WO2019191293A1 - Materials and methods for diagnosing, preventing and/or treating infections by pathogenic agents - Google Patents

Abstract

The invention pertains to biosurfactants that serve as virulence factors in pathogenic microorganisms.

Description

MATERIALS AND METHODS FOR DIAGNOSING, PREVENTING AND/OR TREATING

INFECTIONS BY PATHOGENIC AGENTS

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Patent Application No. 62/648,516, filed March 27, 2018, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Virulence factors are molecules produced by microorganisms that add to their efficacy of infection and typically enable them to do one or more of the following:

1. colonize a niche in the host (e.g., attaching to cells);

2. evade the host’s immune response;

3. overcome the host’s mechanical barriers and establish at the initial point of infection (e.g., adhesins and invasins);

4. suppress the host’s immune defense response (e.g., antiphagocytic factors);

5. entry into and exit out of cells, particularly, if the pathogen is intracellular;

6. obtain nutrition from the host; and

7. promote further initiation and establishment of infection (e.g., toxins, hemolysins, proteases, and other enzymes) causing additional damage to the host.

For a number of clinically important pathogens, there are still gaps in understanding etiopathogenesis and clinical manifestations. Discovering new virulence factors of pathogenic bacteria is a key to understanding the etiology and pathogenesis of infections. These virulence factors can also be important targets for developing novel therapeutic and preventative preparations to protect against infections caused by pathogens.

Many virulence factors have been described, showing molecules of different origin and structure. They belong to various classes of biomolecules, e.g. peptides, proteins, glycopeptides, glycoproteins, and lipopolysaccharides.

In many infections, virulence factors that are responsible for pathologic changes in the initiation, promotion and outcomes are not known. For example, in inhalation anthrax, mechanisms for pathogen entry, profound hemorrhages in intestinal organs, ease of penetration of blood-brain-barrier, hemolysis, and disseminated intravascular coagulation are unknown. These effects cannot be explained only by the potent three-protein virulence factor produced by B. anthracis or by the capsule of B. anthracis.

Bacillus anthracis causes a highly lethal inhalational infection, where systemic bacteremia, extensive pleural effusions, mesenteric lymphadenitis, hemorrhagic necrotizing pneumonia, profound hemorrhagic meningitis, and multiple gastrointestinal submucosal hemorrhagic lesions have been reported (Abramova et al. 1993). Attempts to show these pathologic changes are affected by the toxin complex and the capsule, encoded by pXOl and pX02 respectively, cannot be completely confirmed (Smith and Keppie 1954; Koehler 2002; Candela and Fouet 2005). In inhalational anthrax, no evidence is currently available to describe how these virulence factors cause the above-described clinical manifestations (Warfel et al. 2005; Kau et al. 2005).

Therefore, it is likely that there are other virulence factors of B. anthracis that are presently unknown. Due to limitations in how unknown virulence factors affect the etiopathogenesis of anthrax infection, the development of more effective vaccines and therapeutic modalities is restricted.

Similarly, in systemic (glandular and pneumonic) tularemia, the mechanism of virulence is unknown. Particularly, why Francisella tularensis is unusually efficient at evading host defenses where only 50 cells or less are sufficient to cause human respiratory infection is not explained. Also, no explanation is available for the survival of F tularensis cells in macrophages, production of reactive oxygen species, profound cytokine production, the induction of apoptosis, or hemorrhagic eruption in parenchymal organs.

Francisella tularensis is the intracellular pathogenic microorganism causing tularemia. It is one of the most infectious pathogens known, requiring only a few microbial cells to cause the disease. It has been a basis for highly efficient biological weapons because of its extreme infectivity, ability to evade immune response, and its capability to cause severe illness and death. Without treatment, the clinical course could progress to respiratory failure, shock, and death. Exposure to the pathogen’s aerosol can result in many disease presentations, which include ulceroglandular, glandular, oculoglandular, oropharyngeal, pneumonic, and typhoidal clinical manifestations. In systemic forms of tularemia, bacteremia may be common during infection. Victims of inhalational exposures may develop hemorrhagic inflammation of the airways early during the illness, which may progress to bronchopneumonia.

Presently, the virulence factors that play a role in the etiopathogenesis of the infection are under studied. For example, there is no explanation for specific mechanisms of the unusual efficiency of F. tularensis to evade host defense and why the bacteria survive in macrophages causing significant damage on cellular and tissue levels.

Recent studies of F. tularensis have identified new functions of encoded transcription factors and have expanded our understanding of virulence gene regulation (Jones et al. 2014; ai et al. 201 1). Progress in identifying loci involved in pathogenicity, and analysis of the genome sequence has revealed a few putative virulence factors, including a hypothetical lipoprotein FTT1 103, however with no known mechanism of action (Qin et al. 2008). Furthermore, it was revealed that the type VI secretion system encoded by the Francisella pathogenicity island is critical for the virulence of this organism (Eshraghi et al. 2016). For F. novicida , the type VI secretion system assembly starts at the bacterial poles within infected macrophages: the system’s dynamics and function depend on the general purpose of ClpB unfoldase, which specifically co localizes with contracted sheaths and is required for their disassembly (Brodmann et al. 2017).

As no specific molecules and mechanisms responsible for the pathogen’s infectiousness and clinical manifestations have been described, all attempts to develop an effective tularemia vaccine have been unsuccessful (Id.).

Further, how virulence factors of Yersinia pestis participate in the pathologic effects seen in the plague is unknown. For example, how the known virulence factor, namely, plasminogen activator (allegedly responsible for the pathogen’s entry) enhances the pathogen penetration so efficiently is not known. Also, the factor responsible for a profound hemorrhagic syndrome is not known and a clear understanding of mechanisms of cell and tissue necrosis and edema are absent. Known virulence factors of plague bacterium cannot fully describe mechanisms of these pathologic changes.

Furthermore, in smallpox, the two currently described virulence factors of Variola major virus cannot fully explain the high efficiency of the virus entry in the body or the profound systemic multiorgan dissemination and severe toxemia.

Such examples of unknown pathological mechanisms are numerous. Therefore, undiscovered virulence factors exist in pathogens and identifying such virulence factors is desired.

A“biosurfactant” is a surface-active biomolecule produced by a microorganism. A biosurfactant has hydrophilic and hydrophobic regions, which makes it capable of aggregating at the interface between fluids with different polarities, for example, a hydrocarbon and water. For example, a class of biosurfactants termed lipopeptides, consists of one or more hydrophobic lipid chains attached to hydrophilic peptide sequences containing charged residues. Another class of biosurfactants, termed glycolipids, consists of one or more hydrophilic sugar molecules attached to a hydrophobic lipid chain.

Biosurfactants are complex amphiphilic molecules comprising different structures that include peptides, lipopeptides, glycolipids, glycopeptides, and phospholipids. This amphiphilic peculiarity predetermines a multiplicity of biochemical and biophysical features of biosurfactants. Biosurfactants are produced by a large variety of microorganisms and have evolved as an adaptation and survival mechanism during millions of years of evolution. An important feature of biosurfactants is the principle of assembly. Some biosurfactant synthesis can be encoded by a cluster of genes of a microorganism, but some others could be self- assembled outside of a microorganism by combination of microbial molecules with molecules in the environment. The self-assembly process can be driven by a combination of hydrogen bonding and electrostatic and other interactions. The processes of self-assembly depend on many factors which include oxygen level, concentration of molecules, pH, ionic strength of solution, and temperature.

Due to different chemical structures and biological properties, each type of amphiphilic molecule could serve different roles in a specific ecological niche (Ron and Rosenberg 2001). A natural role of these molecules is to enhance the bioavailability of degradable organic matter, which indicates that amphiphilic molecules are major elements of microbial adaptation, survival and propagation in various environments (Bodour et al. 2003; Viramontes-Ramos et al. 2010; Thavasi et al. 201 1; Ramesh et al. 2010).

BRIEF SUMMARY OF THE INVENTION

The subject invention provides methods for identifying virulence factors of pathogenic microorganisms. The subject invention further pertains to the virulence factors identified by the methods of the subject invention. The invention further provides methods for detecting, preventing and/or treating disease based on the identification of biosurfactants as virulence factors.

In specific embodiments, the molecular target for a therapeutic method according to the subject invention are biosurfactants and/or molecules involved in the synthesis and/or assembly of biosurfactants.

The identification of biosurfactant virulence factors facilitates the use of new therapeutic and prophylactic methods and compositions that reduce the morbidity and mortality of a number of infectious diseases. Accordingly, certain embodiments of the invention provide vaccine compositions comprising a biosurfactant virulence factor and, optionally, one or more adjuvants and/or one or more pharmaceutically-acceptable carriers or excipients.

Methods of treating or preventing infectious diseases in a subject by administering to the subject the pharmaceutical compositions of the invention are also provided. Accordingly, certain embodiments of the invention provide therapeutic compositions comprising compounds that inhibit a biosurfactant virulence factor. In other embodiments, the therapeutic compositions target proteins or other molecules involved in the production, secretion and/or assembly of biosurfactant virulence factors. DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, a new class of virulence factors has been identified. In a specific embodiment, the virulence factors are amphiphiles. In a further embodiment, the subject invention provides a new method for identifying these molecules. In additional embodiments, the subject invention provides materials and methods for diagnosing, preventing, and/or treating infections caused by pathogens having biosurfactant virulence factors.

The invention identifies biosurfactants produced by microorganisms, particularly, pathogenic microorganisms, as virulence factors.

Specifically, exemplified herein are two bacteria that are distant genetically and phenotypically, with unknown virulence factors related to their pathogenicity; these are Bacillus anthracis and Francisella tularensis.

In specific embodiments, virulence factors were identified for Francisella tularensis and Bacillus anthracis. Specifically exemplified is the presence of a gene cluster responsible for rhamno-di-phosphate lipid synthesis in F. tularensis. Furthermore, amphiphilic lipopeptides were identified in B. anthracis.

In accordance with the subject invention, novel targets for development of vaccines, protective antibodies and chemical molecules to inhibit virulence factors are provided. The vaccines can be used as additional components to the protective antigen (PA)-based vaccines to enhance efficiency and reduce the number of booster injections. Either the molecules themselves or molecules involved in the synthesis and/or assembly of amphiphilic virulence factors can be the primary active components of the vaccine, or targets of inhibitors.

Amphiphiles have a low molecular weight; therefore, when using these molecules to elicit a protective immune response, adjuvants can be used. In one embodiment, the molecules can be conjugated to another entity that generates and/or enhances an immune response. In the case of developing protective antibodies for a therapeutic composition, these medications can be manufactured in the form of, for example, monoclonal and polyclonal antibodies or in the form of humanized antibodies.

In the case of developing chemical inhibitors, in one embodiment the therapeutic compounds can target molecules involved in intracellular biosynthesis processes of amphiphiles, as well as extracellular biosynthetic molecules of amphiphiles and amphiphiles themselves both membrane-bound and secreted.

The subject invention provides new avenues for developing protective and therapeutic compositions and methods both for preventing and/or treating infections involving pathogens that employ amphiphilic molecules as virulence, or co-virulence, factors. In certain embodiments, biosurfactants of the invention have superior surfactant properties compared to non-biological surfactants. Particularly, compared to non-biological surfactants, biosurfactants can have superior surface activity, higher tolerance to changes in pH, stability at higher temperatures, higher ionic strength, and higher emulsifying and demulsifying ability.

In particular embodiments, biosurfactants can reduce surface tension of water to at least about: 10 mN/M, 15 mN/m, 20 mN/m, 25 mN/m, 30 mN/m, or 35 mN/m. In other embodiments, biosurfactants disclosed herein can reduce the interfacial tension of water/hexadecane to less than about: 0.5 mN/m, 1 mN/m, 1.5 mN/m, 2 mN/m, 2.5 mN/m, 3 mN/m, 3.5 mN/m, 4 mN/m, 4.5 mN/m, or 5 mN/m.

Critical micelle concentration (CMC) is defined as the concentration of surfactants above which micelles form and all additional surfactants added to the system go to micelles. In specific embodiments, biosurfactants can have CMC several times lower than non-biological surfactants, i.e., for maximal decrease on surface tension, many fold less surfactant is necessary. In particular embodiments, the CMC of biosurfactants is less than about: 100 mM, 50 mM, 10 mM, 5 mM, 1 mM, 500 hM, 100 hM, 10 hM, or 1 hM.

Biosurfactants include, for example, lipopeptides, flavopeptides, lipoproteins, glycolipids, glycopeptides, phospholipids, and fatty acid esters. Glycolipids include rhamnolipids, trehalose lipids, mannosylerythritol lipids or sophorolipids.

In one embodiment, the biosurfactants can comprise one or more glycolipids such as, for example, rhamnolipids, rhamnose-d-phospholipids, trehalose lipids, trehalose dimycolates, trehalose monomycolates, mannosylerythritol lipids, cellobiose lipids, ustilagic acid and/or sophorolipids.

Based on structural differences, different biosurfactants can elicit different physical, chemical, biochemical, and biophysical properties. Properties of biosurfactants that are virulence factors can include one or more of the following:

Some biosurfactants reduce surface tension of liquids and reduce interfacial tension on a border between different phases, which in turn can enhance the microorganism and its toxic substances’ dissemination and penetration into cells and/or intracellular spaces;

Some biosurfactants inhibit inflammation by inhibiting the expression of IFN-g, IL-6, iNOS, nitric oxide, or TLR4 protein in macrophages. These effects can help pathogens survive at initial stages of infection.

Some biosurfactants cause swelling and/or hemolysis of erythrocytes and lysis of other blood, epithelial, and endothelial cells. These effects can promote pathogenesis. Some biosurfactants can inhibit P-glycoprotein, a permeability glycoprotein located in the cells of epithelium and endothelium. These effects help pathogens and toxins penetrate parenchymal organs and the brain resulting in hemorrhages and sepsis in brain and other organs.

Some biosurfactants cause the death of epithelial cells, polymorphonuclear leukocytes, and/or macrophages, and inhibit phagocytosis.

Some biosurfactants form pores in membranes, which can cause extensive blebbing of the plasma membrane.

Some biosurfactants cause a pronounced inflammatory response and contribute to the establishment of a state of sepsis.

Some biosurfactants increase permeability of intestine walls and blood vessel walls causing, or contributing to, a profound hemorrhage.

Some biosurfactants damage the endothelial layer of blood vessels that can additionally lead to capillary leak syndrome, dilation of blood vessels, a decrease in cardiac function, and septic shock.

Some biosurfactants cause pronounced complement activation, which can also be observed later in the course of infection as microorganisms start intensively multiplying in the blood. This can lead to the formation of complement membrane attack complex that leads to additional formation of transmembrane channels, which could cause intensive hemorrhages, and organ and tissue swelling.

Some biosurfactants can trigger destructive endothelial damage, which can lead to disseminated intravascular coagulation (DIC) with loss of function of certain internal organs such as the kidneys, adrenal glands, and lungs due to compromised blood supply and eventual death.

Biosurfactant virulence factors combine amphiphilic structural features with the functions of bioactive molecules that can be assembled into a variety of nanostructures. These structural features allow biosurfactants to interact with cells, tissues, and body fluids, thus making them virulence factors.

The term“microorganism” or“microbe” as used herein refers to organisms recognized in the art as“microorganisms.” Microorganisms contemplated in the invention include viruses, protozoa, algae, bacteria, and fungi including filamentous fungi and yeast.

Further embodiments of the invention provide methods of isolating biosurfactants from microorganisms, particularly, pathogenic microorganisms. For isolation of a biosurfactant, a microorganism is grown under appropriate conditions, for example, appropriate culture medium and temperature. A pathogenic microorganism can also be obtained from a host infected by the pathogenic microorganism. The microorganism so obtained can be processed to isolate one or more biosurfactants. Such methods include acid precipitation, phase separation, direct liquid partitioning, membrane ultrafiltration, foam fractionation, and extraction with an organic solvent. Additional methods of isolating biosurfactants from the cells of pathogenic microorganisms are known in the art and such embodiments are within the purview of the subject invention.

As noted above, biosurfactants isolated according to the methods of the invention are virulence factors. Accordingly, these factors can be used as immunogenic agents, particularly, in the preparation of vaccines. Therefore, certain embodiments of the invention provide one or more biosurfactants (or immunogenic analogs and/or derivatives thereof) isolated from a microorganism in a vaccine composition.

Typically, a vaccine composition of the invention comprises a biosurfactant and an adjuvant.

Immunogenicity of the biosurfactant virulence factors of the invention can be enhanced through the use of adjuvants. Adjuvants augment the immune response to the factor. Exemplary adjuvants include salt-based adjuvants such as alum salts, bacterial-derived adjuvants like lipopolysaccharides and bacterial toxins, adjuvant emulsions that enable the slow release of antigen, agonsitic antibodies to co-stimulatory molecules, Freunds adjuvant, muramyl dipeptides, and recombinant/synthetic adjuvants. The adjuvant can also be a toll-like receptor (TLR) ligand, particularly a TLR-4, such as monophosphoryl lipid A (MPL), or TLR-7 ligand, such as R837. TLR-4 and TLR-7 ligands in combination with a nanoparticle formulation can also be used (Kasturi et al. (201 1) Nature, Vol. 470: 543-560). Thus, TLR-4 and/or TLR-7 ligands can be included in the priming and/or boosting vaccine preparations of the invention.

Alum is the most common salt-based adjuvant used to stimulate immune responses to protein vaccines. However, alum favors Th2-biased responses and does not stimulate cell- mediated immunity.

Mucosal immunity can be induced through the use of bacterial toxins such as cholera toxin (CT) and the E. coli heat labile enterotoxin (LT). Cytokines, such as interferon-. gamma and granulocyte-macrophage colony stimulating factor (GM-CSF), have shown promise as adjuvants in stimulating cellular immune responses (Petrovsky and Aguilar, Immunol. Cell Biol. 82:488- 496 (2004)).

Certain pathogenic microorganisms produce biosurfactants and/or biosurfactant precursors that interact with host cells. Such interaction facilitates infection of host cells by the pathogenic microorganisms. However, when purified biosurfactants are administered to a subject, such biosurfactants bind to host cells and block the binding of pathogenic microorganisms. As such, biosurfactants can be used in preventing diseases by blocking the ability of pathogenic microorganisms to infect target cells. Certain biosurfactants exhibit antimicrobial activity. Such biosurfactants, when produced by the pathogenic microorganisms, can kill competing microbes and facilitate growth of the pathogenic microorganisms. However, when a purified biosurfactant is administered to a subject, such biosurfactant can be used to kill the pathogenic microbial cells. As such, certain biosurfactants can be used in preventing diseases by killing disease-causing microbes.

In certain embodiments, the invention comprises the administration of antimicrobial compounds. New antimicrobial treatments can be used as an alternative to standard antibiotic treatments to enhance the efficiency of the therapy. Either the virulence factor biosurfactants themselves, or proteins or other molecules involved in the synthesis and/or assembly of these amphiphilic molecules can be the target of the antimicrobial activity.

Certain embodiments of the therapies of the invention utilize secretion system inhibitors. New secretion system treatments can be used as an alternative to, or in conjunction with, standard antibiotic treatments to enhance the efficiency of the therapy. Either the biosurfactant molecules themselves or proteins or other molecules involved in the synthesis and/or assembly of these amphiphilic molecules can be the targets of the secretion system inhibitors.

Certain embodiments of the invention provide methods of treating or preventing a disease in a subject, by administering to the subject a therapeutically effective amount of a biosurfactant inhibitor of the invention. Such administration can be performed in the form of a pharmaceutical composition comprising a biosurfactant inhibitor of the invention and one or more pharmaceutically acceptable carriers or excipients.

A new methodology for identifying a virulence factor can utilize the following steps: a) analyzing peculiarities of etiopathogenesis and clinical manifestations of an infection, b) identifying unexplained features of the infection, c) optionally searching for descriptions of specific molecules the existence of which is not explained, d) analyzing characteristics of molecules produced by taxonomically close microorganisms - not necessarily pathogenic, e) predicting existence of molecules of interest in the pathogens, f) optionally studying physiological or pathophysiological effects produced by these molecules in other bacteria, g) searching existing gene and/or protein databases to find the sequences of these molecules in related bacteria, and h) performing, for example, BLAST analyses to find orthologs in the bacteria of interest.

In specific embodiments of the subject invention, novel virulence factors of anthrax and tularemia pathogens, which could play major roles in etiopathogenesis of these infections, were identified. These virulence factors are amphiphilic molecules. It was shown that these virulence factors are amphiphilic molecules belonging to rhamno-di-phospholipids of Francisella tularensis and lipopeptides of Bacillus anthracis. The newly discovered virulence factors— amphiphiles and molecules involved in their synthesis and/or assembly— constitute a new class of therapeutic and prophylactic targets both for infections and diseases (e.g. in cases of bacterial carcinogenesis) for new vaccines, prophylactic and therapeutic antibodies and new chemical drugs: inhibitors of both amphiphiles themselves and molecules involved in their biosynthesis.

In accordance with the subject invention, it is possible to identity novel targets for the development of vaccines, protective antibodies, and chemical molecules to inhibit these virulence factors. Vaccines can be developed as additional components to protective-antigen (PA)-based vaccines to enhance efficiency and reduce the number of booster injections.

In the case of tularemia, in one embodiment, the subject invention provides a subunit or vaccine using the identified amphiphilic molecules. In the cases of both tularemia and anthrax infections, either the molecules themselves or proteins involved in the synthesis of amphiphilic molecules can be the main active components of the vaccines.

When using amphiphiles as vaccines, due to their small molecular size, new vaccines can utilize adjuvants and/or the molecules can be conjugated. In the development of protective antibodies, these medications can be manufactured in the form of monoclonal and/or polyclonal antibodies or in the form of human-specific gamma-globulins. For developing chemical inhibitors, these medications can target the molecules involved in the intracellular biosynthesis, the extracellular biosynthetic molecules, and/or the amphiphiles themselves.

Definitions:

As used herein, the singular forms“a”,“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent that the terms “including”,“includes”,“having”,“has”,“with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term“comprising.”

The phrases“consisting essentially of’ or“consists essentially of’ indicate that the claim encompasses embodiments containing the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim. Use of the term“comprising” contemplates other embodiments that“consist” or“consist essentially of’ recited component s).

The term“about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. When the term“about” is used in the context of quantitative parameters, these parameters can be varied within a range of 0-10% around the value, i.e., X±10%. “Treatment,” or“treating” (and grammatical variants of these terms), as used herein refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. A therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.

“Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions of the invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

As used herein, the term“subject” refers to an animal, such as a mammal. The animal may be for example, pigs, horses, goats, cats, mice, rats, dogs, apes, fish, chimpanzees, orangutans, guinea pigs, hamsters, cows, sheep, birds, e.g., chickens, as well as any other vertebrate or invertebrate.

The methods described herein can be useful in both pre-clinical and clinical human therapeutics and veterinary applications. In some embodiments, the subject is a mammal (such as an animal model of disease), and in some embodiments, the subject is human.

As used herein, an“analog” of a biosurfactant virulence factor is a compound that when administered to a subject elicits a protective immune response against the pathogen that produces the biosurfactant virulence factor. Typically, the analog would also be a biosurfactant and would also typically have one or more of the above-noted characteristics of biosurfactant virulence factors.

As used herein, reference to a“protective” immune response refers to an immune response that reduces or slows the deleterious effects of the pathogen. The protective immune response does not need to be completely protective.

To show that bacterial amphiphilic molecules are present in pathogenic bacteria and are implicated in human infections, we utilized the NCBI BLAST program to confirm the presence of orthologs of molecules participating in biosynthesis of lipopeptides and rhamnolipids. Based on the NCBI protocol for inferring homology from similarities, if a bit score of 50 or higher is obtained, it infers homology. A bit score of 40 or higher is significant, only if the e-value is lower than 1 O ': an e-value lower than 10 ³ indicates homology. An e-value higher than 10 ³ indicates the match is non-significant (Pearson 2013). EXAMPLES

A greater understanding of the present invention and of its many advantages may be had from the following examples, given by way of illustration. The following examples are illustrative of some of the methods, applications, embodiments and variants of the present 5 invention. They are not to be considered as limiting the invention. Numerous changes and modifications can be made with respect to the invention.

Example 1: Amphiphile Synthesis by Francisella tularensis

NCBI BLAST was performed to confirm the presence of potential homologous proteins0 that synthesize rhamno-di-phosphate lipid. The gene cluster for synthesis of rhamno-di- phospholipid in P. aeruginosa contains rhamnosyltransferases wbpX , wbpY, wbpZ, and wbpL (Lam et al. 1999; Wang et al. 2015). NCBI BLAST was performed to compare the protein sequences of WbpX, WbpY, WbpZ, and WbpL against F. tularensis to confirm the presence of a homologous wbp gene cluster in F. tularensis. In this case, homologs of WbpZ, WbpY, WbpX,5 and WbpL were identified in many strains of F. tularensis.

As shown in Table 1, the list of WbpZ from P. aeruginosa PAOl aligned to proteins sequences of F. tularensis has very high bit scores of at least 206 in all the F. tularensis strains. E- values are close to 0, ranging from 2.0x l0 "°to 7.0x l0 ⁸. All the bit scores from the list of WbpY from P. aeruginosa PAOl aligned to protein sequences of F. tularensis were at least 102 and e-0 values range from 7.0x1 O ²⁷ to 2.0x 1 O ²⁴ (see Table 1.).

furthermore, Table 1 shows that all of the bit scores that were the result of the BLAST of WbpZ from P. aeruginosa PAOl aligned to protein sequences of F. tularensis are higher than 50, ranging from 59 to 64. E-values ranging from 5.0x 10 " to 2.0x 1 O ⁹ also indicated homology. wbpL is the fourth required gene for initiation of rhamno-di-phosphate lipid synthesis. The5 BLAST results of WbpL from P. aeruginosa 1244 and P. putida both inferred homology. All the bit scores from the hit of WbpL from P. aeruginosa 1244 aligned to proteins sequences of F. tularensis are higher than 40, ranging from 42.7 to 43.5. E-values are also lower than 10³, ranging from 8.0x 10⁵ to 2.0x 1 O ⁴ (see Table 1). 0 Table 1.

Query protein Max total Query

Putative function Identities

function [Strain] score score cover e-value Accession number

[Strain | %

Protein (D (bit) (bit) %

WbpZ

rhamnosyltransfe WP_104928633 1 ; WP_003020697.1; rase glycosyl transferase 206 2.0x l0^‘u" WP_057113083.1 ; WP_003016713.1;

[Pseudomonas [Francisetla to ^{322 0} 96 to 97 to 1.0x10 38 to 41 WP 025329448.1 ; WP_014548581.1 ; aeruginosa tularensis] 328 “ WP 014549989.1 ; WP 032729564.1; PAOl | Protein WP 071304543.1

ID: NP 254134.1 WP 071304536.1 ; WP_014549984.1;

WbpY WP 003036991.1 ; WP 103039922.1 ; rhamnosyltransfe

glycosyltransferase WP_104928638.1; KFJ38899.1 ; rase 102

family 1 protein 102 to 7.0x 1 O ²⁷ WPJ) 12429561.1 ; WP_004337233.1;

[Pseudomonas to 72 to 2.0^X 10 25 to 26 WPJ01685992 1 ; WP 003030343.1 ;

[Francisella 108

aeruginosa 108

tularensis/ ²⁴ WPJ103025509.1; WP 00302071 1.1; PA01| Protein WP_003016699.1 ; WPJ)571 13084.1; ID: NP 254135.1 WP_061315656.1 ; WP_003034351.1;

WP 014548568.1

WP_ 104928638.1 ; WP 003039922.1;

WbpX WP_003034351.1; WP_071304536.1 ; rhamnosyltransfe

glycosyltransferase WP_003036991.1; FJ38899.1 ; rase

family 1 protein 59 to WP_014548568.1; WP_003030343.1 ;

[Pseudomonas 27 to 5.0x 10·"

59 to 64

IFrancisella 64 to 2.₀xl 0 28 to 31 WP 003020711.1; WP_003025509.1 ; aeruginosa 33

⁵ WP 004337233.1; WP 0613I5656.1 ; tularensis]

PAOIJ Protein WP 01685992.1; WP 571 13084 1 ; ID: NP 254136.1 WP_003016699.1; WP_012429561.1;

WP 014549984.1

WbpL Phospho-N- rhamnosyltransfe acetylmuramoyl- rase pentapeptide-

[Pseudomonas transferase

aeruginosa 12441 43 43 36 9.0x 10⁵ 31 AKE21584.1

[Francisella

protein ID: tularensis subsp.

AAM21928.1 tularensis str. SCHU

S4 substr. NR-28534]

For the list of WbpL from P. putida aligned to proteins sequences of F. tularensis , the presence of homology was confirmed, as all the bit scores are higher than 40 (45) and e-values are lower than I 0 ³ (from 5.0x 10 " to 2.0^c 10 ⁹). Thus, the presence wbp gene cluster similar to that responsible for synthesizing rhamnolipid-like glycolipids from P. aeruginosa were confirmed in many strains of F. tularensis. The secretion of these amphiphiles can play an important role in the virulence of the pathogenic strains, and can be the main virulence factor of this pathogen.

Additionally, other genes that could contribute to the production of amphiphilic molecules production were also confirmed to be present. There are 10 genes encoding glycosyltransferases, around 40 genes encoding proteins involved in lipoprotein particle metabolism, and a gene encoding the lipoprotein releasing system of the outer membrane lipoproteins carrier. Furthermore, fatty acid biosynthesis ligase and a carrier were also confirmed to be present. All these genes suggested that several other amphiphiles are produced intracellularly or secreted extracellularly and might contribute to the main virulence factor - rhamno-di-phosphate lipid.

Example 2: Amphiphile Synthesis in Bacillus anthracis

A NCBI BLAST analysis was performed to confirm the presence of regions of local similarities between the sequences of Bacillus spp. and B. anthracis. The BLAST results show that B. anthracis and other Bacillus spp. share the 4'-phosphopantetheinyl transferase sfp (Tsuge et al. 2005) gene that encodes an activating enzyme for the sSrfA, Itu and Fen multienzyme complex that converts the inactive protein of the corresponding synthetase to the active form (Id:, Nayak et al. 2013). It is required for the synthesis of either iturin, surfactin, or fengycin (plipastatin), the lipopeptide molecules-amphiphiles with potential virulence functions (Table 2).

Further, the presence of homologs of iturin A (Table 2), surfactin (Table 3), fengycin (Table 4), mycosubtilin, bacillomycin, lichenysin and kurstakin clusters were confirmed. The most representative and well-known lipopeptides are divided into three families: surfactin, iturin and fengycin (plipastatin). Iturin family contains iturin A, bacillomycin and mycobustilin (Stein 2005). Thus, the detailed BLASTp results of iturin A, surfactin and fengycin are described in Tables 2, 3, 4 to elucidate the similarities of lipopeptide synthesis between B. anthracis and other Bacillus spp.

Example 3: Iturin A biosynthesis: B. anthracis vs. other Bacillus species

A BLAST was performed to compare the proteins encoded by the Iturin A biosynthesis gene cluster (sfp 4'-phosphopantetheinyl transferase, itu A, ituB, ituC and ituD ) from Bacillus subtilis against B. anthracis to confirm the presence of homologous gene clusters.

As shown in Table 2, all the bit scores in the list are significantly higher than 50. The bit scores of sfp 4'-phosphopantetheinyl transferase are between 55 to 214, while those of iturin synthetases (ItuA, ItuB and ituC) are between 489 and 7845. E-values of sfp 4’- phosphopantetheinyl transferase are all close to 0, ranging from 1.Ox 10⁶⁹ to 9. Ox 1 CT⁹. E-values of iturin synthetases (ItuA, ItuB and ItuC) are essentially 0.0. For malonyl CoA-acyl carrier protein transacylase (ItuD), e-values are range from 6x l0⁸² to 8x l0⁴⁵ and bit scores range from 158 to 254, which suggested homology. The numbers of homologous proteins are usually as high as 64, but only 2 protein IDs with the highest bit scores are shown in the Table. All the results show that homologs were inferred in all the iturin A synthesis genes.

Table 2.

Query protein

Max Total

function Description of putative Query Identities Number Accession

[Strain] score score

function [Strain! cover % ^{e va ue} % of hits number

(bit) (bit)

Protein ID

SfP 4 -

1.0x 1 O ⁶''

phosphopantet phosphopantetheinyl 64 to 63 to WPJ)98759977.1 ;

64 to 214 to 24 to 44 15

heinyl transferase sfp 214 100 WP 098913427.1

6.0x 1 O^'12

transferase [Bacillus anthracis ]

[Bacillus

subtilis[ hypothetical protein

Protein ID: 55 55 65 9.0x 10 '·’ 28 WP 078984753.1

[Bacillus anthracis[

ItuA iturin poly(phosphoribitol) 61 1 to 1507 to WP 098759975.1 ;

62 to 98 0.00 30 to 44 34

synthetase A ligase subunit DltA 2149 4831 WP 098345163.1 [Bacillus [Bacillus anthracis[

subtilis] non-ribosomal peptide

Protein ID: synthase/poly ketide 659 to 1286 to PFL64725.1; API81806.1 51 to 67 0.00 31 to 37 49

synthase [ acillus 1028 4320 WP 098346824.1 anthracis\ _

D-alanine—

poly(phosphoribitol) 912 to 3545 to WP_098345164.1 ;

89 to 99 0.00 29 to 38 46

ligase subunit DltA 1769 7845 WP 098759990 1

ItuB iturin

[Bacillus anthracis[

synthetase B

hypothetical protein

[Bacillus

COJ30 26385 1884 5880 99 0.00 36 1

subtilis\ PFL55225.1

[Bacillus anthracis[

Protein ID:

API81807.1 non-ribosomal peptide

synthase/polyketidc 1033 to 3541 to WP_098345165.1 ;

89 to 99 0.00 32 to 37 47

synthase [ Bacillus 2670 1 1552 WP 047956810.1 anthracis[ _

D-alanine—

ItuC iturin poly(phosphoribitol) 564 to 2005 to WP_098345164.1 ;

82 to 95 0.00 33 to 39 31

synthetase C ligase subunit DltA 1744 3683 WP 098229538.1 [Bacillus [Bacillus anthracis \

subtilis\ non-ribosomal peptide

Protein ID: synthase/polyketide 489 to 803 to WPJ398345165.1 ; API81808.1 synthase [Bacillus 1765 5045

WP 098346824.1

anthracis[ _

[acyl-carrier-protein| 6x I O ⁸²

S-malonyltransferase 244- to 9x 10^' WP_078984754.1 ;

[Bacillus anthracis[ 254 244-254 72-74 ⁷⁸ 40 to 44 6 WP 098758270 1

ItuD malonyl

ACP S-

CoA-acyl

malonyltransferase

carrier protein

[Bacillus anthracis str.

transacylase

BF1] 246 246 74 2x 1 O ⁷⁸ 43 1 EJY90517.1

[Bacillus

malonyl CoA-ACP

subtilis[ 9x 1 O^'78

Protein ID: transacylase [Bacillus to 2x 10^' KOS25621. I ; API81805.1 anthracis[ 243 243 74 ⁷⁷ 43 2 KOS25745.1

ACP S- 4x 1 O ⁷⁵

malonyltransferase 158 - to 8x 10^' WP 098759976.1 ;

Example 4: Surfactin biosynthesis: B. anthracis vs. other Bacillus species

For surfactin synthesis, the presence of significant and representative matches of surfactin synthesis gene cluster were confirmed (see Table 3). The srfA operon {srfA-A, srfA-B , srfA-C and srfA-D ), the 4 -phosphopantetheinyl transferase sfp, the transcriptional regulator ycxD, and the 5 comA regulatory gene, which encode proteins that function together for surfactin biosynthesis, and permease ycxC that encodes a proteins that facilitates membrane transport of surfactin (Eppelmann et al. 2001).

Table 3 demonstrates the potential homologous genes of a complete set of surfactin synthesis gene cluster from B. subtilis against B. anthracis. The bit scores of srfA operon are as 0 high as 5693, and e-values are between 0.00 to 3.0^c 10 ⁹, which are significantly lower than KG⁵.

The query of two component response regulator ComA also shows homology.

As shown in Table 3, bit scores of comA from B. subtilis against B. anthracis are between 73 to 143, and e-values are ranging from 3.0X KT⁴² to 2.0x l0^~15, suggesting homology. Homologous genes of ycxC, the permease, were observed in B. anthracis with bit scores ranging 5 from 55 to 319 (Table 2) and e-values ranging from l.Ox lO ¹⁰⁸ to 5.0xl0⁸. Homologs of ycxD, encoding pyridoxal 5 '-phosphate (PLP) -dependent transcriptional regulator were also identified in Table 2. Bit scores are between 514 to 525 and e-values are essentially 0. All the homologous sequences suggest that homologous proteins of surfactin synthetases are present in B. anthracis. 0 Table 3.

Query protein Max Query

Description of putative

function [Strain] Total Identitie Number Selected

score cover e-value

function [Strain]

Protein ID score (bit) s % of hits accession number

(bit) %

D-alanine-

SrfA-A poly(phosphoribitol)

nonribosomal 51 to 57 51 to 102 WP 098345164.1 ;

100 l.Ox lO ¹⁰ 38 to 41 9

ligase subunit DltA WP 098556033.1 surfactin [Bacillus anthracis |

synthetase A

\Bacillus suhtilis | non-ribosomal peptide

2.0x 10^·"

synthase/polyketide

Protein ID: 52 to 58 52 to 154 100 to 38 to 44 10 WP_097841333.1;

synthase [ Bacillus

ACY29988.1 PFB73867.1

3.0xl0^J

anthracis\

D-alanine--

SrfA-B poly(phosphoribitol) 850 to 1960 to 91 to

28 to 39 45 WP_098345163.1 ; nonribosomal ligase subunit DltA 1904 5693 99 WP 098368002.1 surfactin [Bacillus anthracis[

synthetase B

non-ribosomal peptide

[Bacillus subtilis\

Protein ID: synthase/polyketide 849 to 2385 to 86 to WP 047956810.1;

0.00 28 to 37 48

AEW31038.1 synthase [Bacillus 2129 3783 100 WP 098346824.1

anthracis\

ligase subunit DltA 857 1976 90

surfactin WP 098759990.1

[Bacillus anthracis[

synthetase C

( Bacillus suhtilis | non-ribosomal peptide

Protein ID: synthase/polyketide 674 to 760 to 70 to

0.00 34 to 41 6 WP_098345165.1 ; synthase [Bacillus 821 2804 99

AEW31039.1 WP 047956810.1

anthracis[ SrfA-D surfactin

synthetase 3.0* 10 ⁸

thioesterase [ Bacillus 82 to

[Bacillus subtilis\ 88 to WP_098759978.1 ;

82 to 264 to 3.0 25 to 52 13

rotein ID: anthracis[ 264

P 95

x lO WP_098227789.1 AEW31040.1

DNA-binding response 3.0xl 0^~42

74 to 95 to

regulator [Bacillus 74 to 143 WP_003166783.1 ;

to 25 to 41 36

ComA two 143 100

anthraci | WP_098340506.1 l .Ox l O^'15

component uncharacterized

response regulator transcriptional

[Bacillus subtilis[ 82 82 98 1.0x10 28 1

regulatory protein yhcZ BAR7701 1. 1 Protein ID: [Bacillus anthracis\

AIC45868.1

transcriptional regulator

73 73 99 2.0x 1 O ¹⁵ 25 1

[Bacillus anthracis[ KOS23705.1

EamA/RhaT family

transporter [Bacillus 3 19 319 92 °^{X l 0'} 59 WP_098346679.1 anthracis |

ycxC permease

EamA/RhaT family

[ Bacillus subtilis ]

transporter [Bacillus 58

Protein ID: 58 72 4.0x 10^JJ 26 WP_071736796.1

anthracis[

AEW31043.1

EamA/RhaT family

PEP-dependent

aminotransferase family 515 to 98 to WP_098298378.1 ;

515 to 525 0.00

protein [Bacillus 55 to 56 24

525 100 WP_098757038.1 ycxD PLP- anthracis[

dependent

GntR family

transcriptional

transcriptional regulator 516 5 16 100

regulator [Bacillus 0.00 55 1 KOS27601.1

[Bacillus anthracis[

subtilis[ Protein

ID: AEW3I044.1 conserved hypothetical

protein [Bacillus

514 514 97 0.00 56 1

anthracis str. 'Ames AAT31700.2

Example 5: Fengycin biosynthesis: B. anthracis vs. other Bacillus species

Homologs of the fengycin biosynthesis gene cluster from B. amyloliquefaciens subsp. plantarum str. FZB42 were also identified in B. anthracis.

5 As shown in Table 4, potential homologous genes of all the fengycin synthetases fenA, fenB,fenC,fenD , and fenE were observed with high bit scores ranging from 415 to 5355 and low e-values ranging from 0.00 to 2.0^c 10 ¹²², inferring homology.

Table 4.

Query protein Description of Max Total

Query

function [Strain] putative function Identit Number Selected accession score score e-value

Protein ID [Strainl cover % ies % of hits number

(bit) (bit)

D-alanine—

poly(phosphoribitol) 81 1 to 875 to

FenA nonribosomal 28 to WP_098345163.1 ;

93 to 99 0.0 41

ligase subunit DllA 1792 1792 38

fengycin synthetase WP 098759990.1

[Bacillus anthraci \

A [Bacillus

amyloliquefaciens[ non-ribosomal

Protein ID: peptide

812 to 875 to

ABS74209.I synthase/polyketide ;

81 to 99 0.0

161 1 4407

synthase [Bacillus

anthracis[ D-alanine--

FenB nonribosomal poly(phosphori bitol) 807 to 990 to 28 to

80 to 99 0 0 WPJ398759990.1 ; fengycin synthetase ligase subunit DUA 1813 1813 34

38 WP 098345163.1 B [ Bacillus [Bacillus anthracis \

amyloliquefaciens]

Protein ID: non-ribosomal 1 1 18

898 to 31 to

ABS74208.I peptide synthetase to 80 to 99 0.0 WP 098215639.1 ;

1474 54

36

[Bacillus anthracis[ 3874 WP 04795681 1.1

D-alanine—

1038

poly(phosphoribitol) 787 to 28 to

FenC nonribosomal to 81 to 99 0.0 WP_098759990.1;

ligase subunit DUA 1888 43

42

fengycin synthetase WP 098345163.1

3648

[Bacillus anthracis[

C [Bacillus

amyloliquefaciens [ non-ribosomal

Protein ID: peptide 1014

788 to

ABS74207.I 28 to

synthase/polyketide to 80 to 99 WPJ198345165.1;

0.0

1974 45

40

synthase [Bacillus 5355 WP 098215639.1

D-alanine--

1997

poly(phosphoribitol) 858 to

FenD nonribosomal 29 to WP 098345163.1;

to 84 to 99 0.0 41

ligase subunit DltA 1937 40

fengycin synthetase 4160 WP 098556033.1

[Bacillus anthracis[

D [Bacillus

amyloliquefaciens | non-ribosomal

Protein ID: peptide 1998

858 to 30 to

ABS74206.1 synthase/polyketide to 85 to 99 0.0 44 WP_097841333.1;

2085 37

synthase [Bacillus 7945 WP 047956811.1

D-alanine-- poly(phosphoribitol) 417 to 761 to 0.0 to 28 to

FenE nonribosomal 80 to 92 WP_098345164.1;

ligase subunit DltA 825 31

1949 2.0x 10 ²² 40

fengycin synthetase WP 098759990.1

[Bacillus anthracis[

E [Bacillus

amyloliquefaciens [ non-ribosomal

Protein ID: peptide

415 to 415 to 0.0 to 29 to WP 098345165.1; ABS74205.1 synthase/polyketide 80 to 98 57

837 2818

synthase [Bacillus 2.0* 10 ¹²² 39 WP 047956810.1

Many other potential homologous genes of various strains with similar functional proteins, not shown in the Tables, were also confirmed to be present by NCBI BLAST. Bacillus cereus group, a strain very similar to B. anthracis , was identified in all the lists. The gene encoding hypothetical protein COJ30_26385 [ Bacillus anthracis ] was shown in the ituA, ituB. ituC , iluD, srfA-B, srfA-C, fenA, fenB, fenC, fenD and fenE lists. The gene encoding amino acid adenylation domain protein [ Bacillus anthracis sir. SVA1 1] was observed in the list of ituC and fenB. For the list of fengycin synthetase, several more hits were identified besides the Table. For fengycin synthetases A, C, D, and E, non-ribosomal peptide synthetase DhbF [ Bacillus anthracis str. UR-1], AMP-binding protein [. Bacillus anthracis ], bacitracin synthetase 1 (BA1) [Bacillus anthracis str. A0465], bacitracin synthetase 1 (BA1) [ Bacillus anthracis str. A0488] and bacitracin synthetase 1 (BA1) [ Bacillus anthracis str. Tsiankovskii-I\ were observed. These additional potential homologous proteins indicated that homologs of gene clusters of iturin A, surfactin, and fengycin are present in various species of B. anthracis.

There is further evidence that other amphiphiles are produced by B. anthraci ; we identified genes nprX and nprR, which are the regulators of biosynthesis of kurstakin, a lipopeptide synthesized by Bacillus cereus and in many strains of B. anthracis, with a bit score higher than 183, an e-value close to 0.0 and identities higher than 69%. NprX and NprR complexes trigger production of kurstakin. Another gene, krsE, which encodes the membrane associated protein facilitating transport of kurstakin, was also confirmed to be present in B. anthracis. All the bit scores in the list are higher than 815, e-values average at 0.0, and values of identities are higher than 98% for all the B. anthracis strains. This strongly supports the presence of krsE genes in many B. anthracis strains. However, the presence of homologous krsA, krsB and krsC genes for kurstakin synthesis was not confirmed. Further study is needed to find whether kurstakin-like molecules are produced by B. anthracis.

According to the NCBI GenBank: NC 003997.3, there might be other unidentified amphiphiles produced by B. anthracis. For example, we confirmed the presence of two genes for phospholipid/fatty acids synthase and several glycosyl transferases, suggesting potential glycolipid biosynthesis. Additionally, we confirmed the presence of two antibiotic biosynthesis genes, which tend to bind lipid and form amphiphiles. In addition to iturin, surfactin, fengycin and possibly kurstakin-like lipopeptides, these amphiphiles might be additional contributing factors to the virulence of B. anthracis.

EXEMPLARY EMBODIMENTS

1. A vaccine composition comprising an isolated biosurfactant virulence factor produced by a pathogenic microorganism, or an immunogenic analog of said biosurfactant virulence factor, and a pharmaceutical carrier.

2. The composition of embodiment 1 , further comprising an adjuvant.

3. The composition of embodiment 2, wherein the adjuvant is: alum, a bacterial- derived lipopolysaccharide, a bacterial toxin, an adjuvant emulsion, agonsitic antibodies to costimulatory molecules, Freunds adjuvant, muramyl dipeptide, recombinant/synthetic adjuvants, a toll-like receptor (TLR) ligand selected from monophosphoryl lipid A (MPL), or R837, a TLR-4 and TLR-7 ligand in combination with a nanoparticle formulation, or a cytokines selected from interferon-g and granulocyte-macrophage colony stimulating factor (GM-CSF).

4. The vaccine composition, according to embodiment 1, wherein said biosurfactant is selected from lipopeptides, flavopeptides, lipoproteins, glycolipids, glycopeptides, phospholipids, and fatty acid esters. 5. The vaccine composition, according to embodiment 4, wherein the glycolipid is selected from rhamnolipids, trehalose lipids, mannosylerythritol lipids and sophorolipids.

6. The vaccine composition, according to embodiment 5, wherein the glycolipid is selected from rhamnolipids, rhamnose-d-phospholipids, trehalose lipids, trehalose dimycolates, trehalose monomycolates, mannosylerythritol lipids, cellobiose lipids, ustilagic acid and sophorolipids.

7. The vaccine composition, according to embodiment 1, wherein wherein the biosurfactant is from Francisella tularemis.

8. The vaccine composition, according to embodiment 7, wherein the biosurfactant is a rhamno-di-phosphate lipid.

9. The vaccine composition, according to embodiment 1, wherein the biosurfactant is from Bacillus anthracis.

10. The vaccine composition, according to embodiment 9, wherein the biosurfactant is selected from iturin, surfactin, fengycin (plipastatin), mycosubtilin, bacillomycin, lichenysin and kurstakin

1 1. The vaccine, according to embodiment 10, wherein the biosurfactant is iturin A from Bacillus anthracis, or an immunological analog thereof.

12. The vaccine, according to embodiment 10, wherein the biosurfactant is fengycin from Bacillus anthracis, or an immunological analog thereof

13. The vaccine, according to embodiment 10, wherein the biosurfactant is surfactin from Bacillus anthraci , or an immunological analog thereof.

14. An antimicrobial composition comprising an inhibitor of a biosurfactant virulence factor produced by a pathogenic microorganism.

15. The antimicrobial composition, according to embodiment 14, wherein the inhibitor acts directly on the biosurfactant. 16. The antimicrobial composition, according to embodiment 14, wherein the inhibitor inhibits the synthesis, secretion, and/or assembly of the biosurfactant.

17. The antimicrobial composition, according to embodiment 16, comprising an inhibitor of secretion of the biosurfactant.

18. The antimicrobial composition, according to embodiment 17, wherein the inhibitor of secretion is selected from salicylidene acylhydrazides, thiazolidinones, and fatty acids.

19. The antimicrobial composition, according to embodiment 14, wherein the inhibitor is an antibody.

20. A method of treating or preventing an infection or a disease in a subject, the method comprising administering to the subject the composition of any of embodiments 1-19.

21. The method of embodiment 20, wherein the disease is an infectious disease.

22. The method for embodiment 20, wherein the method induces a protective immune response in a subject.

23. The method, according to embodiment 20, wherein the infection is caused by Francisella tularemis.

24. The method, according to embodiment 20, wherein the infection is caused by Bacillus anthracis.

25. A method for identifying a virulence factor, wherein said method comprises:

(a) analyzing peculiarities of etiopathogenesis and clinical manifestations of an infection,

(b) identifying unexplained features of the infection,

(c) analyzing characteristics of molecules produced by taxonomically close microorganisms,

(d) predicting existence of molecules of interest in the pathogens, (e) optionally studying physiological or pathophysiological effects produced by these molecules in other bacteria,

(f) searching existing gene and/or protein databases to find sequences of these molecules in related bacteria, and

(g) performing analyses to find orthologs in the bacteria of interest.

26. A method of diagnosing an infection by determining a pathogenic microorganism causing the infection by identifying, in a biological sample, a biosufactant virulence factor associated with the pathogenic microorganism.

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Bodour, A., Drees, K. and Maier, R. (2003). Distribution of biosurfactant-producing bacteria in undisturbed and contaminated arid southwestern soils . Applied and Environmental Microbiology , 69(6), pp.3280-3287. (Bodour et al 2003).

Brodmann, M., Dreier, R., Broz, P. and Basler, M. (2017). Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape. Nature Communications, 8, Article number: 15853. (Brodmann et al. 2017).

Candela, T. and Fouet, A. (2005). Bacillus anthracis CapD, belonging to the g- glutamyltranspeptidase family, is required for the covalent anchoring of capsule to peptidoglycan. Molecular Microbiology, 57(3), pp.717-726. (Candela and Fouet 2005).

Dai, S., Mohapatra, N., Schlesinger, L. and Gunn, J. (201 1). Regulation of Francisella tularensis virulence. Frontiers in Microbiology, 1(144). (Dai et al. 2011).

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Kau, J., Sun, D., Tsai, W., Shyu, H., Huang, H., Lin, H. and Chang, H. (2005). Antiplatelet Activities of Anthrax Lethal Toxin Are Associated with Suppressed p42/44 and p38 Mitogen-Activated Protein Kinase Pathways in the Platelets. The Journal of Infectious Diseases, 192(8), pp.1465-1474. (Kau et al. 2005).

Koehler, T. (2002) Bacillus anthracis genetics and virulence gene regulation. Ed: Koehler TM, Anthrax. Current Topics in Microbiology and Immunology, 271, pp.143- 164. (Koehler 2002). Lam, J., Rocchetta, H. and Burrows, L. (1999). Glycosyltransferases of Pseudomonas aeruginosa that assemble the O antigens of A band and B band lipopolysaccharide. Journal of Endotoxin Research , 5(1-2), pp.96-101. (Lam et al. 1999).

Nayak, S., Porob, S., Fernandes, A., Meena, R. and Ramaiah, N. (2013). PCR detection of ansA from marine bacteria and its sequence characteristics from Bacillus tequilensis NIOS4. Journal of Basic Microbiology, 54(2), pp.162-168. (Nayak et al. 2013).

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Claims

CLAIMS We claim:

1. A vaccine composition comprising an isolated biosurfactant virulence factor produced by a pathogenic microorganism, or an immunogenic analog of said biosurfactant virulence factor, and a pharmaceutical carrier.

2. The composition of claim 1, further comprising an adjuvant.

3. The composition of claim 2, wherein the adjuvant is: alum, a bacterial-derived lipopolysaccharide, a bacterial toxin, an adjuvant emulsion, agonsitic antibodies to co-stimulatory molecules, Freunds adjuvant, muramyl dipeptide, recombinant/synthetic adjuvants, a toll-like receptor (TLR) ligand selected from monophosphoryl lipid A (MPL), or R837, a TLR-4 and TLR- 7 ligand in combination with a nanoparticle formulation, or a cytokines selected from interferon-g and granulocyte-macrophage colony stimulating factor (GM-CSF).

4. The vaccine composition, according to claim 1, wherein said biosurfactant is selected from lipopeptides, flavopeptides, lipoproteins, glycolipids, glycopeptides, phospholipids, and fatty acid esters.

5. The vaccine composition, according to claim 4, wherein the glycolipid is selected from rhamnolipids, trehalose lipids, mannosylerythritol lipids and sophorolipids.

6. The vaccine composition, according to claim 5, wherein the glycolipid is selected from rhamnolipids, rhamnose-d-phospholipids, trehalose lipids, trehalose dimycolates, trehalose monomycolates, mannosylerythritol lipids, cellobiose lipids, ustilagic acid and sophorolipids.

7. The vaccine composition, according to claim 1, wherein wherein the biosurfactant is from Francisella tularemis.

8. The vaccine composition, according to claim 7, wherein the biosurfactant is a rhamno-di-phosphate lipid.

9. The vaccine composition, according to claim 1, wherein the biosurfactant is from Bacillus anthracis.

10. The vaccine composition, according to claim 9, wherein the biosurfactant is selected from iturin, surfactin, fengycin (plipastatin), mycosubtilin, bacillomycin, lichenysin and kurstakin

1 1. The vaccine, according to claim 10, wherein the biosurfactant is iturin A from Bacillus anthracis, or an immunological analog thereof.

12. The vaccine, according to claim 10, wherein the biosurfactant is fengycin from Bacillus anthracis , or an immunological analog thereof.

13. The vaccine, according to claim 10, wherein the biosurfactant is surfactin from Bacillus anthracis, or an immunological analog thereof.

14. An antimicrobial composition comprising an inhibitor of a biosurfactant virulence factor produced by a pathogenic microorganism.

15. The antimicrobial composition, according to claim 14, wherein the inhibitor acts directly on the biosurfactant.

16. The antimicrobial composition, according to claim 14, wherein the inhibitor inhibits the synthesis, secretion, and/or assembly of the biosurfactant.

17. The antimicrobial composition, according to claim 16, comprising an inhibitor of secretion of the biosurfactant.

18. The antimicrobial composition, according to claim 17, wherein the inhibitor of secretion is selected from salicylidene acylhydrazides, thiazolidinones, and fatty acids.

19. The antimicrobial composition, according to claim 14, wherein the inhibitor is an antibody.

20. A method of treating or preventing an infection or a disease in a subject, the method comprising administering to the subject the composition of any of claims 1-19.

21. The method of claim 20, wherein the disease is an infectious disease.

22. The method for claim 20, wherein the method induces a protective immune response in a subject.

23. The method, according to claim 20, wherein the infection is caused by Francisella tularemis.

24. The method, according to claim 20, wherein the infection is caused by Bacillus anthracis.

25. A method for identifying a virulence factor, wherein said method comprises:

(a) analyzing peculiarities of etiopathogenesis and clinical manifestations of an infection,

(b) identifying unexplained features of the infection,

(c) analyzing characteristics of molecules produced by taxonomically close microorganisms,

(d) predicting existence of molecules of interest in the pathogens,

(e) optionally studying physiological or pathophysiological effects produced by these molecules in other bacteria,

(f) searching existing gene and/or protein databases to find sequences of these molecules in related bacteria, and

(g) performing analyses to find orthologs in the bacteria of interest.

26. A method of diagnosing an infection by determining a pathogenic microorganism causing the infection by identifying, in a biological sample, a biosufactant virulence factor associated with the pathogenic microorganism.