Compositions and methods of ENT-testosterone for treatment of bacterial infections

Abstract

The present disclosure generally relates to compositions and methods that involve the use Enantiomer-Testosterone (Ent-Testosterone). The disclosed Ent-Testosterone can be used to inhibit quorum sensing in bacteria. The disclosure further compositions and method for treating a subject with bacterial infection or a condition complicated by colonization of bacteria.

Description

TITLECOMPOSITIONS AND METHODS OF ENT-TESTOSTERONE FOR TREATMENT OF BACTERIAL INFECTIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 552,459, filed February 12, 2024, and titled “COMPOSITIONS AND METHODS OF ENT-TESTOSTERONE FOR TREATMENT OF BACTERIAL INFECTIONS”, and U.S. Provisional Patent Application Serial No. 63 / 580,813, filed September 6, 2023, and titled “COMPOSITIONS AND METHODS OF ENT- TESTOSTERONE FOR TREATMENT OF BACTERIAL INFECTIONS”, which are incorporated by reference herein in their entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been submitted in .XML format via Patent Center and is hereby incorporated by reference in its entirety. Said WIPO Sequence Listing was created on August 21 , 2024, XML copy is named 106546-816096-4247_Sequence listing. xml, and is 19 kilobytes in sizeBACKGROUND

[0003] 1. Field

[0004] The present invention relates to Enantiomer-Testosterone (ENT-Testosterone), and composition and methods using the ENT-Testosterone for treatment of bacterial infections.

[0005] 2. Background

[0006] Skin and soft tissue infections by S. aureus pose a considerable public health threat. Seven-to-ten percent of hospitalized patients are afflicted by skin and soft tissue infections and skin infections are the third most common reason for presentation to the emergency room. Seventy five percent of these infections of the skin are caused by Staphylococcus aureus. Adding to the burden of disease posed by these pathogens is the development of antibiotic-resistant strains of bacteria such as methicillin-resistant S. aureus (MRSA). MRSA is the most frequent cause of skin and soft tissue infections in the emergency room setting and a frequent cause of hospital-acquired infections. Additionally, MRSA infections account for >$1 billion annually in total societal costs.

[0007] S. aureus is an opportunistic pathogen, capable of both quiescent colonization and lethal infections. S. aureus encodes numerous virulence factors from cell surface adhesins toextracellular matrix-degrading enzymes that allow it to bind, invade, and damage epithelial cells and cause infections. The most well-described system of virulence regulation is the accessory gene regulator (agr) quorum-sensing system. Agr senses the concentration of autoinducing peptide (AIP) signal generated by S. aureus or by other bacteria in the environment. AIP is synthesized in a stepwise process. First, a propeptide, AgrD, is produced in the cytoplasm. Then, the AgrD propeptide is processed into AIP and secreted into the extracellular space by the integral membrane endopeptidase AgrB. Studies also implicate MroQ in the AIP biosynthesis pathway. When bacteria are at a sufficient density and the concentration of AIP is sufficiently high (“quorum”), AIP binds and activates the AgrC histidine kinase. Once activated, AgrC phosphorylates the response regulator AgrA, which in turn autoinduces expression of the Agr machinery and induces expression of the RNAIII transcript. RNAIII is the primary effector of the system that triggers the increased production of toxins and exo-enzymes in S. aureus. In addition to responding to AIP, AgrC can respond to other environmental conditions, with recent work demonstrating that nitric oxide can inhibit S. aureus through direct inhibition of AgrC. However, host-derived factors may drive S. aureus skin infection in vivo and impact bacterial pathogenesis and the course of skin infection and disease. Since, bacterial skin infection, for e.g., by S. aureus offer limited therapeutic options, there is an unmet need to develop therapeutics for treating skin and soft tissue infections.SUMMARY

[0008] In one aspect, the disclosure encompasses a composition for treatment of a bacterial infection in a subject in need thereof, the composition comprising an Enantiomer- Testosterone (Ent-Testosterone). In some aspects, the composition further comprises a pharmaceutically acceptable carrier. In some aspects, the pharmaceutically acceptable carrier is petroleum jelly.

[0009] In further aspects, provided herein is a method of treating a subject having, or at risk of having, a bacterial infection, the method comprising administering to the subject a composition comprising Ent-Testosterone.

[0010] The disclosure further encompasses a method of treating a subject having, or at risk of having a skin and soft tissue infection (SSTI) by a bacterium, the method comprising administering to the subject a composition comprising Ent-Testosterone.

[0011] In some aspects, the disclosure comprises a method of treating a subject having, or at risk of having a condition complicated by the colonization of bacteria, the method comprising administering to the subject a composition comprising Ent-Testosterone. Further provided herein is a method of limiting damage to a tissue of a subject caused by a bacterialvirulence associated protein, the method comprising: administering to the subject a composition comprising a Ent-Testosterone. In such aspects, the tissue is skin tissue.

[0012] The disclosure further encompasses a method for attenuating virulence of a Staphylococcus spp., the method comprising contacting the Staphylococcus spp. with a composition comprising an effective amount of Ent-Testosterone.

[0013] In some aspects, the condition is atopic dermatitis, folliculitis, pemphigus, bullous pemphigoid, or cutaneous T-cell Lymphoma.

[0014] In some aspects, the bacterial infection is caused by a bacterium of the genus Staphylococcus. In some aspects, the bacterial infection is caused by Staphylococcus aureus. In some aspects, the Staphylococcus aureus is a methicillin resistant Staphylococcus aureus (MRSA), a methicillin-susceptible Staphylococcus aureus (MSSA), a vancomycin- intermediate Staphylococcus aureus (VISA), a vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is a Staphylococcus aureus strain LAC, a Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, a Staphylococcus aureus strain MN EV. In another aspect, the bacterial infection is caused by Staphylococcus epidermidis.

[0015] In some aspects, the subject is a mammal. In some aspects, the subject is a human.

[0016] In some aspects, the Ent-Testosterone reduces the expression of a virulence factor or a virulence-associated protein gene by at least 10% as compared to an untreated control subject. In some aspects, the virulence-associated protein gene is accessory gene regulator (agr) Type I. In some aspects, the virulence-associated protein gene is agr Type II. In some aspects, the virulence-associated protein gene is agr Type III. In some aspects, the virulence- associated protein gene is agr Type IV. In further aspects, the Ent-Testosterone reduces Staphylococcus induced hemolysis by at least 10%; and / or Staphylococcus induced neutrophil killing by at least 5%.

[0017] In some aspects, the Ent-Testosterone reduces the quorum sensing by the bacteria by at least 10% as compared to an untreated control subject. In some aspects, the effective amount of Ent-Testosterone is about 10 nM to about 10 pM. In some aspects, the effective amount of Ent-Testosterone reduces the accessory gene regulator C histidine kinase (agrC).

[0018] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a schematic of agr system coordinated expression of virulence factors in S. aureus.

[0020] FIG. 2 shows the relative expression of PSMa quantified by RT-PCR in Staphylococcus aureus strains LAC (Type I), USA 100(Type II), MW2 (Type III), and MN EV(Type IV) treated with 10nM Ent-Testosterone, 10nM Testosterone, or vehicle. Means ± SEM (error bars) are plotted. *p < 0.05; **p < 0.01 ; ***p < 0.001 , ****p <0.0001 , ns, not significant by two-tailed unpaired t-test.

[0021] FIG. 3 shows representative images of HSD3B6Askinfemale mice epicutaneously infected with a bioluminescent MRSAagr-P3lux reporter strain of MRSA at 1 hr, 2hr, 3hr, and 4hr after treatment with 10nM Ent-Testosterone, 10nM Testosterone, or a vehicle. Female wildtype mice were epicutaneously infected with 1 xio6CFUs agr-P3 S. aureus reporter treated with testosterone or enf-T. Bioluminescence quantified overtime. 700 Testosterone (n=4), ent- T(n=4), vehicle (n=4).

[0022] FIG. 4 shows average bioluminescence in HSD3B6Askinfemale mice epicutaneously infected with a bioluminescent MRSAagr-P3lux reporter strain of MRSA at 1hr to 8hr after treatment with 10nM Ent-Testosterone, 10nM Testosterone, or a vehicle. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.05, determined by unpaired t-test.

[0023] FIG. 5 shows bioluminescence quantified over time in MRSAagr-P3lux strain was with 10nM, 100nM, 1 pM, and 10 pM of Ent-Testosterone, Al PI I, or vehicle.

[0024] FIG. 6 shows bioluminescence quantified over time in MRSAagr-P3lux strain was with 10nM, 100nM, 1 pM, and 10 pM of Ent-Testosterone, or Testosterone.

[0025] FIGs. 7A-7D show enantiomer-testosterone inhibited S. aureus virulence and agr quorum sensing. FIG. 7A shows structure of testosterone and its stereoisomer enantiomer- Testosterone (ent-T). FIG. 7B-7C show wildtype S. aureus 695 (HG003) treated with 100nM of ent-T, AIP-II, or untreated (vehicle). FIG. 7B shows percentage of S. aureus induced hemolysis and FIG. 7C shows percentage of S. aureus induced neutrophil killing (C) (n=3). FIG. 7D shows qRT-PCR for psma expression in atopic dermatitis S. aureus strains treated with 10nM of testosterone or 10nM of ent-T. Relative expression of psma is normalized to housekeeping gene gyrA expression. Means ± SEM (error bars) are plotted. *p < 0.05; **p < 0.01 ; ***p < 0.001 , ****p <0.0001 , ns, not significant by two-tailed unpaired t-test.

[0026] FIGs. 8A-8B show enantiomer-testosterone (ent-T) inhibits S. aureus virulence factor expression and quorum sensing. FIG. 8A shows qRT-PCR of strains of S. aureus obtained from patients with atopic 1105 dermatitis and treated with 10nM or ent-T, the stereoisomer of testosterone (n=3). Expression of target gene RNAIII normalized tohousekeeping gene gyrA expression. Means ± SEM (error bars) are plotted. *p < 0.05 by two- tailed unpaired t-test. FIG. 8B shows representative bioluminescence images of FIG. 4, female wild-type mice epicutaneously infected with 1 xio6CFUs agr-P3 S. aureus reporter treated with testosterone, ent- , vehicle, or untreated. (n=4).

[0027] FIGs. 9A-9D show testosterone and DHT activate quorum sensing readouts in S. aureus, but other hormone classes do not activate quorum sensing. FIG. 9A shows in vitro bioluminescence of agrreporter (HG003 agrP3 lux) after treatment with 10nM of testosterone, DHT, progesterone, or estradiol. FIG. 9B shows in vitro bioluminescence of agr reporter (HG003 agrP3:.lux) after treatment with DHT. FIG. 9C shows in vitro bioluminescence of agr reporter (HG003 agrP3:.lux) after treatment with progesterone. FIG. 9D shows in vitro bioluminescence of agr reporter (HG003 agrP3 lux) after treatment with AIP-I or estradiol.

[0028] FIG. 10A is a series of photographs of mice tape shaved and applied with MRSA on days 0, 1 , and 2.

[0029] FIG. 10B is a series of line graphs showing the bioluminescence of MRSA treatment in vivo (top panel) and the impact of MRSA in vivo on TEWL (transepidermal water loss)- a marker of skin barrier function (bottom panel).

[0030] FIG. 11 A is a series of photographs of male mice showing agrP3 luminance applied with treated with AH2659 agrP3 reporter strain and applied with vehicle, Ent-Testosterone at 10 nanomoles, or Ent-Testosterone at 100 nanomoles at 0, 1 , 2, 3, 4, 5, 6, 7, and 8 hours after treatment.

[0031] FIG. 11 B is a line graph showing agrP3 luminance over at 0, 1 , 2, 3, 4, 5, 6, 7, and 8 hours after treatment with AH2659 agrP3 reporter strain and applied with vehicle, Ent- Testosterone at 10 nanomoles, or Ent-Testosterone at 100 nanomoles treatment.

[0032] FIG. 11C is a bar graph showing average weight of the mice on the day of infection with AH2659 agrP3 reporter strain in the different treatment groups.

[0033] FIG. 11D is a dot graph showing TEWL in the different treatment groups on the day of infection with AH2659 agrP3 reporter strain.

[0034] FIG. 12 is a line graph showing agrP3 luminance over time at increasing concentrations of Ent-Testosterone with or without testosterone.

[0035] The drawing figures do not limit the present disclosure to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain embodiments of the present disclosure.DETAILED DESCRIPTION

[0036] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0037] The present disclosure is partly based on the identification of Enantiomer- Testosterone (ENT-Testosterone) can inhibit bacteria and can be used as a therapeutic for treating bacterial infections, or conditions complicated by bacterial infections. ENT- Testosterone was found to inhibit expression of virulence factors in Type I, Type II, Type III, and Type IV strains of Staphylococcus. Further, ENT-Testosterone was found to reduce quorum sensing by reducing the activity of accessory gene regulator C histidine kinase (agrC). Provided herein are also composition and methods of use of ENT-Testosterone for treatment or prevention of bacterial infections, or conditions complicated by bacterial infections.I. Terminology

[0038] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred aspects and specific language will be used to describe the same. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

[0039] As used in the specification, articles “a” and “an” are used herein to refer to one or to more than one (i.e. , at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0040] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. The term “about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.

[0041] Throughout this specification, unless the context requires otherwise, the word “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements, or steps but not the exclusion of any other integer or step or group of integers or steps.

[0042] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0043] As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

[0044] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0045] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise-indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1 % to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0046] As used herein, “treatment,” “therapy,” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder, or condition.

[0047] As used herein, “prevent” or “prevention” refers to eliminating or delaying the onset of a particular disease, disorder, or physiological condition, or to the reduction of the degree of severity of a particular disease, disorder or physiological condition, relative to the time and / or degree of onset or severity in the absence of intervention.

[0048] The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0049] As used herein, “individual,” “subject,” “host,” and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis, or therapy is desired, for example, humans, pets, livestock, horses, or other animals. As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. In one aspect, the subject may be a rodent, e.g., a mouse, a rat, a guinea pig, etc. In another aspect, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas, and alpacas. In still another aspect, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another aspect, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In some aspects, the subject can be a human. In other aspects, the subject can be a human in need of treating a bacterial infection.

[0050] As used herein “quorum sensing” refers to bacterial signaling and response. Quorum sensing enables a bacterial species to sense its own number and regulate gene expression according to population density. Quorum sensing is cell density-dependent regulation of genes that involves a freely diffusible molecule synthesized by the cell called an autoinducer, or autoinducing peptide (AIP).

[0051] As used herein “accessory gene regulator” or “agr” relates to the global regulator of virulence. The agr system, as show in FIG. 1 , uses a small secreted autoinducing peptide (AIP) to activate a receptor histidine kinase, AgrC, in the bacterial cell membrane. AgrC phosphorylates the transcription factor AgrA, which in turn activates transcription at the P2 and P3 promoters of the operon. P3 activation drives production of the effector of the operon, RNAIII, which regulates expression of over 200 virulence genes that contribute to invasive infection. S. aureus isolates have one of four agr alleles (Type-I, Type-ll, Type-Ill, or Type-IV), each encoding factors that secrete a unique AIP (AIP1 , AIP2, AIP3, or AIP4, respectively) that is detected by a cognate AgrC histidine kinase. Every staphylococcal isolate contains only a single copy of the agr system, and each particular species produces different types of AIP signals through variation in the agrBDCA operon. S. aureus isolates possess one of the four alleles and an isolate that possesses any one of the four alleles can cause human disease.

[0052] As used herein “Enantiomer-Testosterone” or “Ent-Testosterone” or “Ent-17a- hydroxyandrost-4-en-3-one,” used interchangeably, refers to an enantiomer of the primary male sex hormone testosterone. Enantiomers are pairs of stereoisomers, called antipodes,optical antipodes, or optical isomers. They are mirror images of each other, and one can’t be superimposed on the other. Enantiomers have the same connectivity but opposite three- dimensional structures. Testosterone is a steroid sex hormone, produced mainly by the Leydig cells in the testes in men and in smaller amounts by the ovaries in women. Small quantities of the hormone are also synthesized in the cortex of the adrenal glands in both sexes.

[0053] As used herein “expression” or “level” or “level of expression” refers to amount of a particular protein present in the sample. The amount may be a number, proportion, or a percentage of the analyte compared to the control sample. The amount may be an absolute amount or a relative amount.

[0054] As used herein “skin and soft tissue infections” or “SSTI” are bacterial infections of the skin, muscles, and connective tissue such as ligaments and tendons. Non-limiting examples of SSTI include abscesses, cellulitis, folliculitis, MRSA (methicillin-resistant staphylococcus aureus) infection, and Staph infection (Staphylococcus aureus), erysipelas, impetigo, furuncles, and carbuncles.

[0055] The biological sample may be of any biological tissue, fluid, or cell from the subject. The sample can be solid or fluid. The sample can be a heterogeneous cell population. Nonlimiting examples of suitable biological samples include sputum, serum, blood, blood cells (e.g., white cells), a biopsy, urine, peritoneal fluid, pleural fluid, or cells derived therefrom. The biopsy can be a fine needle aspirate biopsy, a core needle biopsy, a vacuum assisted biopsy, an open surgical biopsy, a shave biopsy, a punch biopsy, an incisional biopsy, a curettage biopsy, or a deep shave biopsy. Biological samples may also include sections of tissues, such as frozen sections or formalin fixed sections taken for histological purposes. A sample can be a skin tissue, tissue surrounding an infection, or non-infected tissue. Methods of collecting a biological sample from a subject are well known in the art.

[0056] Sample from the subject can be procured one or more times, before, during and / or after diagnosis. In some aspects, samples can be procured from the subject before, during, and / or after treatment of bacterial infection. In some aspects, sample can be procured before, during and / or after administration of a treatment. Additionally, samples can be procured repeatedly at multiple stages after initial sample procurement.

[0057] In some aspects, control sample can be procured from a healthy subject. In some aspects, the control sample can comprise of non-infected cells. In some aspects, the noninfected cells can be from the same tissue type as the infected cells. For example, if the infected cells are from skin, then the non-infected cells can be from healthy skin tissue. In some aspects, the control can comprise of an average level in a sample from a subject before onset of infection. In some aspects, control sample can be a sample from the subject prior to diagnosis or treatment. In some aspects, the control is a person or persons with similarcharacteristics to the subject with infection. In some aspects, the control can be an average of the combination from different healthy sources (e.g., more than one healthy control subject). In some aspects, the control sample can be pooled sample.

[0058] As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and / or prevention of a disease or disorder (e.g., bacterial infection). As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., bacterial infection).

[0059] In an aspect, “therapeutically effective amount” means an amount of the disclosed composition that (i) treats a disease or disorder (for. e.g., bacterial infection), (ii) attenuates, ameliorates, or eliminates one or more symptoms associated with a disease or disorder (for e.g., bacterial infection), or (iii) delays the onset of one or more symptoms of a disease or disorder (for e.g., bacterial infection). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the type of bacterial infection being treated; the disclosed compositions employed; the disclosed methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed compositions employed; the duration of the treatment; drugs used in combination or coincidental with the disclosed compositions employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the disclosed compositions at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of the disclosed compositions, disclosed pharmaceutical formulations, disclosed therapeutic agents, or a combination thereof can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a sign or symptom associated with a disease or disorder (for e.g., bacterial infection).II. Ent-Testosterone

[0060] The present disclosure encompasses Ent-Testosterone, which can inhibit bacteria. The Ent-testosterone inhibits the expression of virulence factors in pathological microbes such as, for e.g., bacteria in the genus Staphylococcus.

[0061] In some aspects, the Ent-testosterone as disclosed herein has the following chemical structure:

[0062] In certain aspects, Ent-testosterone can block, inhibit, or decrease activity of agrC. In certain aspects, Ent-testosterone described herein may decrease agrC activity compared to agrC activity in the absence of Ent-Testosterone herein. In certain aspects, Ent-testosterone described herein may decrease agrC activity by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to agrC activity in the absence of an Ent- testosterone herein.

[0063] In certain aspects, Ent-testosterone can block, inhibit, or decrease the expression of one or more virulence-associated protein gene or a virulence factor in a bacterium. In some aspects, virulence-associated protein gene is accessory gene regulator (agr) Type I. In some aspects, virulence-associated protein gene is accessory gene regulator (agr) Type II. In some aspects, virulence-associated protein gene is accessory gene regulator (agr) Type III. In some aspects, virulence-associated protein gene is accessory gene regulator (agr) Type IV. In certain aspects, Ent-testosterone described herein may decrease the expression of one or more virulence factor in a bacterium compared to the expression of one or more virulence factor in a bacterium in the absence of Ent-Testosterone herein. In certain aspects, Ent- testosterone described herein may decrease the expression of one or more virulence factor in a bacterium by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to the expression of one or more virulence factor in bacteria in the absence of an Ent-testosterone herein. In some aspects, Ent-testosterone described herein may decrease the expression of one or more virulence factor in a bacterium by at least about 10%.

[0064] In certain aspects, Ent-testosterone can block, inhibit, or decrease quorum sensing by bacteria. In certain aspects, Ent-testosterone described herein may decrease quorumsensing by bacteria compared to the quorum sensing by a bacterium in the absence of Ent- Testosterone herein. In certain aspects, Ent-testosterone described herein may decrease the quorum sensing by bacteria by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to the quorum sensing by a bacterium in the absence of an Ent-testosterone herein. In some aspects, Ent-testosterone described herein may decrease the quorum sensing by a bacterium by at least about 10%.

[0065] In some aspects, Ent-testosterone can block, inhibit, or decrease growth of bacteria. In certain aspects, Ent-testosterone described herein may decrease growth of bacteria compared to growth of bacteria in the absence of Ent-Testosterone herein. In certain aspects, Ent-testosterone described herein may decrease the growth of bacteria by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to the growth of bacteria in the absence of an Ent-testosterone herein. In some aspects, Ent- testosterone described herein may decrease the growth of bacteria by at least about 10%.

[0066] In some aspects, Ent-testosterone can block, inhibit, or decrease infection by bacteria. In certain aspects, Ent-testosterone described herein may decrease infection by bacteria compared to infection by bacteria in the absence of Ent-Testosterone herein. In certain aspects, Ent-testosterone described herein may decrease the infection by bacteria by about 5% to about 99% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) compared to infection by bacteria in the absence of an Ent-testosterone herein. In some aspects, Ent- testosterone described herein may decrease infection by bacteria by at least about 10%.

[0067] In some aspects, the bacteria can be any bacterium that has quorum sensing. In some aspects, the bacteria can be any bacterium that has agrC signaling system. In some aspects, the bacteria is bacteria from the genus Staphylococcus. In some aspects, the bacteria is Staphylococcus aureus, or Staphylococcus epidermidis. In some aspects, the Staphylococcus aureus is a methicillin resistant Staphylococcus aureus (MRSA), methicillin- susceptible Staphylococcus aureus (MSSA), a vancomycin-intermediate Staphylococcus aureus (VISA), or a vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is a Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, or a Staphylococcus aureus strain MN EV.III. Compositions

[0068] In some aspects, provided herein are compositions comprising Ent-testosterone. Compositions of the present disclosure may be a pharmaceutical composition. In some aspects, pharmaceutical compositions herein may comprise Ent-testosterone, and at least one pharmaceutically acceptable carrier and / or excipient. Pharmaceutically acceptable carriers or excipients suitable for the compositions described herein are well known to one of skill in the art of use for preserving and delivering an active ingredient such as hormone to any mammalian subject including humans and other mammals.

[0069] In some aspects, compositions described herein may be administered to a subject in need thereof. In some aspects, the subject is a human. In some aspects, the subject is a subject having, suspected of having, or at risk for a bacterial infection, or a condition complicated by the colonization of bacteria. In some aspects, the subject is having, or is at risk of having a skin and soft tissue infection (SSTI). In some aspects, the subject is having, or is at risk of having atopic dermatitis, folliculitis, pemphigus, or bullous pemphigoid. Compositions disclosed herein can inhibit growth and / or activity of a bacteria. The bacteria can be any bacterium that has agrC signaling system. In some aspects, the bacteria is bacteria from the genus Staphylococcus. In some aspects, the bacteria is Staphylococcus aureus, or Staphylococcus epidermidis. In some aspects, the Staphylococcus aureus is a methicillin resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), a vancomycin-intermediate Staphylococcus aureus (VISA), or a vancomycin- resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is a Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, or a Staphylococcus aureus strain MN EV.

[0070] In some aspects, Ent-testosterone may therefore be formulated into a pharmaceutical composition. The composition may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). The composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). The composition also can be administered via a sustained or delayed release, and / or be eluted combined or eluted from a medical dressing such as, for example, a bandage.

[0071] In specific aspect, the pharmaceutical composition may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, orany form of mixture. For example, the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a nonaerosol spray, a gel, a lotion, and the like. The composition may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.

[0072] In certain aspects, the compositions to be used in the present methods can include pharmaceutically acceptable carriers, excipients, and / or stabilizers in the form of lyophilized formulations or aqueous solutions. In some aspects, acceptable carriers, excipients, and / or stabilizers are nontoxic to recipients at the dosages and concentrations used, and can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG).

[0073] In some aspects, the composition described herein can have liposomes containing the ENT-Testosterone. In some aspects, liposomes for use herein can be generated by the reverse phase evaporation method with a lipid composition having phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). In some aspects, liposomes for use herein can be extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0074] In some aspects, Ent-Testosterone, can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

[0075] In some aspects, the composition herein may be sterile. This can be readily accomplished by, for example, filtration through sterile filtration membranes. In some aspects,the composition herein can be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0076] In certain aspects, the composition described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, or rectal administration, or administration by inhalation or insufflation. A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the ENT-Testosterone into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and / or intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

[0077] In some aspects, emulsion compositions herein can be those prepared by mixing Ent-Testosterone with Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).

[0078] Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and / or Lipiphysan™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (for e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to about 20% oil, for example, between about 5% and about 20%. The fat emulsion can comprise fat droplets between about 0.1 pm and about 1.0 pm, particularly about 0.1 pm and 0.5 pm, and have a pH in the range of about 5.5 to about 8.0.

[0079] In some aspects, the composition herein for inhalation or intranasal administration may include solutions and suspensions in pharmaceutically acceptable, aqueous, or organic solvents, or mixtures thereof, and powders. In some aspects, liquid or solid compositions herein can contain suitable pharmaceutically acceptable excipients as set out above. In other aspects, the compositions can be administered by the oral or nasal respiratory route for local or systemic effect.

[0080] In some aspects, the composition can be in sterile pharmaceutically acceptable solvents can be nebulized by use of gases. In some aspects, nebulized solutions herein canbe breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent or intermittent positive pressure breathing machine. In some embodiments, solution, suspension, or powder compositions herein can be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

[0081] In certain aspects, the composition disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more buffering agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more buffering agents by total weight of the composition.

[0082] In some aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some aspects, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In some aspects, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0083] In certain aspects, the composition disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces, or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more preservatives by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more preservatives by total weight of the composition.

[0084] In certain aspects, the composition disclosed herein may comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergentsmay be synthetic, natural, or semi-synthetic. In some aspects, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In some aspects, the composition disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition. In some aspects, the composition disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more surface-acting reagents or detergents by total weight of the composition.

[0085] In certain aspects, the composition disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine, and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, the composition disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more stabilizers by total weight of the composition. In some aspects, the composition disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more stabilizers by total weight of the composition.

[0086] In some aspects, the compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose, and others known to those or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolarity may be measured using methods commonly known in the art. In some aspects, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L or about 250 mOsm / L to about 320 mOsm / L. In some aspects, a composition herein may have an osmolality ranging from about 100 mOsm / kg to about 1000 mOsm / kg, from about 200 mOsm / kg to about 800 mOsm / kg, from about 250 mOsm / kg toabout 500 mOsm / kg, or from about 250 mOsm / kg to about 320 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg or from about 280 mOsm / kg to about 320 mOsm / kg. In some aspects, a pharmaceutical composition described herein may have an osmolarity of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320 mOsm / L, or about 280 mOsm / L to about 320 mOsm / L. In some aspects, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more tonicity modifiers by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more tonicity modifiers by total weight of the composition.

[0087] In some aspects, concentrations of Ent-Testosterone disclosed herein can be a pre-determined concentration or a standard concentration. In some aspects, Ent-testosterone disclosed herein can be in a concentration of about 1 micrograms / milliliters (mg / ml) to about 500 mg / ml, about 1 mg / ml to about 250 mg / ml, about 1 mg / ml to about 200 mg / ml, about 1 mg / ml to about 150 mg / ml, about 1 mg / ml to about 100 mg / ml, about 1 mg / ml to about 75 mg / ml, about 1 mg / ml to about 50 mg / ml, about 0.1 mg / ml to about 100 mg / ml, or other suitable concentration. In some aspects, the Ent-Testosterone is lyophilized. In some aspects, the Ent- Testosterone is diluted in a suitable solution to a suitable concentration prior to administration.

[0088] In specific aspect, the composition may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. In some aspects, the Ent-Testosterone is formulated for topical application. For example, the formulation may be delivered such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The composition may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like. In some aspects, the composition disclosed herein comprises Ent-Testosterone and a petroleum jelly.

[0089] In certain aspects, the composition for the present disclosure may be contained in a patch that is applied to the area of skin to be treated. As used herein a "patch" comprises at least a topical composition comprising Ent-Testosterone and a covering layer, such that, the patch can be placed over the area of skin to be treated. The patch may be designed to maximize drug delivery through the stratum corneum and into the epidermis or dermis, reduce lag time, promote uniform absorption, and / or reduce mechanical rub-off. In certain aspects, when the intended use comprises the treatment of an infection of skin, the patch is designedto minimize absorption into the circulatory system. Preferably, the patch components resemble the viscoelastic properties of the skin and conform to the skin during movement to prevent undue shear and delamination. Advantages of a patch comprising the topical formulation over conventional methods of administration include (i) that the dose is controlled by the patch's surface area, (ii) constant rate of administration, (iii) longer duration of action (the ability of to adhere to the skin for 1 , 3, 7 days or longer), (iv) improved patient compliance, (v) non-invasive dosing, and (vi) reversible action (i.e. , the patch can simply be removed).

[0090] In one aspect, a patch suitable for use with the present disclosure contains at least: (1) a backing layer and (2) a carrier formulated with a compound of the invention. Examples of patch systems suitable for practicing the invention include, but are not limited to, matrixtype patches; reservoir-type patches; multi-laminate drug-in-adhesive-type patches; and monolithic drug-in-adhesive type-patch. The matrix patch comprises matrix containing a composition disclosed herein, an adhesive backing film overlay, and preferably, but not necessarily, a release liner. In some cases, it may be necessary to include an impermeable layer to minimize drug migration into the backing film. In some aspects, the matrix containing the inventive compound is held against the skin by the adhesive overlay. Examples of suitable matrix materials include but are not limited to lipophilic polymers, such as polyvinyl chloride, polydimethylsiloxane, and hydrophilic polymers like polyvinylpyrrolidone, polyvinyl alcohol, hydrogels based on gelatin, or polyvinylpyrrolidone / polyethylene oxide mixtures. Suitable release liners include but are not limited to occlusive, opaque, or clear polyester films with a thin coating of pressure sensitive release liner (e.g., silicone-fluorosilicone, and perfluorocarbon-based polymers.

[0088] In some aspects, the amount of Ent-Testosterone administered can vary depending on various factors including, but not limited to, the specific microbe for which treatment is needed, the weight, physical condition, and / or age of the subject, and / or the route of administration. Thus, the absolute weight of Ent-Testosterone included in a given unit dosage form can vary widely and depends upon factors such as the Ent-Testosterone used, the species of infectious microbe, age, weight and physical condition of the subject, and / or method of administration. Those of ordinary skill in the art, can readily determine the appropriate amount with due consideration of such factors.

[0089] In some aspects, the composition disclosed herein can be administered to provide sufficient Ent-Testosterone at a dose of, for example, from about 100 ng / kg to about 50 mg / kg to the subject. In some aspects, the compositions when administered provide a dose of from about 10 pg / kg to about 5 mg / kg Ent-Testosterone to the subject, for example, a dose of from about 100 pg / kg to about 1 mg / kg Ent-Testosterone.

[0090] In some aspects, the compositions described herein can further comprise one or more active agents or therapeutic agents. Alternatively, the ENT-testosterone may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic agents. Non limiting examples of additional active agents, or therapeutic agents include but are not restricted to antibiotics, anti-inflammatory agents, pain killers, immunomodulatory agents, and anti-cancer agents. The antibiotic may be a bactericidal antibiotic, a bacteriostatic antibiotic, and / or a combination of two or more antibiotics. Exemplary antibiotics include, for example, a lincosamide (e.g., lincomycin or clindamycin), a penicillin (e.g., nafcillin), a cephalosporin (e.g., ceftaroline, ceftazidime (alone or in combination with avibactam), or ceftolozane (alone or in combination with tazobactam), a glycopeptide (e.g., vancomycin, oritavancin, dalbavancin), a lipopeptide (e.g., daptomycin), an aminoglycoside (e.g., gentamicin), an oxazolidinones (e.g., linezolid, tedizolid, posizolid, or cycloserine), or a tetracycline (e.g., doxycycline). Exemplary antimicrobial therapeutics include, for example, an immunotherapeutic such as, for example, an antimicrobial antibody treatment and / or antimicrobial cytokine treatment.

[0091] In some aspects, the Ent-Testosterone may be administered to a subject before or after the subject manifests a symptom or clinical sign of infection by a bacterium. “Symptom” refers to any subjective evidence of disease or of a patient's condition. “Sign” or “clinical sign” refers to an objective physical finding relating to a particular condition capable of being found by one other than the patient. Treatment that is initiated before a subject manifest a symptom or clinical sign of infection can be considered prophylactic treatment of a subject “at risk” of infection by the bacteria. As used herein, the term “at risk” refers to a subject that may or may not actually possess the described risk. Thus, for example, a subject “at risk” of infectious condition is a subject present in an area where other individuals have been identified as having the infectious condition and / or is likely to be exposed to the infectious agent even if the subject has not yet manifested any detectable indication of infection by the bacteria and regardless of whether the subject may harbor a subclinical amount of the bacteria.

[0092] Accordingly, the administration of the composition can be performed before, during, or after the subject first exhibits a symptom or clinical sign of the condition or, alternatively, before, during, or after the subject first comes in contact with the bacteria. Treatment initiated before the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the likelihood that the subject experiences clinical evidence of the condition compared to a subject to which the composition is not administered, decreasing the severity of symptoms and / or clinical signs of the condition, and / or completely resolving the condition. T reatment initiated after the subject first exhibits a symptom or clinical sign associated with the condition can be considered therapeutic treatment of the subject andmay result in decreasing the severity of symptoms and / or clinical signs of the condition compared to a subject to which the composition is not administered, and / or completely resolving the condition.

[0093] In some aspects, the administration of the composition comprising Ent- Testosterone can block, inhibit or decrease tissue damage in a subject. In some aspects, the tissue damage may be damage to skin, or other soft tissue. In some aspects, the administration of the composition comprising Ent-Testosterone can decrease tissue damage in a subject, by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above, as compared to tissue damage in the subject before administration of the composition comprising Ent-Testosterone (baseline) or a control subject not receiving the treatment. In some aspects, the administration of the composition comprising disclosed Ent-Testosterone decrease tissue damage of at least about 20% in the subject. Tissue damage may be determined using any known methods in the art for measuring cell apoptosis or cell viability, including bioluminescent imaging or flow cytometry.IV. Methods of Treatment

[0094] The present disclosure further provides a method of treating a subject with bacterial infection or a condition complicated by colonization of bacteria.

[0095] In some aspects, the method comprises treating a subject having, or at risk of having, a bacterial infection comprising administering to the subject a composition comprising Ent-Testosterone. Bacterial infection may be caused by bacteria from the genus Staphylococcus. In some aspects, the bacteria is Staphylococcus aureus, or Staphylococcus epidermidis. In some aspects, the Staphylococcus aureus is a methicillin resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), a vancomycin-intermediate Staphylococcus aureus (VISA), or a vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is a Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, or a Staphylococcus aureus strain MN EV.

[0096] In further aspects, the disclosure encompasses a method of treating a subject having, or at risk of having a skin and soft tissue infection (SSTI) by a bacterium, the method comprising administering to the subject a composition comprising Ent-Testosterone. In some aspects, the SSTI is abscesses, cellulitis, folliculitis, MRSA (methicillin-resistant staphylococcus aureus) infection, and Staph infection (Staphylococcus aureus or Staphylococcus epidermidis), erysipelas, impetigo, furuncles, or carbuncles. The bacteria may be bacteria from the genus Staphylococcus. In some aspects, the bacteria is Staphylococcus aureus. In some aspects, the Staphylococcus aureus is a methicillin resistant Staphylococcusaureus (MRSA), a methicillin-susceptible Staphylococcus aureus (MSSA), a vancomycin- intermediate Staphylococcus aureus (VISA), or a vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is a Staphylococcus aureus strain LAC, a Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, or a Staphylococcus aureus strain MN EV.

[0097] In another aspect, the method comprises treating a subject having, or at risk of having a condition complicated by the colonization of a bacteria, by administering to the subject a composition comprising Ent-Testosterone. In some aspects, the condition is atopic dermatitis, folliculitis, pemphigus, bullous pemphigoid, or cutaneous T-cell lymphoma. The bacteria may be bacteria from the genus Staphylococcus. In some aspects, the bacteria is Staphylococcus aureus or Staphylococcus epidermidis. In some aspects, the Staphylococcus aureus is a methicillin resistant Staphylococcus aureus (MRSA), a methicillin-susceptible Staphylococcus aureus (MSSA), a vancomycin-intermediate Staphylococcus aureus (VISA), or a vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is a Staphylococcus aureus strain LAC, a Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, or a Staphylococcus aureus strain MN EV.

[0098] In some aspects, the method disclosed herein comprises treating a subject having, or at risk of having Staphylococcus aureus infection. The method comprises administering to the subject a composition comprising Ent-Testosterone. The Staphylococcus aureus can be a methicillin resistant Staphylococcus aureus (MRSA), a methicillin-susceptible Staphylococcus aureus (MSSA), a vancomycin-intermediate Staphylococcus aureus (VISA), or a vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is a Staphylococcus aureus strain LAC, a Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, or a Staphylococcus aureus strain MN EV.

[0099] In another aspect, the method disclosed herein comprises treating a subject having, or at risk of having a Staphylococcus epidermidis infection. The method comprises administering to the subject a composition comprising Ent-Testosterone.

[0100] In yet another aspect, the method comprises limiting damage to a tissue of a subject caused by a bacterial virulence associated protein by administering to the subject a composition comprising Ent-Testosterone. In such aspects, the tissue is skin tissue or other soft tissue. The bacteria may be bacteria from the genus Staphylococcus. In some aspects, the bacteria is Staphylococcus aureus or Staphylococcus epidermidis. In some aspects, the Staphylococcus aureus is a methicillin resistant Staphylococcus aureus (MRSA), a methicillin-susceptible Staphylococcus aureus (MSSA), a vancomycin-intermediate Staphylococcus aureus (VISA), or a vancomycin-resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is a Staphylococcus aureus strain LAC, a Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, or a Staphylococcus aureus strain MN EV.

[0101] Further provided is a method for attenuating virulence of a Staphylococcus spp., comprising contacting the Staphylococcus spp. with a composition comprising Ent- Testosterone. In some aspects, Staphylococcus spp is Staphylococcus aureus or Staphylococcus epidermidis. In some aspects, the Staphylococcus aureus is a methicillin resistant Staphylococcus aureus (MRSA), a methicillin-susceptible Staphylococcus aureus (MSSA), a vancomycin-intermediate Staphylococcus aureus (VISA), or a vancomycin- resistant Staphylococcus aureus (VRSA). In some aspects, the Staphylococcus aureus is a Staphylococcus aureus strain LAC, a Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, or a Staphylococcus aureus strain MN EV.

[0102] In some aspects of the method, the disclosed bacteria can be any bacterium that has quorum sensing. Any bacterium that has agrC signaling system is contemplated to be treated by methods disclosed herein, such as for example Pseudomonas aeruginosa.

[0103] The subject may be any one at risk or is diagnosed with a bacterial infection, for e.g., Staphylococcus infection. Staphylococcal infections, particularly S. aureus infections, can affect various parts of the body and can include skin infection and conditions such as osteomyelitis, endocarditis, septic arthritis, pneumonia, abscess formation, gastroenteritis, scalded skin syndrome, and toxic shock syndrome. S. aureus strains are considered the leading cause of nosocomial infections in the United States. Staphylococcal infections, particularly S. aureus infections, caused by a strain that is resistant to commonly used antibiotics are particularly serious and life-threatening. A subject with high risk of Staphylococcus infection include, but are not limited to subjects that have conditions, such as diabetes, cancer, vascular disease, eczema, or lung disease, subject with burns, or a subject who has come into close contact with someone who has a Staphylococcus infection. In further aspects, subjects with higher risk of Staphylococcus infection may include subjects in intensive care units, have undergone a surgery, subject undergoing dialysis, subjects using a catheter, breathing tube or a feeding tube, have a medical device inserted in their bodies, such as a pacemaker, artificial joint, or heart valve, have weakened immune system, such as subjects diagnosed with HIV, subjects taking therapeutic agents to prevent organ or tissue transplant rejection, or subjects undergoing chemotherapy. Subjects may be children or infants who attend a daycare, preschool or school. In further aspects, the subject may be one at risk ordiagnosed with Staphylococcus epidermidis. Staphylococcus epidermidis infections can cause wound infections, boils, sinus infections, endocarditis and other inflammations.

[0104] The methods disclosed herein may comprise a composition with an effective amount of Ent-Testosterone. The effective amount can be a therapeutically effective amount. The composition may be administered as dosages to the subject. In some aspects of the method, a suitable, non-limiting example of a dosage according to the present disclosure may be from about 1 ng / kg to about 5000 mg / kg. In general, however, doses employed for adult human treatment typically may be in the range of about 0.0001 mg / kg / day to about 0.0010 mg / kg / day, about 0.0010 mg / kg / day to about 0.010 mg / kg / day, about 0.010 mg / kg / day to about 0.10 mg / kg / day, about 0.10 mg / kg / day to about 1 mg / kg / day, about 1 mg / kg / day to about 200 mg / kg / day, about 200 mg / kg / day to about 5000 mg / kg / day. For example, the dosage may be about 1 mg / kg / day to about 100 mg / kg / day, such as, e.g., about 2-10 mg / kg / day, about 10-50 mg / kg / day, or about 50-100 mg / kg / day. The dosage can also be selected from about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, about 1000 mg / kg, about 1100 mg / kg, about 1200 mg / kg, about 1300 mg / kg, about 1400 mg / kg, about 1500 mg / kg, about 1600 mg / kg, about 1700 mg / kg, about 1800 mg / kg, about 1900 mg / kg, about 2000 mg / kg, about 2100 mg / kg, about 2200 mg / kg, about 2300 mg / kg, about 2400 mg / kg, about 2500 mg / kg, about 2600 mg / kg, about 2700 mg / kg, about 2800 mg / kg, about 2900 mg / kg, about 3000 mg / kg, about 3500 mg / kg, about 4000 mg / kg, or about 5000 mg / kg.

[0105] The Ent-T estosterone of the present disclosure can be administered to the subject daily or more than once daily. In some aspects, the disclosed Ent-Testosterone can be administered every 2, 3, 4, 5, 6, 7, 14, or every 30 days. The disclosed Ent-Testosterone can be administered over a period ranging from about 1 day to about 1 year, from about 1 day to about 1 week, from about 3 days to about 1 month, from about 2 weeks to about 6 months, or from about 2 months to about 4 months. The disclosed Ent-Testosterone can also be administered over a period of about 1 day, about 7 days, about 30 days, about 60 days, about 120 days, or about 180 days or more. In some aspects, the disclosed Ent-Testosterone is administered over a period of about 57 weeks, about 148 weeks, about 208 weeks, or indefinitely.

[0106] Dosage amount and interval may be adjusted individually to provide a sufficient amount to maintain therapeutic or prophylactic effect. For example, Ent-Testosterone thereofmay be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of Ent-Testosterone may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.

[0107] In some aspects, the method of administration of the Ent-Testosterone, results in reduction of tissue damage in a subject. In some aspects, the tissue damage may be damage to skin, or other soft tissue. In some aspects, the method of administration of the Ent- Testosterone can decrease tissue damage in the subject, by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above, as compared to tissue damage in the subject before administration of the Ent-Testosterone (baseline) or a control subject not receiving the treatment. In some aspects, the method of administration of the Ent-Testosterone decrease tissue damage of at least about 20% in the subject. Tissue damage may be determined using any known methods in the art for measuring cell apoptosis or cell viability, including bioluminescent imaging or flow cytometry.

[0108] In one aspect, the method of administration of the Ent-Testosterone, results in reduction of Staphylococcus induced hemolysis in a subject. In some aspects, the method of administration of the Ent-Testosterone can reduce Staphylococcus induced hemolysis in the subject, by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above, as compared to Staphylococcus induced hemolysis in the subject before administration of the Ent-Testosterone (baseline) or a control subject not receiving the treatment. In some aspects, the method of administration of the Ent-Testosterone decrease Staphylococcus induced hemolysis at least about 5% in the subject.

[0109] In another aspect, the method of administration of the Ent-Testosterone, results in reduction of Staphylococcus induced neutrophil killing in a subject. In some aspects, the method of administration of the Ent-Testosterone can reduce Staphylococcus induced neutrophil killing in the subject, by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above, as compared to Staphylococcus induced neutrophil killing in the subject before administration of the Ent-Testosterone (baseline) or a control subject not receiving the treatment. In some aspects, the method of administration of the Ent-Testosterone decrease Staphylococcus induced neutrophil killing at least about 5% in the subject.

[0110] In further aspects, the method of administration of the disclosed Ent-Testosterone, results in reduction of one or more adverse effect caused by bacterial infection, for e.g.,Staphylococcus aureus in a subject, or a disease state in the subject. Non-limiting examples of adverse effects include hemolysis, toxic shock, biofilm formation, and virulence. In some aspects, the method of administration of the Ent-Testosterone can reduce one or more hemolysis, toxic shock, biofilm formation, and virulence in the subject, by at least 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above, as compared to hemolysis, toxic shock, biofilm formation, and / or virulence in the subject before administration of the Ent- Testosterone (baseline) or a control subject not receiving the treatment.

[0111] The methods of the present disclosure may be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, ENT-Testosterone may be administered concurrently with another anti-inflammatory agent, immunomodulatory agent, antibiotic agent, painkiller, or anticancer agent), or they may achieve different effects (for e.g., control of any adverse effects).

[0112] In some aspects, the disclosed methods can further comprise modifying one or more of the administrations of the Ent-Testosterone described herein. For example, modifying one or more of steps of the method of administration can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of Ent-Testosterone administered to a subject, or by changing the frequency of administration of Ent-Testosterone to a subject, or by changing the duration of time one or more of the disclosed the Ent-Testosterone administered to a subject.V. Kits

[0113] The present disclosure provides kits comprising Ent-Testosterone for use with a method of the disclosure. The kits may comprise a composition comprising Ent-Testosterone, and instructions for administering the Ent-Testosterone to a subject in need thereof. The kit could further comprise other therapeutic agents, such as for e.g., an antibiotic, that can be administered in combination with the Ent-Testosterone. In some aspects, the kits can further comprise a sterile, pharmaceutically acceptable carrier, buffer, or other diluent. The kits provided herein generally include instructions for carrying out the methods. Instructions included in the kits may be affixed to packaging material or may be included as a package insert. While the instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an enduser is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” may include the address of an internet site that provides the instructions.

[0114] The disclosed kits may have a single container that contains the disclosed Ent- Testosterone with or without any additional components, or they may have distinct containers for each desired agent. Where combined therapeutics are provided, a single solution may be pre-mixed, either in a molar equivalent combination, or with one component in excess of the other. Alternatively, Ent-Testosterone and other therapeutic agent components of the kit may be maintained separately within distinct containers prior to administration to a patient.

[0115] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.

[0116] The containers of the kit will generally include at least one vial, test tube, flask, bottle, syringe or another container means, into which the disclosed Ent-Testosterone, and any other desired agent, may be placed and, preferably, suitably aliquoted. Where separate components are included, the kit will also generally contain a second vial or other container into which these are placed, enabling the administration of separated designed doses. The kits may also comprise a second / third container means for containing a sterile, pharmaceutically acceptable buffer or another diluent.

[0117] The kits may also contain a means by which to administer the disclosed Ent- Testosterone to an animal or patient, e.g., one or more needles or syringes, or even an eye dropper, pipette, or other such like apparatus, from which the formulation may be injected into the animal or applied to a diseased area of the body. The kits of the present disclosure will also typically include a means for containing the vials, or such like, and other component, in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials and other apparatus are placed and retained.

[0118] Having described several aspects, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present disclosure. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuringthe present disclosure. Accordingly, this description should not be taken as limiting the scope of the present disclosure.

[0119] Those skilled in the art will appreciate that the presently disclosed aspects teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.EXAMPLES

[0120] The following examples are included to demonstrate aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Materials and MethodsMice

[0121] Conventionally raised C57BL6 / J male and female mice aged 6-9 weeks old mice were purchased from the Jackson Laboratory. C57BL6 / J wild-type, K14Cre+ / -(60), and Hsd3b6fl / fl mice were bred and maintained in the specific pathogen-free (SPF) barrier facility at the University of Texas Southwestern Medical Center at Dallas. The generation of Hsd3b6ASkin (K14Cre+ / -; Hsd3b6fl / fl) is described below. Mice were co-housed with 3-5 mice per cage in all experiments. All mice were housed under a 12-hour-light:12-hour-dark cycle. Mice were fed ad libitum with free access to drinking water according to protocols approved by the Institutional Animal Care and Use Committees (IACUC) of UT Southwestern Medical Center.

[0122] Hsd3b6fl / fl (C57BL6 / J), with loxP sites surrounding the first coding exon of Hsd3b6 were generated using CRISPR / Cas9 genome editing with guide RNAs targeting regions of the Hsd3b6 locus (fig. 745 S2A). Guide RNAs were injected into fertilized C57BL / 6J embryos by the Children’s research institute mouse genome engineering facility at UT Southwestern. Healthy blastocytes were implanted in pseudo-pregnant mice. The resulting litter was screened by genomic sequencing to detect insertion of loxP sites and mice were bred tohomozygosity and backcrossed with wild-type C57BL / 6 mice. To generate K14Cre+ / - ;Hsd3b6fl / fl(Hsd3b6 ASkin) mice, Hsd3b6fl / fl mice were crossed 750 with K14Cre+ / - mice to generate K14Cre+ / -;Hsd3b6fl / + mice, K14Cre; Hsd3b6fl / + mice were crossed to Hsd3b6fl / fl mice to obtain experimental mice K14Cre; Hsd3b6fl / fl (Hsd3b6ASkiri) and corresponding controls, Hsd3b6fl / fl. Hsd3b6fl / fl and Hsd3b6ASkin status was determined using PCR primers (Table 1) and resolving on a 3% Agarose gel.Table 1 : Staphylococcus aureus strainsFACS analysis skin

[0123] Ear skin from the epidermis of 8-12 weeks old wildtype, HSD3B6fl / fl and HSD3B6Askinmce were digested with DNase-l at 1-mg / mL (Sigma-Aldrich; DN25-1G), Liberase-TL at 0.32-mg / mL (Sigma-Aldrich; 5401020001), Collagenase-D at 6-mg / mL (Sigma-Aldrich; 11088858001) in RPMI media, minced and incubated for 1 hr at 37°C at 1400 rpm on a thermocycler, and passed through a 70-pm cell separation filter. After washing, the cell pellet was suspended in FACS buffer (PBS with 3% BSA and 2 mM EDTA). The cell suspensions were transferred to a 96 well plate with v-bottom (Corning Inc: Costar; 3894) and processed further. Cell viability was determined with Ghost-Dye-Red-710 (Cytek; SKU 13-0871-T100). To prevent non-specific antibody binding the cells were stained with Fc receptor blocking with anti-mouse CD16 / 32 antibody (BD Biosciences; 553142). Different cell populations were assessed with the following antibodies re-suspended in FACS buffer for 15 minutes in 4°C. Brilliant-Violet (BV)-650-anti-CD45 (Biolegend; 103151), FITC-anti-CD3 (Cytek; SKU 35-0032-U025), BV-421-anti-CD11b (Biolegend;), BV711-anti-F4 / 80 (Biolegend;). The gating strategy used to determine cell populations is as follows: For total leucocytes singlet live cells were gated on CD45+ markers, from which the total T cells were identified as CD45+CD3+ cells, macrophages were identified as CD45+CD11b+F4 / 80+ cells. Cells were acquired by using NovoCyte flow cytometer and analyzed using NovoExpress software.Immunofluorescence microscopy

[0124] Mouse skin samples were fixed in formalin and embedded in paraffin by the UT Southwestern histology core. Samples were deparaffined with xylene followed by rehydration with decreasing concentrations of ethanol. Heat induced antigen retrieval was attained in 10 mM sodium citrate buffer. Sections were washed briefly and blocked for an hour in blocking / permeabilization buffer (PBS+5% Goat serum+0.5%Triton X-100). Sections were incubated in blocking / permeabilization buffer overnight with the following antibodies: anti- HSD3B6 (2.5 pg / mL; Biorbyt; orb592071), anti- cytokeratin-14 (1 pg / mL; Santa Cruz Biotechnology; sc-53253). After a brief wash in PBST (PBS+0.2% Tween-20) sections were incubated with corresponding secondary antibodies: Donkey anti-rabbit alexa-fluor-647 (2 pg / mL; Jackson Immuno; 711-605-152), Donkey anti-mouse alexa-fluor-594 (2 pg / mL; Thermofisher Scientific; A-21203). The slides were then washed briefly in PBST and mounted with 4, 6-diamidino-2-phenylindole (DAPI) containing mounting medium (SouthernBiotech; 0100-20). Images were processed using ZEISS 780confocal microscope.Bacterial strains and Plasmids

[0125] S. aureus strains (Table 1) were streaked on tryptic soy agar (TSA) plates and grown overnight at 37 °C. Single colonies were selected and cultured in TSB at 150 RPM at 37 °C in a shaking incubator overnight followed by a 1:100 subculture at 37 °C in a shaking incubator to obtain mid-logarithmic phase bacteria. For fusion reporter strains, all in vitro cultures were performed using TSB in the presence of 10 pg / mL of chloramphenicol. Bacteria were pelleted, washed, and re-815 suspended in either TSB for in vitro experiments or PBS for in vivo experiments. HG003, AagrC, AagrBD, and AagrA mutant strains were obtained from Dr. Ferric Fang. S. aureus strains from atopic dermatitis skin obtained from Drs. Julie Segre and Heidi Kong. All other strains from the collections of the Horswill and the Harris- Tryon labs.Construction of a lux expressing S. aureus strain

[0126] The integrated luxCDABEG cassette was transduced into S. aureus strains HG003, AagrBD and AagrC obtained from the lab of Ferric Fang using phage 11 generating strains AH6222 (Jux+), AH6224 (Jux+) and AH6223 (Jux+), respectively.In vitro luminescence assays

[0127] S. aureus strains, HG003, AagrBD and AagrC expressing Lux ((p11 ::LL29 / t / xCDABEG) and quorum sensing lux (pAmiAgrP3lux) plasmids (HG003 (AH6225) agr Type I, USA100 (AH430). Type II and MW2 (AH1747) agr Type III) (52, 65, 66) were grown in TSB supplemented with antibiotic selection and subcultured in TSB 1 :200 into fresh TSB containing steroid hormone. Assay completed in Opaque-sided, 96-well, clear bottom, tissueculture treated plates with a final well volume of 200 pL. Bioluminescent signals (photons / 0.1 second acquisition time) were measured by BioTek H1 Synergy plate reader. Experiments completed in triplicate, with agr type specific AlPs AIP-I (Peptide Institute, Inc., Cat# 4515-v), AIP-II (Peptide Institute, Inc., Cat# 4516-v), and AIP-lll(Peptide Institute, Inc., Cat# 4517-v) as positive control.Hemolysis assay

[0128] Hemolysis assay completed as previously described with the following modifications. Overnight cultures of HG003, AagrBD, and AagrC strains were inoculated 1 :200 into 10 mL of TSB containing testosterone, AIP-I, or vehicle alone at concentrations of 10 nM. Cells were grown to mid-log phase (OD600 nm 0.6). Supernatant of 1 ml of culture was filter sterilized using Millex® sterile syringe filters of 0.22 pm pore size (Cat#SLGV033RS). Filtered supernatant diluted 1 :1 with PBS was added to 25 pL of human blood to a 96-well V-bottom plate and incubated with agitation at 37°C for 1 hr. After spinning 1000RPM for 10 min, the supernatant was transferred to a flat-bottom 96-well plate. Absorbance read at 541 nm for hemoglobin using a BioTek H1 Synergy plate reader. % hemolysis calculated using the following formula: (A541 of RBC treated sample-A541 of buffer) / (A541 of H20-A541 of buffer) Buffer (PBS) = baseline, H20 = 100% hemolysis.Neutrophil Killing assay

[0129] S. aureus induced neutrophil killing measured as previously described. HG003, AagrBD and AagrC strains were treated with testosterone, AIP-I or vehicle at concentrations of 10nM and allowed to grow to mid log phase (GD600nm 0.6). Purified Human Neutrophils (IQ Biosciences) were seeded at 1 x 105cells per well into 96-well plate in 90 pL of RPMI. 10 pL of bacterial supernatants were added (final concentration of 10%). After 3h incubation at 37°C, 5% CO2, the plates were centrifuged at 250g, 10 min, and resulting supernatants wereused to measure lactate dehydrogenase (LDH) leakage from damaged cells as the marker of neutrophil lysis with an LDH Cytotoxicity Detection Kit (Invitrogen, Cat# 2570393). Percent neutrophil lysis was calculated using neutrophils incubated with 10% of RPMI as zero percent lysis control, and neutrophils incubated with 0.2% Triton X-100 defined as 100 percent lysis.Quantitative real-time PCR

[0130] HG003, USA100 (AH3684), MW2 (AH843), AagrBD, AagrA, AagrC and atopic dermatitis strains were treated with testosterone and / or respective AlPs at concentrations of 10nM and allowed to grow to mid-log phase (OD600nm 0.6). Cells were pelleted and lysed with lysis matrix B tubes containing 0.1mm silica spheres (MP Lysing Matrix Tubes, Cat#174701) and lysostaphin (Sigma, Cat# L7386) at room temperature, and RNA was purified using the RNeasy Mini Kit (Qiagen, Cat# 74104). RNA was quantified by absorbance at 260 nm, and its purity was evaluated by the ratios of absorbance at 260 / 280 nm. RNA was used as a template to generate cDNA with the High-Capacity Reverse Transcription Kit (Applied Biosystems, Cat#01071619). Quantitative real-time PCR was performed by amplifying cDNA with Power SYBR Green Master Mix (Applied Biosystems, Cat# 2749999) and QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Relative expression values were calculated using the comparative Ct (AACt) method, and transcript abundances were normalized to gyrA transcript abundance. The primer sequences are shown in Table 2.Table 2: Primers for Quantitative RT-PCR and Gene SequencingRNA Sep

[0131] Briefly, cultures of HG003 were grown in TSB with 10nM testosterone, pregnenolone, or DMSO alone in triplicate to an optical density of 0.6 at OD600 nm. Cells were harvested and treated with RNA Protect Bacteria Reagent (Qiagen, Cat# 76526). Cells were lysed using lysostaphin (Sigma, Cat# L7386) and RNA purified using the RNeasy mini kit (Qiagen, Cat# 74104) and sample quality was affirmed via Bio analyzer (Agilent). Ribosomal RNA was depleted using RiboCop for bacterial META Removal Kit (Lexogen). cDNA libraries were generated at the University of Michigan Microbiome core using the CORALL RNA-seq Library Prep Kit (Lexogen). Samples were barcoded, pooled and sequenced in 125x125 paired-end reads on an Illumina HiSeq 2000 sequencer. Raw sequencing reads in fastq format were aligned and annotated to the S. aureus NCTC8325 reference genome with annotated sRNA(70) using QiagenCLC Genomics Workbench default settings (version 21.0.5): mismatch cost, 2; insertion and deletion cost, 3; length and similarity fraction, 0.8. Normalization and differential expression calculations of uniquely mapped bacterial transcripts were performed using CLC. All transcripts with an FDR adjusted p-value <0.05 were considered significant.S. aureus skin infections

[0132] Prior to mouse infection studies, mice were acclimatized to the animal biosafety level 2 (ABSL-2) animal housing facility. Age, strain and sex matched C57BL / 6 male and female mice, Hsd3b6fl / fl and Hsd3b6ASkin were used in the study. A previously described mouse model of epicutaneous S. aureus exposure was followed. Briefly, the dorsal skin ofanesthetized mice (2% isoflurane) were shaved and depilated (Nair cream). After 24 hours, bioluminescent S. aureus strains were grown to mid-log-phase, pelleted and resuspended in PBS to achieve inoculum containing 1xio6CFU. A 100 pL volume of PBS containing 1 xio6CFU with or without 10nmoles 900 of testosterone, AIP-I, or the same volume of vehicle was placed on a sterile gauze pad and attached to the shaved skin with transparent bio-occlusive dressing (Tegaderm; 3M, Henry Schein medicals, Cat#1622W), and secured with adhesive bandages (BAND-AID, Johnson and Johnson, American white cross, Cat#1275033) for 4 days. Photons emitted from luminescent bacteria were collected during an auto exposure using the I VIS Lumina3 imager machine and living image software (Xenogen, Alameda, CA). Bioluminescent image data are presented on a pseudocolor scale (blue representing least intense and red representing the most intense signal) overlaid onto a gray-scale photographic image. Using the image analysis tools in living image software, circular analysis windows (of uniform area) were overlaid onto dorsal regions of infection area, and the corresponding bioluminescence values (total flux) were measured and plotted versus days after infection. Mice were randomly assigned to treatment groups, and at experimental endpoints, mice were humanely euthanized using carbon dioxide inhalation.Measurement of PSM alpha expression

[0133] Relative expression of AgrA-dependent virulence factor phenol-soluble modulin alpha (PSMa) of transcript was quantified by RT-PCR and was compared to the expression of the bacterial housekeeping gene DNA gyrase (gyrA). Means ± SEM are plotted.In vivo experiments

[0134] Female HSD3B6Askinmice were epicutaneously infected with a bioluminescent strain of MRSA, MRSA-lux. Using an in vivo imaging system (IVIS) that detects bacterial bioluminescence in 2-D and 3-D, the course of infection was tracked over time in vivo.Quantification and Statistical Analysis

[0135] Data represent mean ± standard error of the mean. Formal randomization techniques were not used; however, mice were allocated to experiments randomly and samples were processed in an arbitrary order. Mouse skin samples that were determined to be in the anagen hair cycle were excluded. All statistical analyses were performed with GraphPad Prism software, except the bioluminescent imaging data that was analyzed as described above. To assess the statistical significance of the difference between two treatments, two-tailed Student’s t-tests was used. To assess the statistical significance of differences between more than two treatments, one-way ANOVA was used. Outliers within experiments were identified by Grubb’s test and removed. For the RNAseq experiments,expression data was analyzed with CLC. All transcripts with an FDR adjusted p-value <0.05 were considered significant.Example 1. Ent-Testosterone reduced virulence gene expression in multiple strains of Staphylococcus.

[0136] To assess the efficacy of Ent-Testosterone, Staphylococcus aureus strains, LAC (Type I), USA 100 (Type II), MW2 (Type III), and MN EV(Type IV) were treated with 10nM Ent- Testosterone, 10nM Testosterone, or vehicle and relative expression of AgrA-dependent virulence factor phenol-soluble modulin alpha (PSMa) was quantified by RT-PCR and was compared to the expression of the bacterial housekeeping gene DNA gyrase (gyrA) (FIG. 2). Ent-Testosterone was found to inhibit expression of PSMa in all of the Type I, Type II, Type III, and Type IV strains of Staphylococcus aureus, in contrast to Testosterone, which enhanced the expression of virulence factor PSMa.Example 2: Ent-Testosterone reduced quorum sensing in mice in vivo

[0137] The effect of Ent-T estosterone was further tested in vivo. HSD3B6Askinfemale mice were epicutaneously infected with a bioluminescent MRSAagr-P3lux reporter strain of MRSA and treated with 10nM Ent-Testosterone, 10nM Testosterone, or vehicle. Hsd3b6ASKINmice secrete less testosterone in the skin compared to control mice. Using an in vivo imaging system (I VIS) that detects bacterial bioluminescence in 2-D and 3-D, the course of infection overtime was tracked in vivo. Representative image after 1 hr, 2hr, 3hr, and 4hr after treatment is shown in FIG. 3 and the mean bioluminescence was plotted hourly for over 8 hours after treatment (FIG. 4). As shown in FIG. 3 and FIG. 4, while testosterone enhanced bioluminescence, Ent-Testosterone inhibited the bioluminescence over time. These results indicated treatment with Ent-Testosterone inhibited agr induced bioluminescence and therefore suggested that Ent-Testosterone can inhibit quorum sensing.Example 3: Ent-Testosterone dosage

[0138] Further, various doses of Ent-Testosterone were tested in vitro using MRSAagr-P3lux reporter strain of MRSA. MRSAagr-P3lux strain was treated with 10nM, 100nM, 1 pM, and 10 pM of Ent-Testosterone, Testosterone, Al PI I, or vehicle, and bioluminescence was quantified over time, as shown in FIG. 5 and FIG. 6. In all the doses tested, Ent-Testosterone reduced bioluminescence, indicating that Ent-Testosterone inhibited MRSA.Example 4: The enantiomer of testosterone inhibits S. aureus pathogenesis

[0139] T o understand the specificity of the interaction of testosterone with the agr system the impact of a stereoisomer of testosterone, enantiomer-testosterone (ent-T), on S. aureus pathogenesis was tested. Like other unnatural enantiomers, ent-T has the same physiochemical properties as testosterone but with a mirror image orientation at all six chiralcenters that leads to altered rotation of polarized light (FIG. 7A). Enantiomers have been observed to have unique receptor binding and signaling properties. Unlike the other hormones that were tested which displayed neutral effects on the agr system (FIG. 9A-9D), Ent- Testosterone acted as a broad inhibitor of the agr quorum sensing system (FIG. 2, FIG. 7B- 7D, FIG. 8A-8B). Ent-Testosterone was able to decrease S. aureus dependent hemolysis and neutrophil killing in Type-1 agr strains (FIG. 7B-7C). Further, Ent-Testosterone had the capacity to decrease the expression of agr transcriptional readouts in Type-ll, Type-Ill, and atopic dermatitis strains of S. aureus (FIG. 7B-7D, FIG. 8A). Moreover, topical application of Ent-Testosterone to the skin of S. aureus infected mice turns off quorum sensing in vivo (FIG. 3, FIG. 4, FIG. 8B). Thus, ent-T is a global inhibitor of S. aureus quorum sensing that worked across agr types, similar to established bacterially derived regulators of agr, such as noncognate AlPs (FIG. 7B-7C).Example 5: Ent-Testosterone Mediates Quorum Quenching

[0140] The skin barrier of mice was perturbed with tape stripping, to test the effects of ENT- testosterone on an infection model. Previously established epicutaneous skin infection protocol was used. Briefly, mice were anesthetized with isoflurane, tape stripped (TEWL = 20 g / m2 / h) and 24 h later, 107CFU of MRSA SAP 430 in 10OpI was applied to back skin and spread using a swab. The inoculum was allowed to dry for 10 min and mice were returned to their cages. Treatment was undertaken once daily. No Tegaderm was used during the treatment. No difference was observed between control vehicle treatment and ENT in mice treated with MRSA in vivo. However, the TEWL treatment group showed significant difference with control vehicle treatment (FIGs. 10A-10B). This could be because Ent-Testosterone mediates quorum quenching at in vivo sites of infection. It inhibit the production of AlPs a member of a family of signaling molecules used in quorum sensing. Therefore, the proposed Ent-Testosterone could have the potential to reduce AD symptoms, such as itching, redness, swelling, pain, and / or skin barrier break down by inhibiting quorum sensing and thereby barrier recovery without the eradication of S. aureus, which is part of the natural skin microbiome.Example 6: Ent-Testosterone Inhibits Quorum sensing in vivo

[0141] ENT-Testosterone was applied epicutaneosuly at 10 and 100 nanomoles on male mice followed by application of AH2659 agrP3 reporter strain was immediately, and agrkinetics were recorded every hour. A previously described mouse model of epicutaneous S. aureus exposure was followed. Briefly, the dorsal skin of anesthetized mice (2% isoflurane) were shaved and depilated (Nair cream). After 24 hours, bioluminescent S. aureus strains were grown to mid-log-phase, pelleted and resuspended in PBS to achieve inoculum containing 1 xio6CFU. A 100 pLvolume of PBS containing 1 x106CFU was placed on a sterile gauze pad and attached to the shaved skin with transparent bio-occlusive dressing (Tegaderm; 3M,Henry Schein medicals, Cat#1622W), and secured with adhesive bandages (BAND-AID, Johnson and Johnson, American white cross, Cat#1275033). Photons emitted from luminescent bacteria were collected during an auto exposure using the I VIS Lumina3 imager machine and living image software (Xenogen, Alameda, CA).

[0142] As shown in FIGs. 11A-11B, ENT-Testosterone was found to inhibit quorum sensing. No weight changes were observed in between treatment groups on the day of infection (FIG. 11C). There was also no differences in TEWL applied between the treatment groups (FIG. 11D).

[0143] Further, to test whether ENT-Testosterone outcompete testosterone on the skin in high doses, experiments were conducted with increasing concentrations of ENT-Testosterone in combination with testosterone and agrP3 luminance was measured. As shown in FIG. 12, increasing concentrations of ENT-Testosterone was found to reduce HG003 agrP3 luminance in a dose dependent manner, in the presence of Testosterone.Summary of examples

[0144] In summary, the Ent-Testosterone inhibited the expression of virulence factors, and quorum sensing, in vitro and in vivo. These results indicated that Ent-Testosterone can inhibit bacterial virulence, and thus have therapeutic application in treating bacterial infections or condition complicated by the colonization of bacteria.

[0145] In contrast to progesterone and estradiol that had a neutral effect on S. aureus, ENT-Testosterone, the stereoisomer of testosterone, decreased quorum sensing and decreased bacterially induced hemolysis and neutrophil killing. ENT-Testosterone also inhibited quorum sensing during S. aureus infections in vivo. These findings demonstrated that S. aureus responds to testosterone in an enantioselective manner, with testosterone generating activating signals and its chiral counterpart, ENT-Testosterone leading to inhibition of the same phenotypes. Since natural hormones and their enantiomers have been shown to have the same physiochemical properties within membranes, the distinct phenotypes of testosterone and ENT-Testosterone suggested that the effects of these hormones on agr signaling were independent of any predicted impacts of androgen hormones on bacterial membranes.

Claims

CLAIMSWhat is claimed is:

1. A composition for treatment of a bacterial infection in a subject in need thereof, the composition comprising Enantiomer-Testosterone (Ent-Testosterone).

2. A method of treating a subject having, or at risk of having, a bacterial infection, the method comprising: administering to the subject a composition comprising Ent-Testosterone.

3. A method of treating a subject having, or at risk of having a skin and soft tissue infection (SSTI) by a bacterium, the method comprising: administering to the subject a composition comprising Ent-Testosterone.

4. A method of treating a subject having, or at risk of having a condition complicated by the colonization of a bacteria, the method comprising: administering to the subject a composition comprising Ent-Testosterone.

5. The method of claim 4, wherein the condition is atopic dermatitis, folliculitis, pemphigus, bullous pemphigoid, or cutaneous T-cell Lymphoma.

6. The composition or the method of any one of the claims 1-5, wherein the bacterial infection is caused by a bacterium of the genus Staphylococcus.

7. The composition or the method of claim 6, wherein the bacterial infection is caused by Staphylococcus aureus or Staphylococcus epidermidis.

8. The composition or the method of claim 7, wherein the Staphylococcus aureus is a methicillin resistant Staphylococcus aureus (MRSA), a methicillin-susceptible Staphylococcus aureus (MSSA), a vancomycin-intermediate Staphylococcus aureus (VISA), and / or a vancomycin-resistant Staphylococcus aureus (VRSA).

9. The composition or the method of claim 7, wherein the Staphylococcus aureus is a Staphylococcus aureus strain LAC, Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, and / or a Staphylococcus aureus strain MN EV.

10. The composition or the method of any one of the claims 1-9, wherein the subject is a mammal.

11. The composition or the method of claim 10, wherein the subject is a human.

12. The composition or the method of any one of the claims 1-11 , wherein the Ent- Testosterone reduces (a) the expression of a virulence factor or a virulence-associated protein gene by at least 10% as compared to an untreated control subject; (b) Staphylococcus inducedhemolysis by at least 10%; and / or (c) Staphylococcus induced neutrophil killing by at least 5%.

13. The composition or the method of claim 12, wherein the virulence-associated protein gene is accessory gene regulator (agr) Type I.

14. The composition or the method of claim 12, wherein the virulence-associated protein gene is agr Type II.

15. The composition or the method of claim 12, wherein the virulence-associated protein gene is agr Type III.

16. The composition or the method of claim 12, wherein the virulence-associated protein gene is agr Type IV.

17. The composition or the method of any one of the claims 1-16, wherein the Ent- Testosterone reduces the quorum sensing by the bacteria by at least 10% as compared to an untreated control subject.

18. The composition or the method of any one of the claims 1-17, wherein the Ent- Testosterone is about 10nM to about 10pM.

19. The composition or the method of any one of the claims 1-12, wherein the Ent- Testosterone reduces the accessory gene regulator C histidine kinase (agrC).

20. The composition or the method of any one of the claims 1-19, wherein the composition further comprises a pharmaceutically acceptable carrier.

21. The composition or the method of claim 20, wherein the pharmaceutically acceptable carrier is petroleum jelly.

22. A method of limiting damage to a tissue of a subject caused by a bacterial virulence associated protein, the method comprising: administering to the subject a composition comprising Ent-Testosterone.

23. The method of claim 22, wherein the tissue is skin tissue.

24. A method for attenuating virulence of a Staphylococcus spp., the method comprising: contacting the Staphylococcus spp. with a composition comprising an effective amount of Ent- Testosterone.

25. The method of claim 24, wherein the Staphylococcus spp. is Staphylococcus aureus, or Staphylococcus epidermidis.

26. The method of any one of the claims 24-25, wherein the Staphylococcus aureus is a methicillin resistant Staphylococcus aureus (MRSA), a methicillin-susceptible Staphylococcusaureus (MSSA), a vancomycin-intermediate Staphylococcus aureus (VISA), and / or a vancomycin-resistant Staphylococcus aureus (VRSA).

27. The method of any one of the claims 24-26, wherein the Staphylococcus aureus is a Staphylococcus aureus strain LAC, a Staphylococcus aureus strain USA 100, a Staphylococcus aureus strain MW2, and / or a Staphylococcus aureus strain MN EV.

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