Genome signaling identification method adapted to host mycobacterium abscessus and kit

Abstract

The invention belongs to the field of pathogenic microorganism genome biological information analysis, and particularly relates to a set of bioinformatics identification process of a human host specific gene adapted by mycobacterium abscessus and an application kit thereof. Mycobacterium abscessus is ubiquitous in the environment and is an opportunistic pathogenic bacterium. Along with the appearance of circulating clones (DCC) and emerging circulating clones (ECC), mycobacterium abscessus prompts that the mycobacterium abscessus gradually evolves from conditioned pathogenic bacteria widely existing in the environment to pathogenic bacteria suitable for human hosts. The invention provides a set of bioinformatics and molecular biology processes for identifying human host specific genes adapted to mycobacterium abscessus. In addition, the invention relates to 30 genes of a specific adaptive host of the mycobacterium abscessus, and the 30 genes comprise antibiotic drug resistance (eis2, erm (41) and gyrA), cell walls (ubiA and embC), a two-component system (phoP and phoR), global regulatory factors (crp and espR), metal ion regulation (der and ideR) and the like.

Description

Technical Field

[0001] This patent belongs to the field of bioinformatics analysis of pathogenic microorganism genomes, and specifically involves a bioinformatics identification process and application kit for a set of genes specific to Mycobacterium abscessus adapted to human hosts. Background Art

[0002] Currently, the incidence of Mycobacterium abscessus lung infection is increasing significantly around the world. In the UK and the US, its incidence has exceeded that of tuberculosis. [1] . The disease often occurs in immunocompromised patients or those with underlying lung diseases such as cystic fibrosis (CF), bronchiectasis or chronic obstructive pulmonary disease (COPD). The pathogen is Mycobacterium abscessus (MAB). Mycobacterium abscessus belongs to the rapidly growing mycobacteria (RGM) and is ubiquitous in the environment. It is an opportunistic pathogen. Currently, MAB can be divided into three subspecies: Mycobacterium abscessus (M.abscessus), Mycobacterium massiliense (M.massiliense) and Mycobacterium bolletii (M.bolletii). [2] .

[0003] The proliferation of dominant circulating clones (DCC) has shed new light on the global epidemiology of Mycobacterium abscessus (MAB), but the evolutionary dynamics of the global MAB population remain poorly understood. [3-5] . To fill this gap, this application analyzed 5,835 publicly available MAB strain genomes from more than 20 countries. Phylogenetic analysis revealed a diverse global MAB population, including at least 59 clonal branches, called emerging circulating clones (ECC). Bayesian-based coalescent analysis showed that these ECCs appeared after the mid-20th century and have been expanding in parallel. In summary, the emergence of DCC and ECC abscess Mycobacterium suggests that it has gradually evolved from adapting to the environment to adapting to the human host.

[0004] In this case, it is assumed that the Mycobacterium abcessus complex (MABC) population will certainly encounter new selective pressures in the human host, which will be reflected in changes in the selective pressure on specific genes. To test this hypothesis, this application evaluates the selective pressure within the MABC population by studying long-term evolution and recent evolution. Long-term evolution is evaluated by mutations in long branches, whose characteristics can reflect the selective pressures experienced by MABC over thousands of years. [6]Recent evolution was assessed by examining mutations in leaves representing terminal branches, which have accumulated during recent expansions. Since horizontal evolution (recombination) is also an important way for MABC to acquire variation, it may play an important role, especially in long-term evolution, and may interfere with the signal of vertical evolution. Therefore, potential recombinant regions and accessory genomes were first excluded by Gubbins. All vertical evolution analyses were performed on the core genome.

[0005] This application uses dN / dS as a proxy for selection pressure to evaluate the selection pressure of MABC core genes at the "long-term" and "short-term" scales (as shown in Table 1). In order to further clarify the specific genes for Mycobacterium abscessus to adapt to the host, gene enrichment analysis was performed on the genes initially screened, including prediction of transcription factor binding sites, analysis of protein sequence amino acid conserved sites, gene function enrichment analysis, protein functional domain prediction, etc.

[0006] In order to verify the specific adaptability of the above-mentioned Mycobacterium abscessus genes to the host, the present application also conducted morphological identification of clinically isolated and cultured Mycobacterium abscessus. Thus, 30 genes such as phoR were identified. Further research on the functions of these selected genes is expected to reveal the key steps in the evolution of Mycobacterium abscessus from environmental bacteria to human pathogens.

[0007] In view of this, a patent application is filed for identifying a set of (30) specific genes that help Mycobacterium abscessus adapt to its host.

[0008] References

[0009] [1] JMBryant, DM Grogono, D. Greaves, J. Foweraker, I. Roddick, T. Inns, M. Reacher, CS Haworth, MDCurran, SR Harris, SJ Peacock, J. Parkhill, RAFloto. Whole-genome sequencing to identify transmission of Mycobacteriumabscessus between patients with cystic fibrosis: a retrospective cohort study[J].Lancet,2013,381(9877):1551-60.

[0010] [2]J.L.Tan,K.P.Ng,C.S.Ong,Y.F.Ngeow.Genomic Comparisons RevealMicroevolutionary Differences in Mycobacterium abscessus Subspecies[J].FrontMicrobiol,2017.02042.

[0011] [3]J.M.Bryant,D.M.Grogono,D.Rodriguez-Rincon,I.Everall,K.P.Brown,P.Moreno,D.Verma,E.Hill,J.Drijkoningen,P.Gilligan,C.R.Esther,P.G.Noone,O.Giddings,S.C.Bell,R.Thomson,C.E.Wainwright,C.Coulter,S.Pandey,M.E.Wood,R.E.Stockwell,K.A.Ramsay,L.J.Sherrard,T.J.Kidd,N.Jabbour,G.R.Johnson,L.D.Knibbs,L.Morawska,P.D.Sly,A.Jones,D.Bilton,I.Laurenson,M.Ruddy,S.Bourke,I.C.Bowler,S.J.Chapman,A.Clayton,M.Cullen,T.Daniels,O.Dempsey,M.Denton,M.Desai,R.J.Drew,F.Edenborough,J.Evans,J.Folb,H.Humphrey,B.Isalska,S.Jensen-Fangel,B. AMJones, TLKatzenstein, T. Lillebaek, G. MacGregor, S. Mayell, M. Millar, D. Modha, EF Nash, C. O'Brien, D. O'Brien, C. Oh ri,CSPao,D.Peckham,F.Perrin,A.Perry,T.Pressler,L.Prtak,T.Qvist,A.Robb,H.Rodgers,K.Schaffer,N.Shafi,J.van Ingen,M.Walshaw,D.Watson,N.West,J.Whitehouse,CSHaworth,SRHarris,D.Ordway,J.Parkhill,RAFloto.Emergence andspreadofhuman-transmissible multidrug-resistant nontuberculous mycobacterium[J].Science,2016,354(6313):751-7.

[0012] [4]SWChoo,WYWee,YFNgeow,W.Mitchell,JLTan,GJWong,Y.Zhao,J.Xiao.Genomic recognition of clinical isolates of emerging human pathogenMycobacterium abscessus reveals high evolutionary potential[J].SciRep,2014,4(4061).

[0013] [5]N.Commins,MRSullivan,K.McGowen,EMKoch,EJRubin,M.Farhat.Mutationrates andadaptive variation among the clinicallydominantclusters ofMycobacterium abscessus[J].ProcNatlAcad SciU SA,2023,120(22):e2302033120.

[0014] [6]PJPlanet,A.Narechania,L.Chen,B.Mathema,S.Boundy,G.Archer,B.Kreiswirth.Architecture of a Species:Phylogenomics ofStaphylococcus aureus[J].Trends Microbiol,2017,25(2):153-66. Summary of the invention

[0015] The first object of the present invention is to provide a kit for identifying specific genes of Mycobacterium abscessus adapted to the host, the kit comprising 30 gene set reagents or components for identifying Mycobacterium abscessus adapted to the human host. The above kit can identify the 30 specific gene sets of Mycobacterium abscessus adapted to the human host, in order to reveal the key steps in the evolution of Mycobacterium abscessus from environmental bacteria to human pathogens, and help the precise prevention and treatment of Mycobacterium abscessus.

[0016] The second object of the present invention is to provide an application of the above kit in the identification of a set of 30 genes that Mycobacterium abscessus adapts to human hosts or in evolutionary analysis. The above application can improve the accuracy of specific gene identification or evolutionary analysis of Mycobacterium abscessus adapts to human hosts.

[0017] The third object of the present invention is to provide an application of the above-mentioned kit in the preparation of a diagnostic product for 30 specific genes of a pathogen of Mycobacterium abscessus infection.

[0018] The fourth object of the present invention is to provide a method for detecting Mycobacterium abscessus, characterized in that the reagents and / or components for detecting the 30 gene sets include one or more of PCR primers, molecular probes, biosensors and chips.

[0019] The fifth object of the present invention is a method for identifying or evolving genes of Mycobacterium abscessus adapted to its host, characterized in that the method uses the kit described in any one of the first to fourth objects of the present invention to detect 30 defined genes of Mycobacterium abscessus adapted to its host (human).

[0020] The sixth object of the present invention is that the method for detecting the 30 genes includes one or more of the following: a detection method based on gel electrophoresis, a DNA sequencing method, a DNA chip method, a denaturing high performance liquid chromatography method or a mass spectrometry detection method.

[0021] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0022] 1. Calculation of the selection pressure of the host (human) environment adaptability of Mycobacterium abscessus;

[0023] 2. Enrichment analysis of genes specific to the adaptation of Mycobacterium abscessus to the host (human) environment;

[0024] 3. Biological verification of specific genes of Mycobacterium abscessus adapting to the host (human) environment.

[0025] The present invention provides a method and a kit for identifying genes of Mycobacterium abscessus adapted to humans, characterized in that the kit comprises reagents and / or components for detecting a gene set, and the physical locations of the 30 genes are based on the abscessus subspecies GZ002 (NZ_CP034181.1), the massi subspecies CCUG48898 (NZ_AP014547.1) and the bolai subspecies GD91 (NZ_CP065265.1) (such as Figure 1 shown);

[0026] The most important genes are: antibiotic resistance genes eis2, erm(41), and gyrA;

[0027] cell wall genes ubiA and embC;

[0028] two-component system genes phoP and phoR;

[0029] global regulator genes crp and espR;

[0030] Metal ion-regulated genes der and ideR.

[0031] The kit of the present invention can detect a set of 30 genes selected from the host (human) specificity of Mycobacterium abscessus. The 30 host (human) specificity genes of the present invention use dN / dS as a proxy for selection pressure, and the selection pressure of MABC core genes is evaluated at the "long-term" and "short-term" scales. In order to further clarify the specific genes of Mycobacterium abscessus adapting to the host, gene enrichment analysis was performed on the genes initially screened, including transcription factor binding site prediction, protein sequence amino acid conservative site analysis, gene function enrichment analysis, protein functional domain prediction, etc. In order to verify the specific adaptability of the above-mentioned Mycobacterium abscessus genes to the host, morphological identification was performed on Mycobacterium abscessus isolated and cultured clinically. Thereby, three genes such as phoR were identified. The most important genes are: antibiotic resistance (eis2, erm (41), gyrA), cell wall (ubiA, embC), two-component system (phoP, phoR), global regulatory factors (crp, espR) and metal ion regulation (der, ideR), etc. Further studies on the functions of these selected genes are expected to reveal the key steps in the evolution of Mycobacterium abscessus from an environmental bacterium to a human pathogen.

[0032] The above embodiment of the present invention selects the specific genes of Mycobacterium abscessus that adapt to human hosts. After identification and evolutionary analysis, a total of 30 genes are selected. Further research on the functions of these selected genes is expected to reveal the key steps in the evolution of Mycobacterium abscessus from environmental bacteria to human pathogens.

[0033] In some embodiments, the kit described in the present invention is characterized in that: the 30 genes include antibiotic resistance genes eis2, erm (41), gyrA; cell wall genes ubiA, embC; two-component system genes phoP, phoR; global regulatory factor genes crp, espR; metal ion regulatory genes der, ideR, etc.

[0034] The kit described in the patent of the present invention is characterized in that the gene distribution of Mycobacterium abscessus that is strongly selected by the host (human) includes 30 important genes.

[0035] The kit described in the patent of the present invention is characterized in that the reagents and / or components for detecting the 30 gene sets include one or more of PCR primers, molecular probes, biosensors and chips.

[0036] The kit described in the patent of the present invention is used in the identification or evolution analysis of 30 adaptive host genes of Mycobacterium abscessus.

[0037] The present invention is a method for identifying or analyzing the genes of Mycobacterium abscessus adapting to the host or evolution, characterized in that the method uses the kit described in any one of the first to fourth purposes of the present invention to detect 30 defined genes of Mycobacterium abscessus adapting to the host (human).

[0038] The kit described in the patent of the present invention is characterized in that the method for detecting the 30 genes includes one or more of the following: a detection method based on gel electrophoresis, a DNA sequencing method, a DNA chip method, a denaturing high performance liquid chromatography method or a mass spectrometry detection method.

[0039] Beneficial Effects

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1) The 30 genes of Mycobacterium abscessus that are specifically adapted to the host involved in the kit, application and method of the present invention are enriched and analyzed by calculating the selection pressure of the host (human) environment adaptability of Mycobacterium abscessus, and a series of bioinformatics analysis processes such as 30 specific genes of Mycobacterium abscessus host (human) environment adaptation. This bioinformatics analysis pipeline (process) provides advanced analysis technology and means for analyzing other mycobacteria.

[0042] 2) The 30 genes involved in the kit, application and method of the present invention are finally verified by biological methods to be specific genes for adaptation of Mycobacterium abscessus host (human) environment. This will reveal the key steps in the evolution of Mycobacterium abscessus from environmental bacteria to human pathogens, and provide candidate genes for the precise prevention and control of Mycobacterium abscessus. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific implementation methods of the present invention or the solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 Thirty genes that are specific to the adaptation of Mycobacterium abscessus to the human host.

[0045] Note: Top: shows the 30 genes under selection pressure in the DCCs strain; Middle: shows the 30 genes under selection pressure in the ECCs strain; Bottom: shows the 30 genes under selection pressure in the context of long-term selection events.

[0046] Figure 2 The above 30 genes are only under positive selection in the lungs of human hosts and show pure selection in the skin. Note: A. A diagram showing how to identify long-term and recent mutation events; B. Comparison of the selection pressure of 30 genes in ECCs, DCCs, and in the context of long-term selection events; C. Shows the mutation distribution of positively selected genes from samples from skin (extrapulmonary) and intrapulmonary sources; D. The accumulation of non-fixable mutations in positively selected genes emphasizes that genes under recent positive selection (such as phoR, embC, etc.) continue to accumulate mutations in the host.

[0047] Figure 3 Schematic representation of genome-wide selective pressures in Mycobacterium abscessus.

[0048] Note: A. Distribution of genetic selection pressure of the abscess subspecies; B. Distribution of genetic selection pressure of the Massi subspecies; C. Distribution of genetic selection pressure of the Bolai subspecies; D. Distribution of fixed mutations of DCCs in phoR gene; E. Distribution of non-fixed mutations of DCCs in phoR gene; F. Distribution of fixed mutations of ECCs in phoR gene; G. Distribution of non-fixed mutations of ECCs in phoR gene.

[0049] Figure 4 The phenotypic shift from smooth to rough is caused by inactivation of the mps1 or mps2 genes.

[0050] Note: A. To explore the functional integrity of mps1 and mps2 genes and the resulting phenotypic changes caused by insertion / deletion (indels); B. To show the strong selection signal of indel mutations in mps1 and mps2 genes in subspecies; C. To show the distribution of mutations within mps1 gene; D. To show the distribution of mutations within mps2 gene. DETAILED DESCRIPTION

[0051] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0052] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0053] the term

[0054] Dominant circulating clone (DCC) refers to the phenomenon that in a specific environment, cells or pathogens of certain genotypes or phenotypes become the dominant group through natural selection and spread. Dominant circulating clone is an interdisciplinary concept that involves multiple aspects such as gene selection, pathogen transmission and ecosystem dynamics, and has broad application prospects and research value.

[0055] Emerging circulating clones (ECC): 1) more than 10 isolates within a branch; 2) the average genetic distance between strains is less than 25 SNPs; 3) the genetic distance to the adjacent branch is greater than 100 SNPs; 4) the root bootstrap>0.95. A total of 60 ECCs were identified, accounting for about 87% of Non-DCCs.

[0056] Mycobacterium abscessus complex (MABC): It is the most isolated, fast-growing mycobacterium (accounting for about 22.2% to 23.1% of NTM). MABC is widely distributed in the environment and can cause a variety of infections, including lung infections in patients with cystic fibrosis (CF) or bronchiectasis (BE), skin and soft tissue infections, and disseminated infections in immunocompromised individuals.

[0057] dN / dS: In genetics, Ka / Ks or dN / dS represents the ratio between synonymous substitutions (Ka) and synonymous substitutions (Ks). This ratio can determine whether there is selection pressure acting on this protein-coding gene. Nucleotide variations that do not lead to amino acid changes are called synonymous mutations, and vice versa, they are called non-synonymous mutations. It is generally believed that synonymous mutations are not subject to natural selection, while non-synonymous mutations are subject to natural selection. In evolutionary analysis, it is very meaningful to understand the rates at which synonymous and non-synonymous mutations occur. Commonly used parameters include the following: synonymous mutation frequency (Ks), non-synonymous mutation frequency (Ka), and the ratio of non-synonymous mutation rate to synonymous mutation rate (Ka / Ks). If Ka / Ks>1, it is considered to have a positive selection effect. If Ka / Ks=1, it is considered to have a neutral selection. If Ka / Ks<1, it is considered to have a purifying selection effect.

[0058] Structural Domain: It is another structural level between the secondary and tertiary structures. The so-called domain refers to the compact globular structure area that is clearly separated in the protein subunit structure, also known as the jurisdiction. The polypeptide chain first forms a regular secondary structure with adjacent amino acid residues in certain areas, and then the adjacent secondary structure fragments are assembled together to form a super secondary structure. On this basis, the polypeptide chain folds into a tertiary structure that is approximately spherical. For larger protein molecules or subunits, the polypeptide chain is often composed of two or more spatially distinguishable and relatively independent regional structures that are associated to form a tertiary structure. This relatively independent regional structure is called a domain. For smaller protein molecules or subunits, the domain and its tertiary structure often mean the same thing, that is, these proteins or subunits are single domains. The domain itself is tightly assembled, but the relationship between domains is loose. There is often a peptide chain of varying lengths between domains, forming the so-called hinge region. The number of domains in different protein molecules is different, and several domains in the same protein molecule are similar or very different from each other. The number of amino acid residues in common domains ranges from 100 to 400. The smallest domain has only 40 to 50 amino acid residues, while the largest domain can exceed 400 amino acid residues.

[0059] Opportunistic infection: refers to some conditional pathogens that do not cause disease when the human immune function is normal, but can take advantage of the opportunity to invade the human body and cause infectious diseases with obvious clinical symptoms and signs when the human immune function is reduced. Opportunistic infection is also called conditional infection, and the pathogen that causes opportunistic infection is called opportunistic pathogen.

[0060] SMART: A database of protein domains, which contains information on approximately 1,300 domains and covers proteins from multiple databases such as uniprot and ensembl.

[0061] Seqinr: is a bioinformatics package used in R language, which provides a set of powerful tools for processing and analyzing biological sequence data. Whether it is DNA, RNA or protein sequence, seqinr can help researchers extract valuable information from it. The seqinr package provides a series of functions covering all aspects of biological sequence analysis.

[0062] SNPPar: for efficient detection and analysis of homologous SNPs from large whole-genome sequencing datasets (>1000 isolates and / or >100,000 SNPs); SNPPar takes SNP alignments, trees, and annotated reference genomes as input, and uses a combination of simple monophyletic tests and ancestral state reconstruction (ASR, via TreeTime) to assign mutation events to branches and identify homoplasy. Mutations are annotated at the codon and gene levels to facilitate analysis of convergent evolution. Simulated data tests (alignments of 120 Mycobacterium tuberculosis representative of local and global samples) show that SNPPar can detect homologous SNPs with very high specificity (zero false positives in all tests) and high sensitivity (zero false negatives in 89% of tests).

[0063] Example 1 Calculation of the selection pressure of the host (human) environment adaptability of Mycobacterium abscessus

[0064] 1.1 Sample collection

[0065] Assuming that recent positive selection may be specific to the human host lung environment, most samples were collected from sputum, whereas MABC samples from other sources should not have this selection pattern. To test this, selection pressure was assessed in samples from non-lung sources.

[0066] 1.2 Methods

[0067] (1) Quantify the number of independent mutation events associated with each SNP

[0068] Using SNPPar v1.0

[261] Genes that have undergone changes in selection pressure in DCC and ECC were analyzed; the number of independent occurrences of each SNP in the ECC and DCC phylogenetic trees was inferred using the SNPParv1.0 tool. SNPPar uses TreeTime to reconstruct the ancestral sequence of each node and infer mutation events on each branch of the phylogenetic tree.

[0069] (2) Identification of mutant genes

[0070] For SNPs that appeared one or more times independently in the phylogenetic tree, the specific genes that underwent mutation were identified through the annotation process, and non-synonymous and synonymous mutations within each gene were calculated. The dN / dS ratio of all mutation event genes was calculated using the seqinr package in R (version 4.2.2) and evaluated using 3 independent analyses, which are as follows:

[0071] a) Calculate the genetic selection pressure for each subspecies based on the subspecies merger results;

[0072] b) Merging the results for DCC and ECC separately provides insights into the selection pressure on genes in these two categories;

[0073] c) An analysis was performed to incorporate genetic mutation events that occurred prior to DCC and ECC formation, allowing for the assessment of long-term selective pressures.

[0074] 1.3 Results

[0075] The purified genes selected were Figure 1 As shown in the figure, the MAB_4267c gene experienced 30 mutation events (dN / dS: 7.75) during its long-term evolution and was under strong purifying selection in DCCs (dN / dS: 0.12) and ECCs (dN / dS: 0.28). Similarly, MAB_2509c also switched from positive selection (dN / dS: 2.7) to purifying selection (dN / dS: 0.27). The changes in these genes may imply that the existence of some genes is no longer conducive to the living environment in the body, and therefore gradually begins to be marginalized. On the contrary, these genes may be an indispensable force for survival in the environment.

[0076] A recent study published 188 MABC isolates known to have originated in the Brazilian surgical setting ( Figure 2C), these isolates were from postoperative skin and soft tissue lesions, thus serving as an ideal control group. In addition, another 35 MABC strains were labeled as environmental samples in the NCBI database (derived from kitchen sinks, shower heads, and other environmental sampling points, etc.). Using these two groups as controls for sputum samples, additional patterns of selection pressure in the Brazilian clade and environmental samples were found. Among a total of 1,047 parallel mutation events found, five genes in the Brazilian samples were observed to show positive selection trends, such as mprB (dN / dS:3.6), which was significantly different from the selection pattern in the DCCs and ECCs groups, where these five genes were under purifying selection. It is noteworthy that none of the 15 genes previously identified as under positive selection in the DCCs and ECCs groups were mutated in the Brazilian clade, especially because these samples all belonged to the DCC3 subtype, so it was expected that it should at least show the advantages of DCCs on the phoR gene. And the closest genetically related pulmonary clade also showed positive selection signals on phoR ( Figure 2 C) Therefore, this differential result strongly suggests that positive selection of genes such as phoR is specific to the human host lung environment.

[0077] Furthermore, examining the selective pressures from environmental samples, we observed that the mutational patterns in these samples were significantly different from those in lung samples. Analysis of thousands of mutations revealed that genes that had experienced positive selection in DCCs versus ECCs showed signs of purifying selection in these samples. This differential selection signature further validated that the positive selection signature was specific to the human lung environment (respiratory system). Figure 2 C).

[0078] Since non-fixable mutations in bacterial samples collected from human hosts could provide insights into ongoing evolution of MABC within the host, we further examined non-fixable mutations in those lung samples and found that most of the previously identified positively selected genes also showed positive selection signals in non-fixable mutations ( Figure 2 D). Among these genes, phoR was still the gene most significantly under selection pressure. Specifically, 71 strains were identified that accumulated non-synonymous mutations in the phoR gene ( Figure 3 D). Notably, the locations on the gene where these non-fixing mutations occur correspond to the locations of the fixed mutations, indicating a consistent pattern of evolution ( Figure 3D). In addition to phoR, 13 other genes, such as der (36 occurrences in DCCs and 28 in ECCs), embC (26 in DCCs and 43 in ECCs), and ubiA (24 in DCC and 27 in ECC), showed a consistent pattern of mutation accumulation (as shown in Table 1). This consistent accumulation further supports the hypothesis that these genes are under selection in the host environment.

[0079] Figure 1 and Figure 2 The output is reliable. The SNPs present in each strain were annotated to cross-validate the output of SNPPar, and the mutation distribution lollipop plots were presented using the trackViewer package in R (version 4.2.2). Taken together, these findings suggest that the selection pressure acting on MABC has recently shifted. This shift indicates a niche shift of MABC, from the environmental background to the human airway, and further resulted in specific adaptive evolution.

[0080] Table 1. Probability of co-occurrence of ubiA and embc genes

[0081]

[0082] Example 2 Enrichment analysis of specific genes for adaptation of Mycobacterium abscessus to the host (human) environment

[0083] 2.1 Analysis of specific gene enrichment for Mycobacterium abscessus host (human) environment adaptation

[0084] The bioinformatics analysis of the unique genetic variation genes of all recent outbreak clusters (DCC and ECC strains) mainly includes the following aspects: prediction of transcription factor binding sites, analysis of protein sequence amino acid conservation sites, gene function enrichment analysis, protein functional domain prediction, etc.

[0085] The specific analysis operation process is as follows:

[0086] (1) Download the annotation file of the Mycobacterium abscessus genome from the NCBI database. The annotation files used are GZ002 (NZ_CP034181.1), CCUG48898 (NZ_AP014547.1), and GD91 (NZ_CP065265.1). At the same time, the annotation file of the commonly used comparison strain ATCC19977 is integrated. Genes are marked with conventional gene names, and SNPs in unique genetic variations are annotated for gene function. According to whether the SNP changes the type of amino acid encoded by the codon, the SNP is divided into non-synonymous mutations (including nonsense mutations such as premature termination mutations) and synonymous mutations. At the same time, the impact of the insertion and deletion positions on the genes is predicted.

[0087] (2) All protein sequences of Mycobacterium were downloaded from Uniprot (www.uniprot.org / ) and NCBI databases, and PSI-BLAST (Position-Specific Iterated BLAST) was performed on each homologous protein sequence to obtain the conservation information of each amino acid site in each protein sequence in the genome of Bacillus abscessus. The conservation of the site was evaluated using a score ranging from 0 to 4.36. When the conservation score was above 2.5, it was determined to be a conserved site, and the higher the score, the more conservative it was. For each non-synonymous mutation, it was possible to identify whether it was in a conserved site and the degree of conservation.

[0088] (3) Based on the gene function category annotation system, SMART (http: / / smart.embl-heidelberg.de / ) was used to predict the structural and functional domain information of the protein sequence and determine the functional domain where the unique genetic variation is located;

[0089] (4) GO, KEGG, and COG analyses were performed on the private mutation sites of each ECC and DCC using the online software David (https: / / david.ncifcrf.gov / summary.jsp), STRING (https: / / cn.string-db.org / ), and eggnog-mapper (http: / / eggnog-mapper.embl.de). The gene proportions and enrichment factors of each DCC and ECC were visualized using ggplot2 in R (version 4.2.2) (see Figure 3 -A,B,C).

[0090] 2.2 Results of enrichment analysis of genes specific to the adaptation of Mycobacterium abscessus to the host (human) environment

[0091] Natural selection pressure may act on a pathway, and mutations affecting the same pathway may have negative or positive epistasis. Therefore, we further sought to investigate the genetic correlation between genes that have recently been under positive selection in the MABC population. The preliminary analysis of this application uses the method recently published by Maha in MBE, the principle of which is to reveal the interdependence between all genes that are undergoing positive selection. After preliminary testing, the results did not show a dependent pattern of simultaneous or sequential mutations between these positively selected genes; but a particularly interesting finding was that the embC and ubiA genes had mutually exclusive mutation patterns, meaning that mutations in one gene prevented mutations in the other gene from occurring. Further simulations were performed based on the mutation frequencies of these two genes. According to the model of this application, the probability of two genes mutating simultaneously in the same strain should follow a normal distribution. Based on this model, considering the sample size of 1,000, it is expected to observe at least 8 examples of simultaneous mutation strains. However, contrary to expectations, no such situation was detected in the sample. Therefore, the actual situation in vivo is that embC and ubiA will not accumulate adaptive mutations in a strain at the same time.

[0092] Example 3 Biological verification of specific genes of Mycobacterium abscessus host (human) environment adaptation

[0093] 3.1 Biological verification process of specific genes of Mycobacterium abscessus host (human) environment adaptation

[0094] In critically ill patients with clinical disease of Mycobacterium tuberculosis abscessus, the rough type of Mycobacterium tuberculosis abscessus isolated is dominant, that is, the rough type of Mycobacterium tuberculosis abscessus indicates a poor prognosis of the disease. Therefore, studying and revealing the molecular mechanism of the transition of Mycobacterium tuberculosis abscessus from the smooth type to the rough type is crucial for analyzing the role of GPL in pathogenic and pathogenic Mycobacterium abscessus diseases.

[0095] The mechanisms of the smooth-to-rough transition in nontuberculous mycobacterial (NTM) species are diverse and often differ significantly between species. The cell wall of mycobacteria differs from the typical cell walls of Gram-positive and Gram-negative bacteria in that it is characterized by a high lipid content: it is composed primarily of several ubiquitous and species-specific lipids and glycolipids, including surface-exposed GPLs; these lipids are covalently linked and embedded in a layer of arabinan-peptidoglycan complexes. Structurally, GPLs consist of 3-hydroxy and 3-methoxy C 28-30The GPL genome consists of a heterogeneous population of fatty acids amidated with a D-phenylalanine-D-isolysine-D-alanine tripeptide-amino alcohol core. The GPL genome includes a series of large genes: mps1, mps2, mmpS4, mmpL4a, and mmpL4b; of these, mps1 and mps2, encode non-ribosomal peptide synthetases that require the tripeptide-amino alcohol group; of these, three adjacent genes, mmpS4, mmpL4a, and mmpL4b, encode membrane proteins of the machinery responsible for GPL traversing the inner membrane ( Figure 4 AD).

[0096] 3.2 Biological verification results of specific genes of Mycobacterium abscessus host (human) environment adaptation

[0097] The results showed that the pathway genes related to the GPL biosynthesis pathway were also under positive selection pressure, as evidenced by the accumulation of nonsense mutations and indels (indels) in the mps1 and mps2 genes that would lead to loss of function (LOF). Figure 4 AD). Previous studies have shown that impairment of the GPL secretion or synthesis pathway can cause MABC to switch from a smooth phenotype to a rough phenotype, and the rough phenotype is one of the common adaptive changes of MABC strains during chronic infection. However, most phenotypic changes reported previously occurred in the mml gene, while there were fewer observations on the results of changes in the mps1 and mps2 genes. Therefore, in order to verify that the inactivation of the mps1 and mps2 genes would lead to phenotypic changes, the colony phenotypes of three strains with mps1 or mps2 mutations in samples collected from Beijing Chest Hospital were examined, and all of these strains showed a rough phenotype ( Figure 4 A). The control strain with a very close genetic distance (only 3 SNPs difference) but without mps1 and mps2 mutations showed a smooth phenotype, suggesting that the inactivating mutations of mps1 and mps2 are the root cause of the phenotypic changes.

Claims

1. A set of genomic bioinformatics identification process and application kit for Mycobacterium abscessus adapted to human hosts, characterized in that The kit includes reagents and / or components for detecting a gene set, wherein the gene set includes the following 30 genes, the physical locations of which are based on the reference sequence used for Mycobacterium abscessus subsp. abscessus GZ002 (NZ_CP034181.1), subsp. massi CCUG48898 (NZ_AP014547.1) and subsp. boleyi GD91 (NZ_CP065265.1); the important genes are: antibiotic resistance (eis2, erm(41), gyrA); cell wall (ubiA, embC); two-component system (phoP, phoR); global regulators (crp, espR); Metal ion regulation (der, ideR).

2. The kit according to claim 1, characterized in that: The 30 genes include antibiotic resistance genes eis2, erm (41) and gyrA; cell wall genes ubiA and embC; two-component system genes phoP and phoR; global regulator genes crp and espR; metal ion regulatory genes der and ideR, etc.

3. The kit according to claim 2, characterized in that Distribution of genes of Mycobacterium abscessus that are under strong selective pressure from the host (human), the 30 most important genes.

4. The kit according to claim 2, characterized in that The reagents and / or components for detecting the 30 gene sets include one or more of PCR primers, molecular probes, biosensors and chips.

5. Use of the kit according to any one of claims 1 to 4 in the identification or evolution analysis of 30 host-adapted genes of Mycobacterium abscessus.

6. Use of the kit according to any one of claims 1 to 4 in the preparation of a diagnostic product for judging the 30 genes of Mycobacterium abscessus adapted to the host.

7. A method for bioinformatics identification or evolutionary analysis of host adaptation genes of Mycobacterium abscessus, characterized in that: The method uses the kit according to any one of claims 1 to 4 to detect 30 genes defined by the host (human) adapted by Mycobacterium abscessus.

8. The method according to claim 7, characterized in that The method for detecting the 30 genes includes one or more of the following: a detection method based on gel electrophoresis, a DNA sequencing method, a DNA chip method, a denaturing high performance liquid chromatography method or a mass spectrometry detection method.

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