MprF-mediated biosynthesis of lysylphosphatidylglycerol, an important determinant in staphylococcal defensin resistance.
ABSTRACT Frequently bacteria are exposed to membrane-damaging cationic antimicrobial molecules (CAMs) produced by the host's immune system (defensins, cathelicidins) or by competing microorganisms (bacteriocins). Staphylococcus aureus achieves CAM resistance by modifying anionic phosphatidylglycerol with positively charged L-lysine, resulting in repulsion of the peptides. Inactivation of the novel S. aureus gene, mprF, which is found in many bacterial pathogens, has resulted in the loss of lysylphosphatidylglycerol (L-PG), increased inactivation by CAM-containing neutrophils, and attenuated virulence. We demonstrate here that expression of mprF is sufficient to confer L-PG production in Escherichia coli, which indicates that MprF represents the L-PG synthase. L-PG biosynthesis was studied in vitro and found to be dependent on phosphatidylglycerol and lysyl-tRNA, two putative substrate molecules. Further addition of cadaverin, a competitive inhibitor of the lysyl-tRNA synthetases, or of RNase A abolished L-PG biosynthesis, thereby confirming the involvement of lysyl-tRNA. This study forms the basis for further detailed analyses of L-PG biosynthesis and its role in bacterial infections.
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ABSTRACT: We report the use of antimicrobial hemolymph proteins from the model host Galleria mellonella as an inhibitor for various Listeria strains, providing a novel source for antilisterial therapeutics. We also have shown that specific virulence-associated genes known to mediate antimicrobial resistance of Listeria in mam-malian models indicated a similar function in Galleria. The Gram-positive bacterium Listeria monocytogenes is able to cause food-borne infections in humans, such as listeriosis, which develops into fatal sepsis, meningitis, and meningoen-cephalitis (7, 12, 15). Listeriae are ubiquitously distributed in the environment and are resistant to extreme food-manufac-turing processes. Recently, L. monocytogenes contamination in a food plant in Canada resulted in 23 casualties, along with 57 confirmed cases (Public Health Agency of Canada; www.phac -aspc.gc.ca). The pathogenic potential of Listeria is further attributed to a growing number of strains resistant to antimi-crobial compounds and particularly to antibiotics (5, 19). Thus, it has become extremely important to identify novel sources for antilisterial therapeutics of both clinical and industrial interest. The mechanisms of bacterial resistance against cationic an-timicrobial peptides (CAMPs) are not clearly understood, and there are only few reports relating antimicrobial sensitivity of Listeria (5, 19). Identification of the two-component system virR/virS in Listeria revealed its dual role in virulence and resistance against CAMPs (13, 20). It is known that the tran-scriptional regulator VirR independently regulates expression of mprF and the genes comprising the dlt operon, which are well known for providing resistance against CAMPs of both animal and bacterial origin (2, 3, 20, 22). MprF synthesizes the lysylphosphatidyl glycerol membrane phospholipids (22), and the dlt operon (comprising of the genes dltA, dltB, dltC, and dltD)-encoded proteins are mainly responsible for adding D-alanine residues to the cell wall-associated lipotechoic acids (LTAs) (1). Both of these candidate genes maintain the posi-tive charge balance of the bacterial cell wall, facilitating CAMP resistance. Recently, we reported that following innate immune induc-tion, the hemolymph of the lepidopteran greater wax moth Galleria mellonella produces antimicrobials which inhibited L. monocytogenes growth (16). Using the Galleria model, we have represented the comparative virulence attributes of different Listeria species and L. monocytogenes serotypes, similar to the findings with other mammalian models. However, Galleria can resist septic L. monocytogenes infection, and a very high 50% lethal dose (LD 50) value (10 6 CFU/larva) is required for larval mortality. Here we report that the different Listeria species and L. monocytogenes serotypes are sensitive to the induced antimicrobial hemolymph proteins of Galleria. Accordingly, our findings demonstrate a similar role of virulence-associ-ated genes like virR, dltB, and mprF in antimicrobial resistance against Galleria hemolymph, comparable to the earlier findings using mammalian models (1, 13, 22).Applied and Environmental Microbiology 07/2011; 77(12):4237-4240. · 3.83 Impact Factor
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ABSTRACT: Bacterial pathogens colonize human body surfaces soon after birth. In order to survive the constant threat of invasion and infection, the human innate immune system has evolved several efficient mechanisms to prevent harmful microorganisms from traversing epithelial barriers. These include cationic antimicrobial peptides (CAMPs) such as defensins and the cathelicidin LL-37, bacteriolytic enzymes such as lysozyme, antimicrobial fatty acids, toxic oxygen- or nitrogen-containing molecules, the bacteriolytic complement components and further mechanisms with indirect impacts on bacterial multiplication. Staphylococcus aureus is an important human commensal and pathogen. In order to successfully establish an infection, S. aureus has evolved several mechanisms to resist the innate immune system. In this review, we focus on the mechanisms employed by S. aureus to achieve protection against antimicrobial host defense molecules with special emphasis on CAMPs. Lessons from recent studies on antimicrobial host defense molecules and cognate bacterial resistance adaptation should help in the development of more sustainable anti-infective compounds.Future Microbiology 08/2008; 3(4):437-51. · 3.82 Impact Factor
Article: Structure, dynamics, and antimicrobial and immune modulatory activities of human LL-23 and its single-residue variants mutated on the basis of homologous primate cathelicidins.[show abstract] [hide abstract]
ABSTRACT: LL-23 is a natural peptide corresponding to the 23 N-terminal amino acid residues of human host defense cathelicidin LL-37. LL-23 demonstrated, compared to LL-37, a conserved ability to induce the chemokine MCP-1 in human peripheral blood mononuclear cells, a lack of ability to suppress induction of the pro-inflammatory cytokine TNF-α in response to bacterial lipopolysaccharides (LPS), and reduced antimicrobial activity. Heteronuclear multidimensional nuclear magnetic resonance (NMR) characterization of LL-23 revealed similar secondary structures and backbone dynamics in three membrane-mimetic micelles: SDS, dodecylphosphocholine (DPC), and dioctanoylphosphatidylglycerol. The NMR structure of LL-23 determined in perdeuterated DPC contained a unique serine that segregated the hydrophobic surface of the amphipathic helix into two domains. To improve our understanding, Ser9 of LL-23was changed to either Ala or Val on the basis of homologous primate cathelicidins. These changes made the hydrophobic surface of LL-23 continuous and enhanced antibacterial activity. While identical helical structures did not explain the altered activities, a reduced rate of hydrogen-deuterium exchange from LL-23 to LL-23A9 to LL-23V9 suggested a deeper penetration of LL-23V9 into the interior of the micelles, which correlated with enhanced activities. Moreover, these LL-23 variants had discrete immunomodulatory activities. Both restored the TNF-α dampening activity to the level of LL-37. Furthermore, LL-23A9, like LL-23, maintained superior protective MCP-1 production, while LL-23V9 was strongly immunosuppressive, preventing baseline MCP-1 induction and substantially reducing LPS-stimulated MCP-1 production. Thus, these LL-23 variants, designed on the basis of a structural hot spot, are promising immune modulators that are easier to synthesize and less toxic to mammalian cells than the parent peptide LL-37.Biochemistry 12/2011; 51(2):653-64. · 3.42 Impact Factor