Chlamydiae and Rickettsiae have undergone extreme genome reduction, and the majority of genes in each pathogen are critical for their intracellular growth. Therefore, obligate intracellular bacteria rely on their hosts to compensate for the defects caused by genome reduction. Consistent with this hypothesis, previous studies have demonstrated that approximately 74–92% of predicted genes in Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, Orientia, and Chlamydiae are transcriptionally active during bacterial replication within vertebrate and vector host cells. Challenges in generating targeted mutants can be attributed to the essential nature of the gene selected for mutagenesis, its dependence on intracellular replication, and the lack of methods to support extracellular growth. Despite the success of generating random mutants using transposon mutagenesis, generating targeted mutations in specific genes of interest and then complementing them is difficult and highly desirable for obligate intracellular bacteria. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure solves this major problem by providing a method for generating at least one stable, targeted mutant by allelic exchange in Rickettsiales or Chlamydiales bacteria. Advantageously, the present disclosure further provides the ability to disrupt or inactivate multiple genes and then restore at least one intact gene with another allelic exchange mutant, resulting in restored transcription from the inactivated gene from its own promoter. In a preferred form, the disrupted or inactivated gene induces an immune response specific to the bacterium and prevents or at least reduces the bacterium's ability to replicate in an obligate host. Thus, the present disclosure provides attenuated forms of the bacterium useful in immunogenic compositions that, when administered prophylactically, elicit an immune response that reduces the incidence or severity of at least one clinical symptom associated with or attributable to Rickettsiales and Chlamydiales, and, when administered post-infection, reduces the duration or severity of at least one clinical symptom associated with or attributable to Rickettsiales and Chlamydiales.

[0005] As known in the art, "sequence identity" refers to the relationship between two or more polypeptide (polyamino acid) sequences or two or more polynucleotide sequences, i.e., between a reference sequence and a given sequence to which it is compared. Sequence identity is determined by comparing a given sequence to a reference sequence (as determined by matches between strings of these sequences) after optimally aligning the two sequences to achieve the highest degree of sequence similarity. After such alignment, sequence identity is ascertained position by position. For example, if the nucleotide or amino acid residue at a particular position is identical, then the sequences are "identical" at that position. The percentage of sequence identity is determined by dividing the total number of positions with such positional identity by the total number of nucleotides or residues in the reference sequence. Sequence identity may be determined using techniques such as, but not limited to, "Computational Molecular Biology, Lesk, A.N., ed., Oxford University Press, New York (1988)," "Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993)," "Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey (1994)," "Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987)," "Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991)," and "Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073." (1988), the teachings of which are incorporated herein by reference.Preferred methods for determining sequence identity are designed to obtain the largest match between the sequences tested. Methods for determining sequence identity are codified in publicly available computer programs that can determine the sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990)), the teachings of which are incorporated herein by reference. The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, SF et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align a given sequence with a reference sequence using default gap weighting to obtain the highest level of sequence identity between the given sequence and the reference sequence. For example, if a given polynucleotide has a nucleotide sequence that has at least 85%, preferably 90%, more preferably 95% "sequence identity" with a reference nucleotide sequence, this means that the nucleotide sequence of the given polynucleotide is identical to the reference sequence, except that the given polynucleotide sequence may contain up to 15, preferably up to 10, more preferably up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence.In other words, in a polynucleotide having a nucleotide sequence that is at least 85%, preferably 90%, more preferably 95% identical to a reference nucleotide sequence, up to 15%, preferably 10%, more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or up to 15%, preferably 10%, more preferably 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5'-terminal or 3'-terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, either individually interspersed among the nucleotides of the reference sequence or interspersed within one or more contiguous groups of the reference sequence. Similarly, if a given polypeptide has an amino acid sequence that is at least 85%, preferably 90%, more preferably 95% identical to the reference amino acid sequence, this means that the amino acid sequence of the given polypeptide is identical to the reference sequence, provided that the given polypeptide sequence may contain up to 15, preferably up to 10, more preferably up to 5 amino acid modifications for every 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, more preferably up to 5% of the amino acid residues in the reference sequence can be deleted or substituted with other amino acids, or up to 15%, preferably up to 10%, more preferably up to 5% of the total number of amino acid residues in the reference sequence can be inserted into the reference sequence. These modifications of the reference sequence can be made at the amino- or carboxy-terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, either individually interspersed among the residues of the reference sequence or interspersed within one or more consecutive groups of the reference sequence. Preferably, non-identical residue positions differ by conservative amino acid substitutions. However, conservative substitutions are not included in the match when determining sequence identity.

[0006] As used herein, "sequence homology" refers to a method for determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced, if necessary. However, in contrast to "sequence identity," conservative amino acid substitutions are counted as matches when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology to a reference sequence, 85%, preferably 90%, and more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match another amino acid or nucleotide or contain conservative substitutions. Alternatively, up to 15%, preferably up to 10%, and more preferably up to 5% of the total number of amino acid residues or nucleotides (excluding conservative substitutions) in the reference sequence can be inserted into the reference sequence. Preferably, a homologous sequence contains at least 50, preferably at least 100, more preferably at least 250, and even more preferably at least 500 consecutive nucleotides.

[0007] A "conservative substitution" refers to the replacement of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties (e.g., size, hydrophobicity, etc.) such that the overall functionality is not significantly altered.

[0008] As will be appreciated by those skilled in the art, the immunogenic compositions of the present disclosure can include known injectable, physiologically acceptable sterile solutions for preparing ready-to-use solutions for parenteral injection or infusion, and isotonic aqueous solutions, such as saline or its corresponding plasma protein solutions, can be readily utilized. Additionally, the immunogenic and vaccine compositions of the present disclosure can include diluents, isotonic agents, stabilizers, or adjuvants. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers can include albumin and alkali salts of ethylenediaminetetraacetic acid, among others.

[0009] In one aspect, the immunogenic compositions of the present disclosure may further comprise additional components, antigens, pharmaceutically acceptable carriers, veterinarily acceptable carriers, adjuvants, preservatives, stabilizers, or combinations thereof.

[0010] As used herein, "adjuvant" can include aluminum hydroxide, aluminum phosphate, savonin, Quil A, cyclic GMP-AMP, Montanide gel, QS-21 (Cambridge Biotech Inc., Cambridge, Massachusetts, USA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Alabama, USA), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsions may be based in particular on light liquid paraffin oil (European Pharmacopoeia type), isoprenoid oils (such as squalane or squalene oils obtained by oligomerization of alkenes, especially isobutene, decene), esters of acids or alcohols containing linear alkyl groups (more particularly vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate), glyceryl tri(caprylate / caprate), or propylene glycol dioleate), or esters of branched-chain fatty acids or alcohols (especially isostearate). These oils are used in combination with emulsifiers to form emulsions. The emulsifier is preferably a nonionic surfactant, particularly esters of sorbitan, mannides (e.g., anhydromannitol oleate), glycols, polyglycerols, propylene glycol, oleic acid, isostearic acid, ricinoleic acid, or hydroxystearic acid (which may be ethoxylated), as well as polyoxypropylene-polyoxyethylene copolymer blocks, particularly Pluronic products (especially L121). See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp. 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).For example, SPT emulsion, described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach," edited by M. Powell and M. Newman, Plenum Press, 1995, and emulsion MF59, described on page 183 of the same book, can be used. Further examples of adjuvants are compounds selected from polymers of acrylic or methacrylic acid or copolymers of maleic anhydride and alkenyl derivatives. Suitable adjuvant compounds are, in particular, polymers of acrylic or methacrylic acid crosslinked with polyalkenyl ethers of sugars or polyalcohols. These compounds are called carbomers (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art can also refer to U.S. Pat. No. 2,909,462, which describes an acrylic polymer crosslinked with a polyhydroxylated compound having at least three, and preferably not more than eight, hydroxyl groups, in which the hydrogen atoms of at least three of the hydroxyl groups have been replaced by unsaturated aliphatic radicals having at least two carbon atoms. Preferred radicals are those containing 2 to 4 carbon atoms, such as vinyl, allyl, and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. Additional suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi), block copolymers (CytRx, Atlanta, GA, USA), SAF-M (Chiron, Emeryville, CA, USA), monophosphoryl lipid A, avridine lipid-amine adjuvant, heat-labile enterotoxin from Escherichia coli (recombinant or otherwise), cholera toxin, IMS 1314, or muramyl dipeptide, among others.

[0011] Preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. More preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 μg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of about 750 μg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose.

[0012] In addition, compositions of the present disclosure may include one or more pharmaceutically or veterinarily acceptable carriers. As used herein, a "pharmaceutically or veterinarily acceptable carrier" includes any or all of solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial agents, antifungal agents, isotonic agents, or absorption delaying agents.

[0013] A pharmaceutically acceptable vehicle is understood to refer to a compound or a combination of compounds contained in a pharmaceutical composition or vaccine that, for example, facilitates the administration of the active compound, extends its lifespan and / or efficacy in the body, increases its solubility in solution, or improves its storage stability, without causing side effects. These pharmaceutically acceptable vehicles are well known and can be adapted by those skilled in the art depending on the nature and mode of administration of the selected active compound.

[0014] For example, the immunogenic composition or vaccine according to the present disclosure can be administered in an amount of about 0.1 to 1000 μg per kg of animal or human body weight, either once or in several divided doses spaced apart over time. Dosage ranges or values ​​include, but are not limited to, 0.5 to 800 μg, 1 to 1000 μg, 1 to 500 μg, 1 to 300 μg, 1 to 200 μg, 1 to 150 μg, 1 to 125 μg, 1 to 100 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, and 35 μg per kg of animal or human body weight. , 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 125 μg, 150 μg, 200 μg, 250 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, and 1000 μg are envisioned. In another preferred embodiment, the dosage is set independently of the body weight of the animal or human.

[0015] In accordance with the present disclosure, the immunogenic compositions or vaccines of the present disclosure can include at least one additional pathogen other than E. chaffeensis, making it a combination vaccine or immunogenic composition. In such embodiments, administration of an effective amount of the vaccine or immunogenic composition of the present disclosure provides effective protection, including immunity against infection caused by a rickettsial or chlamydial bacterium and at least one additional pathogen, including reducing the severity or incidence of clinical symptoms of the infection.The other pathogen is preferably Actinobacillus pleuropneumoniae, adenovirus, alphavirus (e.g., eastern equine encephalitis virus), Bordetella bronchiseptica, Brachyspira species (preferably Brachyspira hyodysenteriae, Brachyspira piosicoli), Brucella suis (preferably biotypes 1, 2 and 3), classical swine fever virus, Clostridium species (preferably Clostridium difficile, Clostridium perfringens types A, B and C, Clostridium novi, Clostridium septicum, Clostridium tetani), coronavirus (preferably porcine respiratory coronavirus), Erythrozoonosis Suis, Erysipelothrix rhusiopathiae, Escherichia coli, Haemophilus parasuis (preferably subtypes 1, 7 and 14), hemagglutinating encephalomyelitis virus, Japanese encephalitis virus, Lawsonia intracellularis, Leptospira species (preferably Leptospira australis, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagicae, Leptospira interrogans, Leptospira pomona, Leptospira thalassovi), Mycobacterium species (preferably Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium bovis), Mycoplasma hyopneumoniae (M hyo), Pasteurella multocida, porcine cytomegalovirus, porcine parvovirus, porcine reproductive and respiratory syndrome (PRRS) virus, porcine circovirus, pseudorabies virus, rotavirus, Salmonella spp. (preferably Salmonella typhimurium, Salmonella cholerae suis), Staphylococcus hyicus, Staphylococcus spp., Streptococcus spp. (preferably Streptococcus suis), porcine herpesvirus, swine influenza virus, swinepox virus, vesicular stomatitis virus, vesicular exanthema of swine virus, Leptospira hardjo, Mycoplasma hyosinoeae, poliovirus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), or a combination thereof.

[0016] The immunogenic compositions of the present disclosure may further include one or more other immunomodulatory agents, such as interleukins, interferons, or other cytokines.

[0017] The immunogenic compositions of the present disclosure may further comprise gentamicin and merthiolate.

[0018] While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of skill in the art, the present invention contemplates compositions containing from about 50 μg to about 2000 μg of adjuvant. Accordingly, as used herein, an immunogenic composition further refers to a composition containing from about 1 μg / ml to about 60 μg / ml of an antibiotic or immunomodulator, more preferably less than about 30 μg / ml of an antibiotic or immunomodulator.

[0019] According to a further aspect, at least one additional administration of the immunogenic composition of the present disclosure is administered to a subject in need thereof. The second or subsequent administration of the immunogenic composition of the present disclosure is administered at least seven days after the first or previous administration. Preferably, the immunogenic composition of the present disclosure is administered together with an immunostimulant. Preferably, the immunostimulant is administered at least twice. The second or subsequent administration of the immunostimulant is preferably administered at least three days, more preferably at least five days, and even more preferably at least seven days after the first or previous administration. Preferably, the immunostimulant is administered at least 10 days, preferably 15 days, more preferably 20 days, and even more preferably at least 22 days after the initial administration of the immunogenic composition of the present disclosure. A preferred immunostimulant is, for example, keyhole limpet hemocyanin (KLH), preferably emulsified with incomplete Freund's adjuvant (KLH / ICFA). It should be understood that any other immunostimulant known to those skilled in the art can also be used. As used herein, the term "immunostimulant" refers to any substance or composition capable of eliciting an immune response, e.g., an immune response against a specific pathogen, preferably without initiating or increasing a specific immune response. Also, administration of the immunostimulant in an appropriate dose is taught.

[0020] In a further aspect, the present disclosure provides methods for generating at least one stable targeted mutant by allelic exchange in the genera Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, Orientia, and Chlamydia.

[0021] In yet another aspect of the present disclosure, Rickettsiales and Chlamydiales mutated using the methods of the present disclosure are useful in immunogenic compositions against Rickettsiales and Chlamydiales pathogens. In preferred embodiments, such immunogenic compositions are effective in reducing the incidence or severity of at least one clinical symptom of an infectious disease resulting from or associated with infection with a Rickettsiales or Chlamydiales pathogen. In some preferred embodiments, such immunogenic compositions are administered prophylactically to reduce the incidence and / or severity of clinical symptoms. In particularly preferred embodiments, clinical symptoms are prevented in animals administered such immunogenic compositions prior to infection or challenge with a Rickettsiales and / or Chlamydiales pathogen. In other embodiments, such immunogenic compositions are administered after infection with a Rickettsiales and / or Chlamydiales pathogen. In such situations, clinical symptoms associated with or resulting from the infection are reduced in incidence, severity, and / or duration.

[0022] In a further embodiment, the stable targeted mutation disrupts the function of at least one gene.

[0023] In yet another embodiment, the function of a disrupted gene can be restored.

[0024] In one embodiment of the present disclosure, stable targeted mutants are generated in Ehrlichia chaffeensis by disrupting two genes and then restoring function from one gene. This same method has also been successfully used in Ehrlichia species, including Ehrlichia canis, and Anaplasma species, including Anaplasma phagocytophilum.

[0025] In another aspect, the present disclosure provides methods for targeted gene disruption or mutation in Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, Orientia, and / or Chlamydia species to generate attenuated bacterial organisms that are beneficial in inducing effective host immune responses to confer protection against virulent diseases caused by those same species.

[0026] In yet another aspect, methods are provided for inducing an immune response in a human or animal, comprising administering an immunogenic composition or vaccine of the present disclosure to an animal or human in need thereof. Additionally, methods are provided for reducing the incidence and / or severity of at least one clinical symptom associated with or resulting from infection with a pathogen from the genera Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, Orientia, and Chlamydia. In some forms, the infection is caused by or associated with Ehrlichia chaffeensis, Ehrlichia canis, Anaplasma platys, Anaplasma marginale, and / or Anaplasma phagocytophilum, and the immunogenic composition or vaccine of the present disclosure comprises at least one of these species, and the targeted gene disruption or mutation is directed against these species. Such methods generally comprise administering an immunogenic composition of the present disclosure to an animal in need thereof to induce an immune response against infection and the subsequent clinical symptoms in an animal infected or challenged with species of the genera Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, Orientia, and Chlamydia after administration of the immunogenic composition of the present disclosure. In some forms, the immunogenic compositions of the present disclosure comprise modified live species of Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, Orientia, or Chlamydia.

[0027] In one embodiment, the immunogenic composition of the present disclosure comprises Ehrlichia chaffeensis, Ehrlichia canis, Anaplasma phagocytophilum, or Anaplasma marginale that has undergone targeted mutagenesis as described herein. When Ehrlichia chaffeensis is the mutant species, the immunogenic composition of the present disclosure preferably comprises an attenuated Ehrlichia chaffeensis bacterium containing a mutation that inactivates the bacterium and inhibits its ability to replicate. In some preferred embodiments, the mutation is an insertion or deletion. In some embodiments, the insertion or deletion is anywhere within a gene selected from the Ech_0379 gene or the Ech_0660 gene, thereby inactivating gene function. In some embodiments, the Ehrlichia chaffeensis genome comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95, 96, 97, 98, 99, or even 100% sequence identity to Ech_0660 (SEQ ID NO: 35). Preferably, the sequence comparison is with a non-mutated version of the same region of the Ehrlichia chaffeensis genome. When Ehrlichia canis is a mutant species, the immunogenic compositions of the present disclosure preferably comprise an attenuated Ehrlichia canis bacterium comprising a mutation that inactivates a bacterial gene, inhibiting its ability to replicate. In some preferred embodiments, the mutation is an insertion or deletion. In some embodiments, the insertion or deletion is anywhere within the Ecaj_0381 gene, thereby inactivating gene function. In some forms, the Ehrlichia canis genome comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95, 96, 97, 98, 99, or even 100% sequence identity to SEQ ID NO: 54. Preferably, the sequence comparison is with a non-mutated version of the same region of the Ehrlichia canis genome. When the Anaplasma phagocytophilum is a mutant species, the immunogenic compositions of the present disclosure preferably comprise an attenuated Anaplasma phagocytophilum bacterium that includes a mutation that inactivates a bacterial gene, inhibiting its ability to replicate.In some preferred embodiments, the mutation is an insertion or deletion. In some embodiments, the insertion or deletion is anywhere within the APH_0634 gene, thereby inactivating gene function. In some embodiments, the genome of the Anaplasma phagocytophilum APH_0634 gene comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95, 96, 97, 98, 99, or even 100% sequence identity to SEQ ID NO: 55. Preferably, the sequence is compared with a non-mutated version of the same region of the Anaplasma phagocytophilum genome.

[0028] The immunogenic compositions or vaccines and methods provided by the present disclosure are not limited to Ehrlichia chaffeensis, Ehrlichia canis, and Anaplasma phagocytophilum, but also include any member of the orders Rickettsiales or Chlamydiales, including, but not limited to, the orders Pelagibacteres, the family Pelagibacteridae (including subgroups Ib, II, IIIa, IIIb, IV, and V), the genus Pelagibacter, the families Promomitochondriales, the families Anaplasmata, species of the genera Ehrlichia, Anaplasma, Wolbachia, and Neorickettsia, species of the family Midichloriaceae, Midichloria, the family Rickettsiaceae, and Rickettsia. Additionally, the immunogenic compositions or vaccines of the present disclosure may further include, but are not limited to, Ehrlichia ruminantium, Ehrlichia canis, Anaplasma marginale, Anaplasma platys, Ehrlichia muris, and combinations thereof.

[0029] In a further aspect of the present disclosure, the immunogenic composition of the present disclosure comprises a homolog of the Ehrlichia chaffeensis ECH_0660 gene mutated using the methods of the present disclosure. In a preferred embodiment, the homolog is selected from the group consisting of Ehrlichia, Anaplasma, Rickettsia, Neorickettsia, Orientia, and Chlamydia. In another preferred embodiment, the gene homolog is Ecaj_0381 of Ehrlichia canis, and preferably has at least 70%, 75%, 80%, 85%, 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95, 96, 97, 98, 99, or even 100% sequence identity to GenBank #CP 000107.1. In another preferred embodiment, the gene homolog is Erum_3930 from Ehrlichia ruminantium, preferably having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity to GenBank #CR 767821.1. In another preferred embodiment, the gene homolog is APH_0634 from Anaplasma phagocytophilum, preferably having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity to GenBank #CP 000235.1. In another preferred embodiment, the gene homolog is AMH_581 of Anaplasma marginale, preferably having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95, 96, 97, 98, 99, or even 100% sequence identity to GenBank #CP 000030.1.In another preferred form, the gene homolog is EMUR_02070 of Ehrlichia muris AS145, preferably having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95, 96, 97, 98, 99, or even 100% sequence homology to GenBank #CP 006917.1.

[0030] The immunogenic compositions according to the present disclosure may be administered intravenously, intramuscularly, intranasally, intradermally, intratracheally, intravaginally, intravenously, intravascularly, intraarterially, intraperitoneally, orally, intrathecally, or by direct injection into any target tissue. Depending on the desired duration and therapeutic effect of treatment, the immunogenic compositions according to the present disclosure may be administered in various doses once or several times, or continuously, for example, daily for several days, weeks, or months.

[0031] In another aspect, the immunogenic compositions of the present disclosure are administered to an animal at least 2 weeks old in need thereof. More preferably, the animal to which the immunogenic compositions of the present disclosure are administered is between 2 weeks and 1 year of age, and even more preferably between 3 weeks and 6 months of age. Of course, other ages and ranges are also contemplated, such as 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, and 20 weeks or more. Alternatively, the immunogenic compositions of the present disclosure are administered at least 2 weeks prior to exposure to the rickettsial or chlamydial bacteria.

[0032] In some forms, the immunogenic compositions of the present disclosure are administered after infection with a Rickettsiales or Chlamydiales bacteria, in such situations, administration of the immunogenic compositions of the present disclosure reduces the duration or severity of clinical symptoms associated with or resulting from the infection.

[0033] In another aspect, the disclosure provides an immunogenic composition comprising a bacterium of the order Rickettsiales or Chlamydiales containing a targeted allelic exchange variant and a component selected from the group consisting of a veterinarily acceptable carrier, a pharmaceutically acceptable carrier, an adjuvant, a preservative, a buffer, an antibiotic, a cell culture supernatant, an immunomodulator, and any combination thereof. In some embodiments, the bacterium of the order Rickettsiales or Chlamydiales is selected from the group consisting of Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, Orientia, and Chlamydia species. In some embodiments, the bacterium of the Ehrlichia species is selected from the group consisting of Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia ruminantium, Ehrlichia muris, and / or Ehrlichia muris-like agents. In some embodiments, the bacterium of the Anaplasma species is selected from the group consisting of Anaplasma phagocytophilum, Anaplasma platys, and Anaplasma marginale. In some forms, the targeted allelic exchange variant inactivates a gene. In some forms, the inactivation is due to an insertion or deletion. In some forms, the inactivation reduces the bacterial replication capacity. In some forms, the targeted allelic exchange variant is present in Ehrlichia chaffeensis Ech_0230, Ech_0379, or Ech_0660. In some forms, the targeted allelic exchange variant is present in Ehrlichia canis Ecaj_0381. In some forms, the targeted allelic exchange variant is present in Anaplasma phagocytophilum APH_0634.

[0034] In another aspect, the present disclosure provides a method for reducing at least one of the incidence and severity of clinical symptoms caused by a Rickettsiale or Chlamydiale bacteria, comprising administering an immunogenic composition comprising a Rickettsiale or Chlamydiale bacteria containing a targeted allelic exchange variant and an ingredient selected from the group consisting of a veterinarily acceptable carrier, a pharmaceutically acceptable carrier, an adjuvant, a preservative, a buffer, an antibiotic, a cell culture supernatant, an immunomodulator, and any combination thereof. In some forms, the administering step uses an administration method selected from the group consisting of intravenous, intramuscular, intranasal, intradermal, intratracheal, intravaginal, intravenous, intravascular, intraarterial, intraperitoneal, oral, intrathecal, or direct injection into any target tissue. In some forms, the administration of the immunogenic composition is referred to as a first administration, and this first administration is followed by a second administration. In some forms, the second administration is administered at least seven days after the first administration. In a preferred embodiment, the reduction in incidence is in animals administered the immunogenic composition, and the reduction in incidence is at least 10% compared to a group of animals not administered the immunogenic composition. In a preferred embodiment, the reduction in severity is assessed in a single animal administered the immunogenic composition and compared to animals not administered the immunogenic composition. In a preferred embodiment, the reduction in severity is at least 10% in animals administered the immunogenic composition compared to animals not administered the immunogenic composition that are subsequently infected or challenged with a Rickettsiales or Chlamydiales bacterium. In some embodiments, the reduction in severity in a group of animals administered the immunogenic composition is at least 10% compared to a group of animals not administered the immunogenic composition. In some embodiments, the Rickettsiales or Chlamydiales bacterium is selected from the group consisting of Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, Orientia, and Chlamydia. In some embodiments, the bacterium of the genus Ehrlichia is selected from the group consisting of Ehrlichia chaffeensis, Ehrlichia ruminantium, Ehrlichia muris, and Ehrlichia canis.In some embodiments, the Anaplasma bacterium is selected from the group consisting of Anaplasma phagocytophilum, Anaplasma platys, and Anaplasma marginale. In some embodiments, the targeted allelic exchange variant inactivates a gene. In some embodiments, the inactivation is due to an insertion or deletion. In some embodiments, the inactivation reduces the replicative capacity of the bacterium. In some embodiments, the targeted allelic exchange variant is present in Ehrlichia chaffeensis strain Ech_0230, Ech_0379, or Ech_0660. In some embodiments, the targeted allelic exchange variant is present in Ehrlichia canis strain Ecaj_0381. In some embodiments, the targeted allelic exchange variant is present in Anaplasma phagocytophilum strain APH_0634. In some embodiments, the component is an adjuvant selected from the group consisting of saponin, cyclic GMP-AMP, montanide gel, and any combination thereof. [Brief explanation of the drawings]

[0035] [Figure 1]

[0033] Figure 1 is a schematic diagram of the strategy used to generate targeted allelic exchange mutants in E. chaffeensis to inactivate genes Ech_0230 and Ech_0379 and restore the inactivated gene function of Ech_0379. A and A' in the diagram refer to the 5' and 3' homology arms. [Figure 2A] Figure 1 shows a schematic representation of the plasmid map of pHR-Ech_0230-tuf-aadA, including identification of homologous arms. The plasmid sequence data for this construct has been deposited in GenBank (accession number #MF068805). [Figure 2B] Figure 1 shows a schematic representation of the plasmid map of pHR-Ech_0379-tuf-aadA, including identification of homologous arms. Plasmid sequence data for all constructs have been deposited in GenBank (accession number #MF068806). [Figure 2C] Schematic representation of the plasmid map, including identification of homologous arms. Plasmid sequence data for this construct has been deposited in GenBank (accession number MF068807). [Figure 3A] Figure 1 shows targeted allelic exchange mutagenesis to disrupt the Ech_0230 gene. The genomic segment spanning the region selected for preparing the allelic exchange construct is shown, including the restriction enzyme sites (EcoRI (E) and ClaI (C)) used to map the insertion. The genomic coordinates of the restriction enzyme sites and the size of the insert (tuf-aadA) determined the expected DNA size for PCR and Southern blot analysis. [Figure 3B] Figure 1 shows the amplicons resolved following three different PCRs (L, 1 kb and molecular weight DNA markers; wild-type (W), PCR with wild-type genomic DNA as template; mutant (M), PCR with mutant genomic DNA as template) using primers targeting the allelic insertion (primers identified as 1 and 4) and genomic regions upstream and downstream of the inserted DNA (primers 2 and 3). [Figure 3C] Figure 1 shows PCR DNA sequence verification of the insertion site in the targeted mutant. In the upper panel, the DNA sequence generated from the amplicon indicated above and to the left of the black arrow represents the sequence from the E. chaffeensis genome, and the sequence above and to the right of the black arrow represents the sequence inserted into the gene disruption mutant. In the lower panel, the DNA sequence generated from the amplicon indicated above and to the left of the black arrow represents the sequence from the E. chaffeensis genome, and the sequence above and to the right of the black arrow represents the sequence inserted into the gene disruption mutant. The sequence boundaries at the 5' and 3' insertion junctions are identified by small black arrows. Also, in the upper panel, the upper sequence is SEQ ID NO: 42, and the lower sequence is SEQ ID NO: 48. In the lower panel, the upper sequence is SEQ ID NO: 43, and the lower sequence is SEQ ID NO: 49. [Figure 3D] This is a photograph of Southern blot analysis (S-blot) of genomic DNA (W and M) digested with ClaI (C) or EcoRI (E). Blot analysis was performed using the aadA gene segment as a probe. [Figure 4A]Figure 1 shows targeted allelic exchange mutagenesis to disrupt the Ech_0379 gene. The genomic segment spanning the region selected for preparing the allelic exchange construct is shown, including the restriction enzyme sites (ClaI (C) and HindIII (H)) used to map the insertion. The genomic coordinates of the restriction enzyme sites and the size of the insert (tuf-aadA) determined the expected DNA size for PCR and Southern blot analysis. [Figure 4B] Figure 1 shows the amplicons resolved following three different PCRs (L, 1 kb and molecular weight DNA markers; wild-type (W), PCR with wild-type genomic DNA as template; mutant (M), PCR with mutant genomic DNA as template) using primers targeting the allelic insertion (primers identified as 1 and 4) and genomic regions upstream and downstream of the inserted DNA (primers 2 and 3). [Figure 4C] Figure 1 shows PCR DNA sequence confirmation of the insertion site in the targeted mutant. In the upper panel, the DNA sequence generated from the amplicon indicated above and to the left of the black arrow represents the sequence from the E. chaffeensis genome, and the sequence above and to the right of the black arrow represents the sequence inserted into the gene disruption mutant. In the lower panel, the DNA sequence generated from the amplicon indicated above and to the left of the black arrow represents the sequence from the E. chaffeensis genome, and the sequence above and to the right of the black arrow represents the sequence inserted into the gene disruption mutant. The sequence boundaries at the 5' and 3' insertion junctions are identified by small black arrows. Also, in the upper panel, the upper sequence is SEQ ID NO: 44, and the lower sequence is SEQ ID NO: 50. In the lower panel, the upper sequence is SEQ ID NO: 45, and the lower sequence is SEQ ID NO: 51. [Figure 4D] Photographs of Southern blot analysis of genomic DNA (W and M) digested with ClaI (C) and HindIII (H). Blot analysis was performed using the aadA gene segment as a probe. [Figure 5A] Targeted allele exchange mutagenesis to restore the Ech_0379 gene is shown, as in Figure 3, except that the upper diagram showing the genome segment represents the genome from the Ech_0379 mutant. [Figure 5B]Figure 1 shows an Ech_0379 gene-restored mutant culture expressing mCherry. Restored mutant organisms cultured in ISE6 cells were assessed for mCherry expression by confocal microscopy using a 40x magnification lens. mCherry expression is indicated by the arrow. [Figure 5C] Figure 1 shows the resolved amplicons following three different PCRs using primers targeting the genomic regions upstream and downstream of the allelic insertion (primers identified as 1 and 4) and the inserted DNA (primers 2 and 3) (L, 1 kb+ molecular weight DNA marker; wild-type (W), PCR with wild-type genomic DNA as template; mutant (M), PCR with mutant genomic DNA as template). [Figure 5D] Figure 5B shows targeted allele exchange mutagenesis to restore the Ech_0379 gene. This figure is similar to Figure 3C, except that the restriction enzyme and probe used in the Southern blot experiment (shown in Figure 5E) were Cla I and a DNA fragment representing the Ech_0379 gene, respectively. Also, in the upper figure, the upper sequence is SEQ ID NO: 46, and the lower sequence is SEQ ID NO: 52. In the lower figure, the upper sequence is SEQ ID NO: 47, and the lower sequence is SEQ ID NO: 53. [Figure 5E] This figure shows a photograph of Southern blot analysis of genomic DNA (W and M) digested with ClaI (C) or EcoRI (E). The aadA gene segment was used as a probe illustration for the blot analysis. Lanes M1 and M2 represent data from genomic DNA recovered from E. chaffeensis Ech_0379 mutant cultures recovered from DH82 and ISE6 cultures, respectively. Similarly, lanes R1 and R2 represent data from genomic DNA recovered from E. chaffeensis Ech_0379 revertant cultures recovered from DH82 and ISE6 cultures, respectively. [Figure 6A]Transcriptional analysis of RNA recovered from wild-type and allelic exchange mutant E. chaffeensis organisms assessed by RT-PCR. RT-PCR products from wild-type (W) and Ech_0230 mutant (M) organisms were resolved (L, 1 kb and molecular weight DNA markers were resolved; +, genomic DNA from wild-type E. chaffeensis was used as template; -, negative control reaction in which no template was added). [Figure 6B] Similar to Figure 6A, except that the analysis was performed using RNA recovered from Ech_0379 disruption (M) and restoration (R) mutant organisms. Positive controls for this experiment included genomic DNA as templates from W, M, and R (a 0.38 kb amplicon is expected for W and R DNA templates in PCR, and a 1.6 kb product is expected for M DNA as template). [Figure 6C] Transcriptional analysis of RNA recovered from wild-type and allelic exchange mutant E. chaffeensis organisms assessed by RT-PCR is shown. Mutations that inactivate Ech_0379 and restore gene activity did not alter gene expression from neighboring genes. Semiquantitative RT-PCR analysis was performed on wild-type, gene-inactivated, and gene-rescue mutants, Ech_0378, Ech_0379, and Ech_0380, at 30, 35, and 40 PCR cycles; data from 35 cycles are presented. W, M, and R had similar amounts of amplicons for Ech_0378 and Ech_0380; Ech_0379 amplicons were also similar for W and R but absent for M. [Figure 7]

[0039] Figure 1 shows phenotypic characterization of the Ech_0379 gene in the antiporter-deficient E. coli strain EP432. RT-PCR analysis was performed targeting the Ech_0379 transcript in EP432. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present disclosure provides a method for providing a stable immunogenic bacterium capable of generating an immunogenic response in a host, the method preferably comprising the steps of a targeted disruption in the genome of a bacterial strain, followed by allelic exchange.

[0037] The present disclosure provides immunogenic compositions or vaccines comprising the immunogenic bacteria disclosed herein. The immunogenic bacteria for use in the immunogenic compositions or vaccines of the present disclosure can be killed, modified killed, modified live bacteria, recombinant proteins, proteins, or combinations thereof. In a preferred form, the modified live immunogenic bacteria is attenuated.

[0038] Methods of preventing or treating at least one of rickettsiosis, ehrlichiosis, Rocky Mountain spotted fever, human monocytic ehrlichiosis, granulocytic anaplasmosis, and / or anaplasmosis are provided. The methods of the disclosure generally include administering to a human or animal in need thereof an immunogenic composition or vaccine disclosed herein.

[0039] Also provided are methods for reducing the incidence or severity of clinical symptoms associated with at least one of rickettsiosis, ehrlichiosis, Rocky Mountain spotted fever, human monocytic ehrlichiosis, granulocytic anaplasmosis, and / or anaplasmosis, comprising administering an immunogenic composition or vaccine disclosed herein to a human or animal in need thereof. Clinical symptoms generally include, but are not limited to, fever, headache, chills, fatigue, muscle aches, abdominal pain, nausea, vomiting, diarrhea, confusion, conjunctival injection (red eyes), rash, and combinations thereof. Preferably, clinical symptoms associated with at least one of rickettsiosis, ehrlichiosis, Rocky Mountain spotted fever, human monocytic ehrlichiosis, granulocytic anaplasmosis, and / or anaplasmosis are reduced in incidence and / or severity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to an animal or human not administered the immunogenic composition or vaccine of the present disclosure. Comparisons to groups of animals or humans are also contemplated herein.

[0040] The recipient of the products and methods of the present disclosure can be a human or an animal. The animal is preferably selected from, but not limited to, pigs, cows, goats, horses, dogs, deer, coyotes, cats, poultry, and other related wild and domestic animals. In a preferred embodiment, the recipient is a human, dog, cow, horse, or pig.

[0041] A modified or altered native nucleotide sequence is understood to mean any nucleotide sequence obtained by mutagenesis according to techniques well known to those skilled in the art and which comprises modifications to the normal sequence according to the present disclosure, for example mutations in the regulatory and / or promoter sequences of the polypeptide expression, which in particular result in a modification of the rate of expression of the polypeptide or a modulation of the replication cycle.

[0042] In the present disclosure, nucleotide, polynucleotide, or nucleic acid sequence is understood to mean both double-stranded or single-stranded DNA in the form of monomers and dimers (so-called tandems) and the transcription products of said DNA.

[0043] It should be understood that the present disclosure does not relate to genomic nucleotide sequences obtained in their natural environment, i.e., in a natural state. The present disclosure relates to sequences that can be isolated, purified, or partially purified beginning with separation methods (e.g., molecular size exclusion or affinity ion exchange chromatography, or other separation techniques based on solubility in different solvents), or that can be implemented in vectors beginning with genetic engineering methods (e.g., amplification, cloning, and subcloning). Additionally, the sequences of the present disclosure have been modified from those found in nature to include mutations induced by site-directed mutagenesis or other attenuation techniques, such as serial passage, further indicating that the sequences of the present disclosure have been created by the hand of man and are not found in nature.

[0044] The attenuated modified live vaccines or immunogenic compositions of the present disclosure are constructed by human hands and therefore do not contain nucleotide or amino acid sequences found in nature. Therefore, the immunogenic compositions or vaccines of the present disclosure are significantly different from those found in nature. Because the immunogenic compositions or vaccines of the present disclosure have additional elements, such as mutations in the sequence or viral inactivation, that provide functionally different properties from naturally occurring Ehrlichia chaffeensis, they are similar to claim 2 in Example 5 for examples of naturally occurring products that are patent-eligible under 35 U.S.C. § 101 issued by the U.S. Patent Office in 2014.

[0045] Approximately 1 kb of Ehrlichia chaffeensis (E. chaffeensis) genomic DNA segments upstream and downstream of the previously defined random insertion mutation sites in the Ech_0230 and Ech_0379 genes were obtained by PCR and cloned into a plasmid vector. The promoter segment of the E. chaffeensis elongation factor Tu gene, Tuf-2 (Ech_0407), was similarly cloned into a separate plasmid in front of the aadA gene coding sequence (the aadA gene confers resistance to spectinomycin and streptomycin). The Tuf-2 gene promoter (tuf) was selected for aadA protein expression because it drives the expression of Tu, a highly conserved and constitutively expressed protein required for polypeptide elongation in the protein translation machinery. Furthermore, our bioinformatics analysis and transcriptional mapping by primer extension experiments suggested that tuf is a strong promoter involved in the transcription of genes, most of which encode 30S and 50S ribosomal proteins and have multiple transcription start sites (not shown). The aadA gene was selected because it works well in conferring antibiotic resistance in E. chaffeensis and Anaplasma species. The tuf-aadA segment was engineered into homologous recombination constructs Ech_0230 and Ech_0379 (pHR-Ech_0230tuf-aadA and pHR-Ech_0379tuf-aadA). Linear DNA fragments from the constructs containing the 5' and 3' homologous arms of the gene separated by the tuf-aadA segment were generated by PCR for use in generating targeted mutants.To generate a rescue mutagenesis template to reverse the targeted gene mutation in the Ech_0379 gene, a 0.5-kb fragment downstream from the mutation site in the gene was obtained by PCR from the E. chaffeensis genome and engineered into the pHR-Ech_0379tuf-aadA construct to generate the modified construct: pHR-res-Ech_0379-Amtr-mCh-Gent, which contains the entire Ech_0379 gene ORF at its 5' end, followed by the Amtr promoter, mCherry, and gentamicin resistance cassette (Gent) ORFs (Amtr-mCh-Gent), and a 1-kb genomic segment containing the 3' portion of the Ech_0379 gene. Gent was codon-optimized for efficient translation in E. chaffeensis. Next, a linear fragment was generated from the rescue construct, beginning with the Ech_0379 gene and containing a 5' homologous arm containing the Amtr-mCh-Gent segment and the 3'-terminal genomic segment downstream of the Ech_0379 insert, serving as the 3' homologous arm. Linear DNA fragments for disrupting the Ech_0230 or Ech_0379 gene were electroporated into host-cell-free wild-type E. chaffeensis organisms recovered from ISE6 tick cells, followed by reinfection of the ISE6 tick cells. These mutants were selected for their ability to grow for several weeks in medium containing spectinomycin and streptomycin and then infected into the macrophage cell line DH82, where they were continuously propagated for several months. For rescue mutation experiments, linear DNA fragments of the Ech_0379 gene restoration template were similarly electroporated into E. chaffeensis organisms containing mutations within the Ech_0379 gene. E. chaffeensis cultures with restored Ech_0379 genes were then selected for their ability to grow in gentamicin-containing medium. After recovery of E. chaffeensis cultures grown in antibiotic-containing medium, target gene inactivation in Ech_0230 or Ech_0379 was confirmed by two insertion-specific PCR analyses: (1) the genomic region 5' to the allelic exchange site and insertion-specific DNA, and (2) the genomic region 3' to the insert DNA and allelic exchange site.Clonal purity was then confirmed by additional PCR analysis targeting genomic regions upstream and downstream of the allelic exchange insertion site. The integrity of the PCR product was confirmed by PCR-DNA sequence analysis. The generation of Ech_0379 gene-restored mutants was also assessed for Cherry protein expression by fluorescence microscopy (Figure 5B). The presence of the mutation in the E. chaffeensis genome was also confirmed by Southern blot analysis for both gene-disrupted and gene-restored mutants. RT-PCR analysis revealed that Ech_0230 and Ech_0379 transcripts were present in wild-type E. chaffeensis but absent in gene-disrupted mutant organisms. Complemented mutants, like wild-type E. chaffeensis, were positive for Ech_0379 transcripts. Furthermore, we tested whether allelic exchange mutants that inactivate and restore Ech_0379 gene activity could cause polar effects in altering gene expression from neighboring genes. This analysis was performed by semiquantitative RT-PCR using three sets of PCR cycles (30, 35, and 40). Regardless of the PCR cycle number, the RT-PCR products of Ech_0378 and Ech_0380 were similar in the wild-type, gene-inactivated mutant, and gene-rescued mutant. The RT-PCR product of Ech_0379 was absent only in the gene-inactivated mutant but similar in the wild-type and gene-rescued mutant (Figure 6C). There was no evidence supporting the presence of off-target inserts generated during all three mutation experiments. The Ech_0379 gene open reading frame was completely restored in front of its own promoter from the complementing allele exchange mutation experiment, and therefore, its gene structure was similar to that of wild-type E. chaffeensis, except for the additional expression of mCherry and gentamicin resistance proteins. This modified E. chaffeensis, which resembles the wild-type in that it has a complete genome, will be useful for novel studies in real-time pathogen monitoring by fluorescence imaging in vitro and in vivo, similar to previous studies published for Borrelia burgdorferi.

[0046] This specification discloses the content of the present invention including the best mode using examples, and enables those skilled in the art to implement the present invention (including the production and use of any device or system, and the implementation of any method incorporated in the described content). The patented technical scope of the present invention is specified by the claims in the claims, and other embodiments conceivable by those skilled in the art may also be included therein. Such other embodiments shall be within the technical scope specified by those claims if they include components that are not different from the language of each claim, or if they include equivalent components that are not substantially different from the language of each claim.

[0047] Examples

[0048] Example 1

[0049] Materials and Methods

[0050] <In Vitro Culture of E. chaffeensis> The E. chaffeensis Arkansas isolate was passaged in the ISE6 tick cell line, an I. scapularis embryonic cell line (Elwell, C., Mirrashidi, K., and Engel, J., Nat. Rev. Microbiol.; 14, 385-400 (2016)). Also, the canine macrophage cell line (DH82) was used to culture E. chaffeensis according to a previously reported protocol (Walker, D.H., Paddocl, C.D. and Dumler, J.S., Med Clin North Am; 92, 1345-1361 (2008)).

[0051] <Construction of Recombinant Plasmids and Segments> All primers used for the preparation of recombinant plasmid constructs developed for targeted mutagenesis experiments are listed in Table 1. Also, the plasmids used and prepared in this experiment are listed in Table 2.

[0052] Table 1A

[0053] Table 1B

[0054]

Table 1C

[0055]

Table 1D

[0056] Table 2

[0057] The detailed molecular steps followed when generating constructs for allelic exchange mutagenesis experiments are shown in Figure 1. Platinum® Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA) was used to generate constructs in all PCR experiments. The Ech_0230 and Ech_0379 genes of E. chaffeensis were used to generate allelic exchange mutations, similar to the random mutations within these genes using the Himar1 random mutagenesis method previously reported by the present inventors. The mutants grew normally in in vitro culture. Approximately 2.0 kb genomic DNA segments spanning approximately 1 kb on either side of the previously identified mutation insertion sites in the gene (designated A and A' in Figure 1) were amplified from the E. chaffeensis genome (GenBank #CP000236.1; the Himar1 mutation insertion sites for Ech_0230 and Ech_0379 are 219,097 and 374,461, respectively). The genomic coordinates of the amplified segments of the Ech_0230 and Ech_0379 genes are 218,060–220,133 and 373,265–375,810, respectively. The amplicons were first cloned into the pCRM™2.1-TOPO TA vector (Life Technologies, Rockville, MD, USA) according to the manufacturer's instructions. Additionally, a 0.37-kb DNA segment of the Tuf-2 gene (Ech_0407) promoter (tuf) was amplified from the E. chaffeensis genome for use in constitutive expression of the aadA gene product, which confers resistance to spectinomycin and streptomycin (genomic coordinates of this segment are 396:385 to 396:751). The aadA gene open reading frame (ORF) was obtained by PCR from the pCis mCherry-SS Himar A7 plasmid (Sahni, SK, Narra, HP, Sahni, A, and Walker, DH; Future Microbiol; 8, 1265-1288 (2013).).The tuf promoter and aadA ORF were also cloned into a separate pCRM™2.1-TOPO TA vector, which was then used to generate a linear fragment of the tuf-aadA segment (Fragment 1) for incorporation into the final target gene disruption mutagenesis construct. Subsequently, a linear fragment (Fragment 2) was generated from the entire plasmid containing the gene fragment (pHR-Ech_0230 and pHR-Ech_0379, respectively) using Ech_0230 or Ech_0379 gene-specific primers designed to split the gene fragment into two equal halves located at each end of the linear fragment while maintaining the plasmid backbone in the center. Next, linear fragment 1 and linear fragment 2 were ligated together according to the Gibson Assembly protocol (New England Biolabs, Ipswich, MA, USA) to generate the final homologous recombination plasmid constructs, in which the gene segment was disrupted by insertion of the tuf-aadA segment. The final constructs were designated pHR-Ech_0230tuf-aadA and pHR-Ech_0379tuf-aadA, respectively (Figure 2A and Figure 2B) (GenBank application numbers 2012015 and 2012023; not yet accepted). Linear fragments from these constructs containing both the 5' and 3' homologous arms of each gene-disrupted segment along with the tuf-aadA cassette were then generated by PCR (Figure 3A). The amplicons were then resolved on a 1% agarose gel (Figure 3B). For use in allelic exchange mutagenesis experiments to generate targeted gene disruption, DNA was gel isolated and concentrated to 1 μg / μl in nuclease-free water.

[0058] To construct the Ech_0379 gene function rescue template, a 0.5 kb fragment downstream from the 3' end of the mutation site was generated by PCR using E. chaffeensis genomic DNA as a template (genomic coordinates 374,462 to 374,837). The Amtr-mCherry (Amtr-mCh) DNA segment, comprising the Anaplasma marginale transcription factor (Tr) gene promoter and mCherry ORF, was amplified using the pCis mCherry-SS Himar A7 plasmid as a template. The gentamicin resistance gene coding sequence (Gent) was commercially codon-optimized based on the frequently occurring codons in the E. chaffeensis genome (GenScript, Piscataway, NJ, USA) (GenBank #KB977452). The Gent segment was then used to clone downstream of the Amtr-mCh fragment, generating the Amtr-mCh-Gent fusion fragment. The 3'-terminal 0.5 kb Ech_0379 segment was then ligated to the 5'-terminal of the Amtr-mCh-Gent fragment by overlapping PCR. The final amplicon was then cloned into the Ech_0379tuf-aadA-HR1 construct using a Gibson assembly cloning strategy, replacing the tuf-aadA segment with the Amtr-mCh-Gent segment, including the 3'-terminal 0.5 kb Ech_0379 ORF segment. The final Ech_0379 rescue plasmid construct, pHR-res-Ech_0379-Amtr-mCh-Gent, contains the full-length Ech_0379 ORF restored in front of its own promoter, followed by Amtr-mCh-Gent and a 1-kb genomic segment at the 3' end of the Ech_0379 gene (Figure 2C) (GenBank application no. #2012033; not yet accepted). This construct was then used as a template to generate a linear fragment by PCR.The linear fragment produced contains, at the 5´ end, its own promoter and the complete ORF, followed by the entire Ech_0379 gene, then the Amtr-mCh-Gent segment and an additional 3´ end 1 kb segment downstream of the Ech_0379 gene mutation site (Figure 5A). The PCR product was purified by the QIAquick PCR Pruification Kit (Qiagen, Hilden, Germany) and concentrated to 1 μg / μl in nuclease-free water as described above for use in allelic exchange mutagenesis experiments to restore the integrity of the gene in E. shaeferensis organisms with Ech_0379 gene disruption.

[0059] <Purification of cell-free E. shaeferensis organisms> Five milliliters of E. shaeferensis cell culture from an approximately 80–90% confluent ISE6 cell culture flask was used to produce host cell-free E. shaeferensis microorganisms. Briefly, the infected cell suspension was harvested by centrifugation at 15,000 g for 10 minutes at 4°C, the supernatant was discarded, then 1.5 ml of 0.3 M ice-cold sucrose solution and 100 μl of autoclaved rocking tumbler grit #1 (60 / 90 grit silicon carbide, Lynden, WA, USA) were added to the cell pellet, and vortexed at maximum speed for 30 seconds using a tabletop vortex device to release the bacteria from the infected host cells. The cell suspension was then centrifuged at 200 g for 10 minutes at 4°C to pellet the host cell debris. The supernatant was carefully recovered into a 3 ml syringe and passed through a 1.6 μm filter (Whatman Ltd., Piscataway, NJ, USA). The filtrate containing E. shaeferensis was pelleted by centrifugation at 15,000 g for 10 minutes at 4°C. The cell pellet was washed twice with 0.3 M ice-cold sucrose solution resuspended in 45 μl of 0.3 M ice-cold sucrose solution and immediately used for electroporation experiments. <a

[0060] <Transformation of E. shaeferensis and clonal isolation of mutants> 3 - 10 μg of linear DNA fragments purified from the allergen - exchange mutagenesis - induced plasmid construct (outlined above) were added in an amount of 45 μl to a cell - free E. shaeferensis organism, gently mixed, and the contents were transferred to an electroporation cuvette with a 1 mm gap (Bio - Rad Laboratories, Hercules, California, USA). The cuvette was incubated on ice for 15 minutes and then subjected to electroporation at settings of 2,000 volts, 25 μF, and 400 Ω (Bio - Rad Laboratories, Gene Pulser Xcell(TM), Hercules, California, USA). The electroporated cells were transferred to a microcentrifuge tube containing 0.5 ml of FBS and 1 ml of non - infected ISE6 cell suspension containing approximately 1×10 individuals of ISE6 cells. This mixed sample was centrifuged at 5,000 g for 5 minutes, incubated at room temperature for 15 minutes, then the cells were resuspended in 5 ml of culture medium, and the entire contents were transferred to a T25 flask containing confluent ISE6 cells and incubated in a humidified incubator at 34°C for 24 hours. Then, 100 μg / ml each of spectinomycin and streptomycin were added to the culture medium. Incubation was continued at 34°C for several weeks to select mutants. Generally, mutants were detected by PCR analysis after 2 - 3 weeks, but the evaluation continued for several weeks beyond this point. Similar experiments were conducted to obtain Ech_0379 gene - restored mutants, except that a medium containing 80 μg / ml of gentamicin was used 24 hours after electroporation. Ech_0379 gene - restored mutant cultures were also evaluated by examining the cultures using a Nikon Diaphot inverted microscope (Nikon, Melville, New York, USA) to detect the expression of mCherry. After identification, antibiotic - resistant cultures were transferred to DH82 cell cultures for further growth and maintenance. Also, liquid nitrogen stocks were made and stored within the first 2 weeks after the establishment of the mutant strains.

[0061] <Confirmation of the presence of E. shaeferensis mutants> Cultures of E. chaffeensis that grew well in the presence of antibiotics were screened for allelic exchange mutations by genomic DNA analysis using insert-specific PCR. Total genomic DNA was recovered from the cultures using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Three PCR analyses were performed using the purified genomic DNA (Figure 4B). The first and second PCR analyses (Figure 4B, first panel) targeted (1) the genomic region 5' to the allelic exchange site and the insert-specific DNA (Figure 4B, second panel), and (2) the insert DNA and the 3' region of the genome to the allelic exchange site. The third PCR analysis (Figure 4B, third panel) was designed to test the clonal purity of the mutants. Primers used in this analysis targeted the genomic regions upstream and downstream of the allelic exchange insertion site (Figure 4C). PCR products were resolved on a 0.9% agarose gel to identify the specific predicted-length amplicon and then subjected to sequencing analysis to further confirm the integrity by mapping the genomic junction of the insertion from both ends of the amplicon. Mutations and clonal purity were then confirmed by Southern blot analysis (Figure 4D). Genomic DNA from mutant and wild-type organisms was subjected to restriction enzyme digestion using ClaI, EcoRI, or HindIII restriction enzymes. The digested DNA was resolved on a 1% agarose gel and transferred to a nylon membrane (Roche Diagnostics, Indianapolis, IN, USA). Insertion-specific aadA gene segment probes were used in Southern blot hybridization experiments to identify the location of the inserted DNA in the Ech_0230 and Ech_0379 targeted disruption mutants. Additionally, Ech_0379 gene segment probes were used to identify the location of the genomic insertion in Ech_0379 targeted gene insertion and restorer mutant clones according to standard procedures for DNA blot analysis.

[0062] <RNA analysis by RT-PCR to verify transcription loss and restoration> Total RNA from wild-type and mutant E. shafeensis organisms grown in ISE6 or DH82 cell culture was isolated using the TRI Reagent total RNA isolation method according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). The RNA samples were then treated with RQ1 DNase (Promega, Madison, WI, USA) at 37°C for 60 minutes to remove residual genomic DNA. Primers targeting Ech_0230 or Ech_0379 ORF were used for RT-PCR analysis, and the presence of specific amplicon was evaluated by 0.9% agarose gel analysis and by subjecting the products to DNA sequencing. Semi-quantitative RT-PCR analysis was performed as described above for the evaluation of mRNA expression from genes Ech_0378, Ech_0379, and Ech_0380 using equal amounts of E. shafeensis RNA recovered from wild-type mutants, Ech_0379 gene disruption mutants, and Ech_0379 gene restoration mutants (Figure 5B, Figure 5C, Figure 6A, and Figure 6B). This analysis was carried out at 30, 35, and 40 cycles (Figure 6C). Southern blot was used for confirmation (Figure 5E).

[0063] Example 2

[0064] Materials and Methods

[0065] <In Vitro Cultivation of E. canis and Anaplasma phagocytophilum> E. canis and A. phagocytophilum were passaged in the ISE6 tick cell line, an I. scapularis embryonic cell line (Munderloh, U.G. et al. J. Clin. Microbiol. 37, 2518-2524 (1999), and Cheng, C. & Ganta, R.R. Curr. Protoc. Microbiol. Chapter 3, Unit 3A 1 (2008)).

[0066] <Construction of Homologous Recombinant Plasmids and Segments> All primers used to generate recombinant plasmid constructs were developed for targeted mutagenesis experiments following a similar protocol as described for E. chaffeensis, except that pathogen gene target-specific primers were used. Similarly, the detailed molecular steps followed when generating constructs for allelic exchange mutagenesis experiments were similar to those following standard molecular methods described for E. chaffeensis (Sambrook, J. & Russell, DW. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. (2001)). Briefly, approximately 2.0 kb genomic DNA segments spanning approximately 1 kb on each side of the previously identified mutation insertion site of the gene were amplified from E. canis or A. phagocytophilum gene homologs, as in the case of the E. chaffeensis gene Ech_0660. The E. canis and A. phagocytophilum Ech_0660 homologous sequences are provided in this disclosure as SEQ ID NOs: 54 and 55, respectively. First, the amplicon was cloned into a plasmid vector. An Ehrlichia or Anaplasma gene promoter driving an antibiotic selectable marker gene, along with a fluorescent reporter gene, is then inserted into the final targeted gene disruption mutagenesis construct, splitting the bacterial sequence into approximately equal halves. Linear fragments are then generated by PCR from the entire recombinant plasmid containing the E. canis or A. phagocytophilum-specific disrupted gene segment. The amplicon is then purified and concentrated to 1 μg / μl in nuclease-free water for use in allelic exchange mutagenesis experiments to generate the targeted gene disruption.

[0067] Purification of cell-free E. canis and A. phagocytophilum organisms The purification method for recovering host cell-free organisms of E. canis or A. phagocytophilum film is essentially the same as that of E. shafeiensis described above in Example 1, but about 80-90% infected confluent ISE6 cell culture flasks containing E. canis or A. phagocytophilum film are used respectively, and it is different in that specific host cell-free organisms are prepared according to the protocol in "Felsheim, R.F. et al. BMC Biotechnol. 6, 42 (2006)".

[0068] <Transformation of E. shafeiensis and Clonal Isolation of Mutants> 3 μg of the purified linear DNA fragment from the allelic exchange mutagenesis plasmid construct (outlined above) was added in an amount of 45 μl to the host cell-free E. shafeiensis organisms, gently mixed, and the contents were transferred to an electroporation cuvette with a 1 mm gap (Bio-Rad Laboratories, Hercules, California, USA). The cuvette was incubated on ice for 15 minutes and then subjected to electroporation at settings of 2,000 volts, 25 μF, and 400 Ω (Bio-Rad Laboratories, Hercules, California, USA, Gene Pulser Xcell(TM)). The electroporated cells were combined with 0.5 ml of FBS and approximately 1×10 in tick cell culture infection medium 6Transferred to a microcentrifuge tube containing 1 ml of non-infected ISE6 cell suspension containing individual ISE6 cells. This mixed sample was centrifuged at 5,000 g for 5 minutes, incubated at room temperature for 15 minutes, then the cells were resuspended in 5 ml of culture medium, and the entire content was transferred to a T25 flask containing confluent ISE6 cells and incubated in a humidified incubator at 34 °C for 24 hours. Thereafter, 100 μg / ml each of spectinomycin and streptomycin were added to the culture medium. Incubation was continued at 34 °C for several weeks to select mutants. Generally, mutants were detected by PCR analysis after 2 - 3 weeks, but the evaluation continued for several weeks beyond this point. Similar experiments were conducted to obtain Ech_0379 gene revertant mutants, except that medium containing 80 μg / ml of gentamicin was used 24 hours after electroporation. Also, Ech_0379 gene revertant mutant cultures were evaluated by examining the cultures using a Nikon Diaphot inverted microscope (Nikon, Melville, NY, USA) to detect the expression of mCherry. After identification, antibiotic-resistant cultures were transferred to DH82 cell cultures for further growth and maintenance. Also, liquid nitrogen stocks were prepared and stored within the first 2 weeks after the establishment of the mutant strains.

[0069] <Confirmation of the presence of mutants of E. canis or A. phagocytophilum> Cultures of E. canis or A. phagocytophilum that grew well in the presence of antibiotics were screened for allelic exchange mutation positivity by genomic DNA analysis by insertion-specific PCR. The protocol was the same as that described for E. shafenesis.

[0070] <RNA analysis by RT-PCR to verify transcriptional loss and restoration> Total RNA from wild-type and mutant E. canis or A. phagocytophilum organisms was isolated according to the TRI Reagent total RNA isolation method and treated with RQ1 DNase. Pathogen-specific primers were used for RT-PCR analysis, and the presence of specific amplicons was assessed by agarose gel analysis and by subjecting the products to DNA sequencing (Sambrook, J. & Russell, DW. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. (2001)).

[0071] Example 3

[0072] Materials and Methods

[0073] In vitro culture and recovery of cell-free E. chaffeensis

[0074] The wild-type and mutant strains of the Arkansas isolate of E. chaffeensis were propagated in the canine macrophage cell line DH82. The isolation and purification of cell-free E. chaffeensis wild-type and mutant strains were performed as follows. Briefly, bacterial infection rates in DH82 cells were assessed by Diff-Quik staining. At 72 h postinfection, when infection reached approximately 80–90%, cultures from four T-150 confluent flasks were harvested and centrifuged at 500 × g for 5 min. The cell pellet was resuspended in 1× phosphate-buffered saline (PBS) containing protease inhibitors (Roche, Indianapolis, IN, USA), and the cells were homogenized on ice by 15–20 strokes using a 23 g needle in a 10 mL syringe. The homogenization efficiency (80–90% lysis) was examined under a light microscope. The whole cell lysate was centrifuged at 500 × g for 5 min at 4°C. The resulting supernatant containing cell-free Ehrlichia organisms was filtered through a 2 μm sterile membrane filter (Millipore, Billerica, MA, USA). Cell-free Ehrlichia from the filtrate were pelleted by centrifugation at 15,000 × g for 15 minutes. The pellet was suspended in PBS and then layered on top of a 30% diatrizoate meglumine and sodium solution (Renografin) MD-76R (Mallinckrodt Inc, St. Louis, MO, USA). The suspension was centrifuged at 100,000 × g for 1 hour at 4°C in an S50-ST swinging bucket rotor (Beckman, Indianapolis, IN, USA). The cell-free Ehrlichia pellet was washed at 15,000 × g for 15 minutes and used for experiments.

[0075] Bacterial mRNA enrichment and sequencing

[0076] Bacterial RNA enrichment, cDNA library construction, and RNA sequencing were performed as follows. Briefly, RNA from wild-type and mutant strains was isolated from purified Ehrlichia cells using TRIzol Reagent (Sigma-Aldrich, St. Louis, MO, USA). Bacterial RNA was then enriched by treating the RNA samples with DNase I (Invitrogen, Carlsbad, CA, USA) and removing host 18S rRNA, 28S rRNA, and polyadenylated mRNA using the MICROBEnrich Kit (Ambion, Foster City, CA, USA). The quantity and integrity of bacterial RNA before and after enrichment were assessed using a NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and an Agilent2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). mRNA was isolated from total RNA samples using a Ribo-Zero Magnetic Kit and then fragmented into short fragments according to the manufacturer's protocol (Epicentre, Madison, WI, USA). cDNA was then synthesized using the mRNA fragments as templates. Wild-type and mutant cDNA libraries were generated using the TruSeq RNA Sample Prep Kit (Illumina, Ingolstadt, Germany). Sample libraries were quantified using an Agilent 2100 Bioanalyzer. The quality of the sample libraries was assessed by real-time PCR (ABI StepOnePlus) before the samples were subjected to sequencing on an Illumina HiSeq™ 4000 (Beijing Genomics Institute, Philadelphia, PA, USA).

[0077] Bioinformatics analysis

[0078] The original image data was converted into raw sequence data by base calling. The raw reads were subjected to quality assessment to determine whether they were suitable for mapping. Low-quality bases (<20) were excluded from analysis. Next, the raw reads were filtered to exclude adapter sequences and low-quality reads. Then, SOAPaligner / SOAP2 was used to align the clean reads to the complete E. chaffeensis Arkansas strain genome according to the original annotated GenBank accession number CP000236.1. This accession number was selected and used because it has been widely used in our previous publications and by other researchers as well to reference the gene names and numbers listed there. A maximum of five mismatches was allowed in the alignment, which is the standard cutoff used for alignment analysis. The aligned data was used to calculate the distribution of reads on the reference gene and determine gene coverage. The alignment results were evaluated for quality checks and then proceeded to DGE analysis. Gene expression levels were calculated using the RPKM method, which normalizes the total read length and the number of sequence reads. The thresholds for determining significant differences in gene expression were a p-value < 0.05, a false positive rate (FDR) ≤ 0.001, and an absolute Log2 ratio ≥ 1. The FDR uses the exact p-value as a measure of control in multiple sample comparisons of RNA-seq data. False positive and false negative error corrections were performed using the Benjamini-Yekutieli method.

[0079] Quantitative real-time reverse transcription PCR

[0080] Quantitative real-time reverse transcription PCR (qRT-PCR analysis) based on SYBR Green detection was performed to verify the gene expression changes observed in the RNA-seq data analysis. Transcript levels were also determined using the wild-type, ECH_0379 mutant, ECH_0490 mutant, and ECH_0660 mutant RNAs used to generate the RNA-seq data. Transcript levels were determined by quantitative RT-PCR with SYBR Green analysis using the SUPERSCRIPT® III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA, USA). RNA from all replicates was reverse transcribed using SuperScript III, followed by quantitative PCR in 25 μL reactions containing 0.5 μM each of forward and reverse primers. Thermal cycling conditions were 40 cycles of 94°C for 15 seconds, 60°C for 30 seconds, and 74°C for 15 seconds. Thirteen randomly selected differentially transcribed genes were used in validation experiments using a StepOnePlus™ real-time PCR instrument (Applied Biosystems, Foster City, CA, USA), and data were analyzed using StepOne Software v2.3. E. chaffeensis 16S rRNA was quantified by real-time RT-PCR and used to normalize RNA concentrations between different RNA batches before conducting validation experiments. For qRT-PCR data, relative changes in expression were calculated using delta-delta Ct (ΔΔCt) calculations, and fold changes were determined by averaging replicate values ​​and standard errors of gene expression. Semiquantitative one-step RT-PCR (Life Technologies, Carlsbad, CA, USA) targeting the E. chaffeensis genes ECH_0490 and ECH_0492, located near the transposon mutation downstream of the ECH_0490 gene, was performed with 30 cycles of amplification using gene-specific primers described in a previous study (PLoS One; 10, e0132657 (2015)). Briefly, RNA from wild-type and ECH_0490 mutants was used as templates for RT-PCR.One tube without reverse transcriptase or template RNA served as a negative control. One tube with DNA as a template served as a positive control. The thermal cycler conditions were 50°C for 1 hour, 35°C for 94 seconds, 30°C for 55 seconds, and 30 cycles of 30°C for 72 seconds for the reverse transcription step. A final 2-minute extension step at 72°C was part of the reaction.

[0081] The rickettsial pathogen Ehrlichia chaffeensis causes human monocytic ehrlichiosis, a tick-borne disease. Mutations within specific genomic locations of the pathogen are useful for understanding pathogenesis and developing attenuated vaccines. Our previous research has shown that mutations at different genomic sites in E. chaffeensis have various effects on their growth and attenuation in vertebrate and tick hosts. Here, we used RNA deep sequencing technology to evaluate the transcriptional effects of three mutations. RNA sequencing was useful for detecting 66–80% of wild-type and mutant E. chaffeensis transcripts. Mutations in the antiporter gene (ECH_0379), which suppresses growth in vertebrate hosts, resulted in the downregulation of many transcriptional genes. Similarly, mutations downstream of the ECH_0490 coding sequence had little effect on the pathogen's in vivo growth but resulted in significant changes in its transcriptome. This mutation led to enhanced expression of several host stress response genes. Although ECH_0660 mutations cause rapid pathogen clearance in vertebrate hosts and aid in the generation of defense responses, they had minimal impact on the transcriptome. The transcriptome data provide novel insights into the impact of mutations on global gene expression and how they contribute to pathogen resistance and / or clearance from the host.

[0082] Ehrlichia chaffeensis is a tick-borne intracellular bacterial pathogen that causes human monocytic ehrlichiosis (HME) and also infects dogs, deer, goats, and coyotes. Mutations at specific genomic locations result in changes in gene expression, affecting the pathogen's ability to infect and persist in the host. The E. chaffeensis genome evolves within the host cellular environment, potentially developing mechanisms to dampen the host immune response. E. chaffeensis genes associated with pathogenesis are likely highly activated within the host microenvironment, and consistent with this hypothesis, differential gene expression is known to occur in response to host cellular defenses. Progress has been made toward identifying genes important for Ehrlichia survival within the host cellular environment. However, to date, only a few abundantly expressed genes have been identified as relevant to pathogenesis. Uncovering genes involved in pathogenesis and virulence and documenting their differential expression will aid in the discovery of novel proteins that may be valuable targets for therapeutic intervention and vaccine development against HME.

[0083] Genetically mutated intracellular pathogens are important resources for studying microbial pathogenesis and can also aid in vaccine development. Our previous work demonstrated the feasibility of transposon-based mutagenesis in E. chaffeensis. We also found that several insertion mutations resulting in transcriptional inactivation of membrane protein genes attenuated pathogen growth in vertebrate hosts. Insertions within the coding regions of the ECH_0379 and ECH_0660 genes provided varying levels of protection against infection in vertebrate hosts. In this study, we hypothesized that the specific genomic location of the mutations affects global gene expression, contributing to altered pathogen survival, infection progression, and replication in the host cellular environment. To test this hypothesis, we evaluated the effects of three mutations previously reported by Cheng et al. on global gene transcription. Two mutants were selected, one carrying mutations within the coding region of the ECH_0660 gene, encoding a phage-like protein (ECH_0660), and the other within the coding region of the ECH_0379 gene, encoding an antiporter protein (ECH_0379). The insertion mutation in the ECH_0660 gene is located at nucleotide position 213 of the 555-base-long open reading frame. Similarly, the mutation in the ECH_0379 gene is located at nucleotide position 682 of the 1056-base-long open reading frame. The third insertion mutant, ECH_0490, has an insertion mutation 166 nucleotides downstream from the stop codon of the ECH_0490 gene.

[0084] High-throughput RNA sequencing (RNA-seq) technology has proven to be a reliable and robust tool for determining global transcriptome activity in obligate intracellular bacteria. Comparative genomic studies have identified several classes of virulence factors involved in the secretion, transport, and regulation of host immune responses of molecules between pathogens and host cells. However, studies focusing on Ehrlichial gene expression have been primarily limited to outer membrane protein genes, type IV secretion system (T4SS) genes, tandem repeat protein (TRP) genes, and ankyrin repeat genes (Anks). Among them, genes encoding T4SS proteins and p28-OMP proteins have been found to be important for virulence.

[0085] The obligate intracellular nature of E. chaffeensis makes obtaining cell-free ehrlichia from host cells problematic. Technical limitations in isolating ehrlichial RNA from highly abundant host RNA pose an obstacle to profiling pathogen transcripts. To overcome this drawback, we employed an effective cell lysis strategy followed by density gradient centrifugation. Furthermore, we enriched ehrlichial RNA by efficiently removing polyadenylated RNA (poly(A) RNA) and eukaryotic and prokaryotic ribosomal RNA from the host and bacterial RNA mixture. Sequencing of the enriched RNA aided in the detection of transcripts for 66–80% of the annotated E. chaffeensis genes according to the annotated genome (GenBank #CP000236.1). Comparison of transcript levels from wild-type and mutant strains revealed the highest degree of regulation in immunogenic and secreted protein genes, especially in the ECH_0490 and ECH_0379 mutants, whereas only minor changes were observed in the ECH_0660 mutant.

[0086] result

[0087] Isolation and purification of cell-free E. chaffeensis from host cells

[0088] A major problem when conducting transcriptome studies of intracellular pathogens is the difficulty of isolating host cell-free bacteria and subsequently recovering high-quality bacterial RNA. Rickettsia organisms, including E. chaffeensis, account for only a small fraction of the total RNA isolated. Due to the presence of highly abundant host cell RNA, recovering bacterial RNA is a challenge in conducting RNA-seq analysis experiments. In this study, we first purified host cell-free bacteria from infected host cells (canine macrophage cell line DH82) using an efficient cell lysis method combined with a density gradient centrifugation protocol. Host cell lysis was performed to efficiently destroy host cells without significantly damaging the bacteria. E. chaffeensis organisms have a diameter of approximately 0.5–1 μm. Therefore, we filtered the infected host cell lysate through a 2 μm membrane to remove most of the host cell debris. High-speed Renograffin density gradient centrifugation of the resulting E. chaffeensis cell suspension helped pellet the bacteria, but host cell debris remained in the top layer of the solution. After total RNA isolation and DNase treatment, bioanalyzer analysis revealed that host 28S and 18S RNA remained at high concentrations in the recovered RNA, despite prior fractionation of host cell-free bacteria. Bacterial mRNA enrichment was performed by depleting host poly(A) RNA and eukaryotic ribosomal RNA using a bacterial RNA enrichment protocol, resulting in nearly undetectable levels of host 28S and 18S RNA. The absence of contaminating E. chaffeensis genomic DNA in the purified RNA samples was confirmed by real-time quantitative PCR using E. chaffeensis 16S rRNA gene primers. The absence of DNA sequences in the raw RNA-seq data was also confirmed by aligning 20 randomly selected E. chaffeensis intergenic non-coding DNA sequences (data not shown).

[0089] Ubiquitous transcription of genes in E. chaffeensis mutants

[0090] RNA sequencing of E. chaffeensis wild-type and mutant strains using an Illumina HiSeq. 4000 system generated 75–130 million reads. Transcriptome data were deposited under NCBI BioProject ID: PRJNA428837 and SRA accession number: SRP128532 (see website nlm.nih.gov / sra / SRP128532). Despite efficient depletion of host ribosomal RNA, only a fraction of the reads (less than 19 percent) mapped to the E. chaffeensis genome. Mapping of the reads (≥10 reads / gene) identified approximately 66–80% of the genes expressed by the Ehrlichia genome based on the annotated genome (GenBank #CP000236.1). The transcriptome of the wild-type organism (n = 3) contained transcripts of approximately 920 genes out of a total of 1158 genes, and similarly, 888, 895, and 768 gene transcripts (n = 3) were identified in the mutant organisms ECH_0660, ECH_0379, and ECH_0490, respectively (Table 3). 2 =0.9) and mutant ECH_0379 (R 2 =0.93), ECH_0490(R 2 =0.68), and ECH_0660(R 2 Replicate RNA-seq data showed a high degree of expression correlation. 2 =0.18), and wild type vs. ECH_0490 (R 2 =0.38), the scatter plot expression data showed a negative correlation. Notably, the expression plot of wild type versus ECH_0660 showed a positive correlation (R 2 =0.96). Only transcripts with a transcriptome per kilobase per million mapped reads (RPKM) ≥ 1 were considered for differential expression analysis.

[0091] [Table 3]

[0092] The global transcriptome of E. chaffeensis

[0093] The transcript distribution in wild-type E. chaffeensis included 481 transcripts represented by five or fewer transcripts, followed by hypothetical protein transcripts (178) and 127 ribosomal protein gene transcripts (14%), representing 19% of the transcriptome. The next most abundant transcripts were those for major outer membrane proteins (22 transcripts). Conserved domain protein transcripts encoded by 14 genes are associated with the NADH dehydrogenase I complex. Other highly expressed genes included molecular chaperones, ATP synthases, putative membrane proteins, cytochrome c oxidases, GTP-binding proteins, putative lipoproteins, translation elongation factors, ABC transporters, and DNA polymerases. These all account for 0.5–1.7% of the transcriptome.

[0094] The ECH_0379 mutation causes transcriptional downregulation of various genes involved in antiporter activity, phage proteins, and genes involved in transport and transcription functions.

[0095] Differential gene expression (DGE) was measured by comparing RPKM expression values ​​between the mutant and wild-type strains. A p-value <0.05, a false discovery rate (FDR) ≤0.001, and concordance of expression values ​​between replicates were considered significant. Gene expression changes were not significant between the mutant and wild-type strains for housekeeping genes. Based on these criteria, 41 genes were determined to be primarily downregulated and two genes were upregulated in the ECH_0379 mutant compared with the wild-type strain (Table 4). The most prominent genes that showed significant decreases in transcript levels were those encoding antiporter proteins, ABC transporters, and an ATP-dependent Clp protease (ECH_0367). Four antiporter protein genes: monovalent cation / proton antiporter (ECH_0466), Na(+) / H(+) antiporter subunit C (mrpC) (ECH_0469), potassium uptake protein TrkH (ECH_1093), and nitrogen regulatory protein NtrY (ECH_0299) showed significantly reduced transcription levels. In addition, transcripts for two membrane transporters, namely, the cation ABC transporter permease protein transcript of gene ECH_0517 and another ABC transporter permease protein transcript of gene ECH_0972, were downregulated. Three genes encoding phage-like proteins, namely, phage prohead protease (ECH_0032), phage portal protein (ECH_0033), and phage major capsid protein (ECH_0830), were also downregulated in this mutant. Transcripts of six genes involved in transcription, namely, DNA replication and restoration protein RecF (ECH_0076), formamidopyrimidine-DNA glycosylase (ECH_0602), dimethyladenosine transferase (ECH_0648), GTP-binding protein EngA (ECH_0504), leucyl-tRNA synthetase (ECH_0794), and endonuclease III (ECH_0857), were also downregulated in this mutant.Also downregulated are enzymes involved in metabolic processes, such as glutamate cysteine ​​ligase (GCL) (ECH_0125), DNA / pantothenate (PMF) metabolic flavoprotein (ECH_0374) (ECH_0392), ATPase, AGF1 (UPGS), uroporphyrinogen III synthase (ECH_0480), diaminopimelate decarboxylase (DAPDC) (ECH_0485), biotin-acetyl-CoA-carboxylase ligase (BACL) (ECH_0848), and argininosuccinate lyase (ASL) (ECH_0937). Transcripts of eight hypothetical protein genes; ECH_0021, ECH_0161, ECH_0264, ECH_0289, ECH_0725, ECH_0879, ECH_0913, and ECH_1053, were also present among the down-regulated genes in this mutant.

[0096] [Table 4A]

[0097] [Table 4B]

[0098] Differential transcriptional regulation of T4SS and p-28OMP gene cluster genes in mutant ECH_0490

[0099] In the ECH_0490 mutant, 37 genes were significantly down-regulated and 17 genes were up-regulated (Table 5). Four of the down-regulated genes belonged to the T4SS: ECH_0494 (VirB3), ECH_0496 (VirB6), ECH_0498 (VirB6), and ECH_0499 (VirB6); and type I secreted membrane fusion protein ((T1SS_HlyD) ECH_0970). Also down-regulated were molecular chaperone genes, such as cold shock protein (CSP) (ECH_0298) and ATP-dependent Clp protease, as well as the ATP-binding subunit ClpA (ClpA). Transport proteins were also downregulated, including the protein export membrane protein (SecF) (ECH_0095), the preprotein translocase (SecY) (ECH_0428), the potassium uptake protein (TrkH) (ECH_1093), and the nitrogen regulatory protein (NtrY) (ECH_0299). Additionally, metabolic enzymes involved in the biosynthetic process, such as tetrahydropyridine-2-carboxylic acid N-succinyltransferase (dapD) (ECH_0058), quinone oxidoreductase (ECH_0385), metalloendopeptidase (MEP) (ECH_0644), peptide deformylase (PDF) (ECH_0939), serine / threonine phosphatase (PSP) (ECH_0964), pyrophosphatase (PPi) (ECH_1014), and orotate phosphoribosyltransferase (OPRTase) (ECH_1108), were also down-regulated. Transcription and translation-related genes, such as elongation factor (EF-Tu) (ECH_0515), aminoacyl-tRNA synthetase (IARS) (ECH_0538), DNA-binding protein (HU) (ECH_0804), 3′-5′ exonuclease domain (ECH_1011), and DNA-binding response regulator (ECH_1012), were also downregulated.

[0100] [Table 5A]

[0101] [Table 5B]

[0102] [Table 5C]

[0103] The upregulated protein genes in this mutant included seven genes belonging to the transmembrane protein category. Four of these belong to the p-28 OMP gene cluster (ECH_1143 (OMP-p28), ECH_1146 (OMP-p28-2), ECH_1136 (OMP-1B), and ECH_1121 (OMP-1N)). In addition, two putative membrane protein genes (ECH_0009, ECH_0230) and an immunodominant surface protein gene (ECH_0039) were upregulated. Also upregulated were the transcriptional genes for the heat shock protein ATP-dependent Clp protease, ClpA (ECH_0567), the ATP-binding chaperone, ClpB (ECH_0367), and the stress response-related RNA polymerase sigma factor (RpoH) (ECH_0655). Transcripts of two genes encoding iron-sulfur proteins {BolA family protein (ECH_0303) and FeS cluster assembly scaffold (IscU) (ECH_0630)} were also upregulated. Differential expression of six hypothetical protein genes was observed, including ECH_0166, ECH_0251, ECH_0450, ECH_0531, ECH_0753, and ECH_0878.

[0104] Mutation of the ECH_0660 gene resulted in minimal transcriptional changes.

[0105] Dramatic gene expression changes were observed in both the ECH_0379 and ECH_0490 mutants, whereas the ECH_0660 mutant transcriptome showed only minor changes compared to the wild type. Only five significantly differentially expressed genes were observed in this mutant (Table 6). The nitrogen regulatory protein (NtrY) (ECH_0299) and the ABC transporter permease protein (ECH_0972) were downregulated, whereas the heme exporter protein CcmA (ECH_0295) and chaperonin (ECH_0364) were upregulated. We also identified several differentially expressed genes common to ECH_0379 and ECH_0490 (Table 7). Ribonuclease D (ECH_0300) and potassium uptake protein (ECH_1093) were commonly downregulated in ECH_0379 and ECH_0490. The T4SS protein VirB4 gene was downregulated in the ECH_0490 mutant, whereas this gene was upregulated in the ECH_0379 mutant. Conversely, ClpB was downregulated in the ECH_0379 mutant and upregulated in the ECH_0490 mutant.

[0106] [Table 6]

[0107] [Table 7]

[0108] Validation of RNA-seq data by quantitative real-time reverse transcription PCR

[0109] Quantitative real-time quantitative reverse transcriptase-PCR (qRT-PCR) analysis was performed on 13 randomly selected genes identified as differentially transcribed according to the RNA-seq data. To generate qRT-PCR data, RNA samples were first normalized to the constitutively expressed E. chaffeensis gene encoding 16S RNA, as described in Cheng et al. Transcript abundance was verified for seven down-regulated genes in ECH_0466 and the ECH_379 mutant, including mrpC, ClpB, ECH_0033, NtrY, TrkH, and ECH_0972. Similarly, six up-regulated genes from the ECH_0490 mutant, including four transcripts belonging to the OMP gene cluster (OMP-p28, OMP-1B, OMP-1N, OMP-p28-2) and one each from the ClpB and RpoH genes, were also verified by qRT-PCR. Similarly, downregulation of ECH_0299 and ECH_0972 gene transcripts was confirmed in the ECH_0660 mutant by qRT-PCR.

[0110] Consideration

[0111] Isolating cell-free bacterial RNA from highly abundant host RNA is the first challenge in transcriptional profiling of intracellular pathogens. Rickettsiales require cultivation within host cells, followed by purification before RNA can be extracted for transcriptome evaluation experiments. To demonstrate the effects of three transposon mutations on E. chaffeensis transcription, we first developed a method for isolating and purifying host-cell-free E. chaffeensis organisms, and then isolated RNA from the E. chaffeensis organisms for next-generation sequencing (NGS) analysis. To isolate cell-free E. chaffeensis, we first performed an efficient host cell lysis protocol, followed by whole-cell lysate filtration and subsequent Renograffin density gradient centrifugation. The second challenge was obtaining host-cell-free RNA for transcriptome profiling. Previous studies have reported that bacterial RNA enrichment methods only yielded bacterial RNA read enrichments of 3–10%. The host-cell-free bacterial isolation and bacterial RNA purification steps implemented in this study enabled a higher enrichment of E. chaffeensis RNA. In our current study, we were able to enrich for bacterial RNA, which helped generate up to 19% of high-mapping RNA reads. Notably, deep RNA-seq analysis helped map 80% of E. chaffeensis genes expressed within infected macrophage host cells.

[0112] Among the highly expressed genes, the p28-OMP multigene cluster was dominant in the transcriptome. The E. chaffeensis p28-OMP multigene locus contains 22 tandemly arranged genes encoding bacterial immunodominant proteins. The presence of all 22 transcripts in the RNA-seq data suggests that this gene cluster is one of the most abundantly expressed genes. These observations are consistent with our previous proteomics study, which reported the expression levels of the p28-OMP gene. NADH dehydrogenase I complex genes were also highly expressed in E. chaffeensis. NADH dehydrogenase inhibits host cell apoptosis by counteracting the phagosomal NOX2 response. T4SS effector proteins in some pathogenic bacteria are thought to be important for manipulating host gene expression to weaken the host immune response. The contribution of T4SS effectors to virulence has previously been reported in Rickettsiales, including A. marginale, A. phagocytophilum, E. canis, and E. chaffeensis. RNA sequencing identified several transcripts encoding T4SS proteins, including VirB3, B4, B6, B8, B9, B10, and B11. Furthermore, the chaperone protein genes DnaK, DnaJ, GroE, and ClpB were also highly expressed in both wild-type and mutant strains. The presence of such proteins involved in cellular homeostasis and oxidative stress response has also been reported in other Rickettsiales, suggesting that if the pathogen proteome is similarly altered by the transcriptome reported in this study, their gene products are also important for the stress response in E. chaffeensis. Indeed, our current study suggests that stress response proteins are important for E. chaffeensis. Other highly expressed protein genes include those encoding housekeeping ribosomal proteins involved in protein synthesis, putative membrane proteins, ABC transporters, and lipoproteins, all of which are likely important for pathogen protein synthesis, transport, trafficking, and effector secretion into host cells.ATP synthase subunits, cytochrome c oxidase, DNA polymerase, GTP-binding proteins, and translation elongation factors involved in energy metabolism, cell division, and transcriptional regulation were also present among the highly expressed genes in wild-type and mutant organisms. The transcriptome coverage was higher than previously reported for E. chaffeensis in ISE6 and AAE2 tick cells. This is important for both the enhanced detection of intracellular pathogen transcripts and the observed gene expression levels. The higher transcriptome coverage is likely a result of deep RNA sequencing by next-generation sequencing compared to microarray analysis. This global set of highly expressed genes may represent products involved in E. chaffeensis virulence, replication, and survival in the host cell environment. Four transcripts encoding ankyrin repeat proteins, known to mediate protein-protein interactions, were also identified in the transcriptome. Notably, the transcriptomes from wild-type and mutant organisms contain 216 transcripts encoding hypothetical proteins of unknown function. Because these genes were present in the core transcriptome, the inventors predicted that these genes were important sets of transcribed genes for E. chaffeensis replication.

[0113] Transcription from numerous genes in the ECH_0379 mutant was found to be reduced compared to the wild type. Genes representing antiporters, ABC transporters, chaperones, metabolic enzymes, and transcriptional regulators were among the down-regulated genes (Table 4). The inventors predicted that mutations in antiporter protein genes caused the metabolic decline. Antiporter and transporter proteins play important roles in the transport of ions and solutes across bacterial cell membranes. Antiporters are integral membrane proteins that mediate the transport of Na across phospholipid membranes. + and / or K + H +The E. chaffeensis genome contains several genes with homology to antiporter proteins or their subunits, suggesting that they are required for the pathogen's intrachromosomal replication and survival in the host. In particular, antiporters help maintain the pH, salt, and temperature conditions of the bacterium. The present inventors have identified monovalent cations / H +We observed a significant reduction in the transcription of antiporter genes, such as antiporter subunit C (ECH_0469) and ECH_0466. Disrupting antiporter function or interfering with their expression can affect pathogen growth in vivo. Indeed, mutations in the ECH_0379 gene inhibited the growth of the organism in both the host (dog) and reservoir (white-tailed deer). ABC transporters are also involved in the uptake of ions and amino acids and may play an important role in the pathogen's ability to infect and survive in the host cellular environment. The ECH_0379 mutant exhibited reduced levels of transcriptional activity of the ABC transporter-encoding genes ECH_0517 and ECH_0972, which function at various stages of infection pathogenesis. These proteins promote pathogen survival in the host microenvironment. This mutation likely inhibits the transport mechanism, thereby affecting the pathogen's ability to infect and survive in host cells. This mutation may also cause altered transcription of genes involved in physiological responses, such as regulating metabolic activity of pathogens. The inventors also found downregulation of several transcripts encoding metabolic enzymes, namely glutamate-cysteine ​​ligase, DNA / pantothenate metabolic flavoprotein family protein, ATPase, uroporphyrinogen-III synthase, diaminopimelate decarboxylase, biotin-acetyl-CoA-carboxylase ligase, and argininosuccinate lyase. Generally, pathogen survival within an intracellular environment depends on the ability to obtain nutrients from host cells. Pathogenic bacteria obtain nutrients from host cells by using metabolic pathways and virulence-related factors to subvert the host immune system. Downregulation of transcripts from the aforementioned genes in the ECH_0379 mutant may disrupt the bacterial metabolic response and ability to obtain nutrients from the host. The mutation also caused reduced expression of genes encoding DNA replication and restoration proteins, formamidopyrimidine-DNA glycosylase, dimethyladenosine transferase, and leucyl-tRNA synthetase, which may also contribute to defects in intracellular growth and survival of the pathogen.Our previous work suggests that despite the mutant's growth being suppressed, it did not provide complete protection against wild-type infection. If changes in the transcriptome correlate with changes in the proteome, the changes in protein expression in the mutant organism compared to wild-type E. chaffeensis may result in altered host responses, thus reducing the effectiveness of the host in mounting a defensive host response when exposed to the mutant organism.

[0114] Pathogenic bacteria produce T4SS effectors that attenuate host cell gene expression, contributing to bacterial virulence. RNA-seq data suggest reduced expression of various T4SS component protein gene transcripts in the ECH_0490 mutant. We also observed reduced transcription of chaperone proteins and several genes involved in the transcription and translation machinery, as well as exonuclease and DNA-binding regulator gene transcripts, in the ECH_0490 mutant. Conversely, ClpB (a major stress-responsive heat shock protein) and RpoH (a stress-responsive RNA polymerase transcription subunit) showed increased transcription in the mutant.

[0115] Chaperone proteins play an important role in protein disaggregation and helping pathogens overcome host cell-induced stress. ClpB reactivates aggregated proteins that accumulate under stress conditions and is abundantly expressed in the replicative stage of E. chaffeensis. Preventing or reducing protein aggregation and associated protein inactivation during bacterial growth within host cells may be beneficial for enhancing pathogen survival. RpoH, a transcriptional regulator of RNA polymerase, is also important for continued pathogen growth because it promotes the expression of stress response proteins. Consistent with expectations, increased expression of ClpB and RpoH was observed in this study for the ECH_0490 mutant. Enhanced expression from these two key genes likely allows the mutant to grow similarly to wild-type E. chaffeensis in vertebrate and tick hosts, as reported in our previous studies. Outer membrane proteins perform various functions, such as invasion, transport, immune response, and adhesion, that are essential for the survival of Ehrlichia species, including E. chaffeensis and E. ruminantium, in their hosts. The ECH_0490 mutant exhibited increased OMP abundance compared to wild-type organisms. We identified seven transmembrane genes encoding immunodominant P28 / OMP family proteins (OMP_p28, OMP_p28-2, OMP-1B, and OMP-1N) and membrane proteins (ECH_0039, ECH_0009, and ECH_0230) to be upregulated. Significant changes in outer membrane protein abundance correlated with global changes in membrane structure, thereby altering the pathogen's susceptibility to host defenses. The transcriptional changes observed in the ECH_0490 mutant did not adversely affect the pathogen, as the mutant grew similarly to the wild-type pathogen in both white-tailed deer (reservoir host) and dogs (accidental host) and its tick host, Amblyomma americanum. Evaluation of the transcriptional activity of the genes ECH_0490 (lipoic acid synthase) and ECH_0492 (putative phosphate ABC transporter), located upstream and downstream of the transposon insertion mutation, respectively, suggests that this mutation does not affect the transcription of these genes.The diverse changes in the transcriptome of this mutant suggest that it affects global gene expression without affecting the area surrounding the mutation site, and that it does not adversely affect pathogen survival in the vertebrate or tick host.

[0116] The most notable observation was the minimal variation evident in the transcriptome of the ECH_0660 mutant compared to wild-type E. chaffeensis. Importantly, mutations within the ECH_0660 gene cause severe growth defects in vertebrate hosts in vivo. Furthermore, infection with this mutant mounts a strong host response, conferring protection against wild-type pathogen infection challenges. In this study, only minor changes in gene expression were observed in this mutant compared to the wild-type. While the subtle changes in gene expression included genes encoding putative nitrogen regulatory proteins, ABC transporters, heme export proteins, and GroES, the changes were significantly less than those described in the previous two mutants. Collectively, these data suggest that mutations in the ECH_0660 gene result in reduced transcriptional changes. Assuming that the proteomes of wild-type and mutant E. chaffeensis strains are altered in a similar way to the transcriptome, the ECH_0660 mutant proteome is highly similar to that of the wild-type bacterium. The high similarity between this mutant and the wild-type allows the vertebrate host to recognize this mutant as more similar to the wild-type organism, thereby inducing a strong host response that mimics wild-type infection. The replication defect previously reported for this mutant is due to the loss of gene expression from fewer genes, such as ECH_0659 and ECH_0660, while maintaining most of the transcriptome similar to the wild-type.

[0117] conclusion

[0118] Deep RNA sequencing studies in intracellular bacteria remain challenging. The RNA-seq data reported here provide the first snapshot of comparative transcriptomics of E. chaffeensis. Sequencing of abundant bacterial RNA from wild-type and mutant strains yielded high gene coverage. Mutations in the ORF of the ECH_0379 gene caused dramatic downregulation of genes leading to metabolic decline, which contributed to the attenuation of the mutant in the vertebrate host. Mutations downstream of the protein-coding sequence of the ECH_0490 gene induced global changes in gene expression, while upregulation of stress response regulatory genes helped the mutant survive in both the vertebrate and tick host. Mutations within the coding sequence of the ECH_0660 gene resulted in little transcriptional change, thus maintaining transcriptome integrity similar to that of the wild type. While the transcriptome data suggest altered protein expression, additional experimental validation from protein analysis studies is required to confirm the results. Taken together, this study provides the first detailed description of transcriptomic data for E. chaffeensis and suggests that the observed variations in the pathogen's ability to survive within the host and in the host's ability to induce defenses against the pathogen are the result of global changes in gene expression, which in turn affect changes in the pathogen's proteome.

Claims

1. 1. An immunogenic composition comprising: a bacterium of the order Rickettsiales containing a targeted allelic exchange mutant; an ingredient selected from the group consisting of a veterinarily acceptable carrier, a pharmaceutically acceptable carrier, an adjuvant, a preservative, a buffering agent, an antibiotic, a cell culture supernatant, an immunomodulator, and any combination thereof; The targeted allelic exchange mutant comprises a gene that has been disrupted and its genetic integrity restored, the disrupted gene being a gene that induces a bacterium-specific immune response and prevents or at least reduces the bacterium's ability to replicate in an obligate host, and the genetic integrity of the gene is restored by inserting the entire ORF region of the disrupted gene and a promoter driving an antibiotic selection marker gene into the location of the disrupted gene.

2. 2. The immunogenic composition of claim 1, The immunogenic composition, wherein the bacteria of the order Rickettsiales are selected from the group consisting of Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, and Orientia.

3. 2. The immunogenic composition of claim 1, The immunogenic composition, wherein the targeted allelic exchange variant attenuates the bacterium and / or inactivates a gene.

4. 4. The immunogenic composition of claim 3, An immunogenic composition characterized in that the gene plays a role in assisting replication.

5. 3. The immunogenic composition of claim 2, The targeted allelic exchange mutant is (a) Ehrlichia chaffeensis Ech_0379 or Ech_0660, or a homologous gene from another strain of the same species or another Ehrlichia species; (b) Ecaj_0381 of Ehrlichia canis, or a homologous gene from another strain of the same species or another Ehrlichia species; (c) APH_0634 of Anaplasma phagocytophilum or a homologous gene from another strain of the same species or another Ehrlichia species; (d) Ehrlichia ruminantium Erum_3930, or another Ehrlichia ruminantium strain or another Ehrlichia species and its homologous genes; (e) AMH_581 of Anaplasma marginale, or a homologous gene from another strain of the same species or another Anaplasma species; (f) EMUR_02070 of Ehrlichia muris AS145 or a homologous gene from another strain of the same species or another Ehrlichia species; An immunogenic composition characterized in that it is present at a location selected from the group consisting of:

6. 2. The immunogenic composition of claim 1, The immunogenic composition, wherein the component is an adjuvant selected from the group consisting of saponin, cyclic GMP-AMP, montanide gel, and any combination thereof.

7. 2. The immunogenic composition of claim 1, An immunogenic composition further comprising an antigen derived from another pathogen.

8. 2. The immunogenic composition of claim 1, The bacterium comprises SEQ ID NO:35, SEQ ID NO:54, or a sequence having at least 90% sequence identity to SEQ ID NO:

55.

9. 1. A method for reducing at least one of the incidence and severity of clinical symptoms caused by Rickettsiales bacteria, comprising: administering to the non-human animal at least once an immunogenic composition, The immunogenic composition comprises: a bacterium of the order Rickettsiales containing a targeted allelic exchange mutant; an ingredient selected from the group consisting of a veterinarily acceptable carrier, a pharmaceutically acceptable carrier, an adjuvant, a preservative, a buffer, an antibiotic, a cell culture supernatant, an immunomodulator, and any combination thereof, and having the steps of: the targeted allelic exchange mutant comprises a gene that has been disrupted and its genetic integrity restored; The method is characterized in that the disrupted gene is a gene that induces a bacterium-specific immune response and prevents or at least reduces the bacterium's ability to replicate in an obligate host, and the genetic integrity of the gene is restored by inserting the entire ORF region of the disrupted gene and a promoter driving an antibiotic selection marker gene into the location of the disrupted gene.

10. 10. The method of claim 9, The method of claim 1, wherein the immunogenic composition is administered using an administration method selected from the group consisting of intravenous, intramuscular, intranasal, intradermal, intratracheal, intravaginal, intravenous, intravascular, intraarterial, intraperitoneal, oral, intrathecal, direct injection into any target tissue, and any combination thereof.

11. 10. The method of claim 9, The method, wherein the bacterium of the order Rickettsiales is selected from the group consisting of Ehrlichia, Anaplasma, Neorickettsia, Rickettsia, and Orientia species.

12. 10. The method of claim 9, The method, wherein the targeted allelic exchange mutant inactivates a gene.

13. 10. The method of claim 9, The targeted allelic exchange mutant is (a) Ech_0379 or Ech_0660 of Ehrlichia chaffeensis, or a gene homologue having 85% or more homology to Ech_0379 or Ech_0660 and belonging to the same organism or another strain of Ehrlichia species; (b) Ecaj_0381 of Ehrlichia canis, or a gene homologue having 85% or more homology to Ecaj_0381 and belonging to the same organism or another strain of Ehrlichia species; (c) APH_0634 of Anaplasma phagocytophilum or a gene homologue having 85% or more homology to APH_0634 and belonging to the same organism or a strain of another Anaplasma species; (d) Ehrlichia ruminantium Erum_3930, or another Ehrlichia ruminantium strain or another Ehrlichia species and its homologous genes; (e) AMH_581 of Anaplasma marginale, or a homologous gene from another Anaplasma marginale strain of the same species or another Anaplasma species; (f) EMUR_02070 of Ehrlichia muris AS145 or a homologous gene from another Ehrlichia muris strain of the same species or another Ehrlichia species; The method of claim 1, wherein the compound is present at a position selected from the group consisting of:

14. 10. The method of claim 9, The method, wherein the bacterium comprises SEQ ID NO:35, SEQ ID NO:54, or a sequence having at least 90% sequence identity to SEQ ID NO:

55.

15. 10. The method of claim 9, The method of claim 1, wherein the non-human animal is selected from the group consisting of pigs, cows, goats, horses, dogs, deer, coyotes, cats, and poultry.

16. 14. The method of claim 13, The targeted allelic exchange mutant is (a) within Ehrlichia chaffeensis Ech_0379 or Ech_0660, or a gene homologue having 90% or greater identity to Ech_0379 or Ech_0660 and belonging to the same organism or another strain of Ehrlichia species; (b) Ecaj_0381 of Ehrlichia canis, or a gene homologue having 90% or more homology to Ecaj_0381 and belonging to the same organism or another strain of Ehrlichia species; (c) APH_0634 of Anaplasma phagocytophilum or within a gene homologue having 90% or more homology to APH_0634 and belonging to the same organism or a strain of another Anaplasma species. The method of claim 1, wherein the compound is present at a position selected from the group consisting of:

17. 14. The method of claim 13, The targeted allelic exchange mutant is (a) within Ehrlichia chaffeensis Ech_0379 or Ech_0660, or a gene homologue having 95% or greater identity to Ech_0379 or Ech_0660 and belonging to the same organism or another strain of Ehrlichia species; (b) Ecaj_0381 of Ehrlichia canis, or a gene homologue having 95% or more homology to Ecaj_0381 and belonging to the same organism or another strain of Ehrlichia species; (c) APH_0634 of Anaplasma phagocytophilum or within a gene homologue having 95% or more homology to APH_0634 and belonging to the same organism or a strain of another Anaplasma species. The method of claim 1, wherein the compound is present at a position selected from the group consisting of:

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