Insight into Invertebrate Defensin Mechanism of Action: OYSTER DEFENSINS INHIBIT PEPTIDOGLYCAN BIOSYNTHESIS BY BINDING TO LIPID II*

Three oyster defensin variants (Cg-Defh1, Cg-Defh2, and Cg-Defm) were produced as recombinant peptides and characterized in terms of activities and mechanism of action. In agreement with their spectrum of activity almost specifically directed against Gram-positive bacteria, oyster defensins were shown here to be specific inhibitors of a bacterial biosynthesis pathway rather than mere membrane-active agents. Indeed, at lethal concentrations, the three defensins did not compromise Staphylococcus aureus membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-N-acetylmuramyl-pentapeptide cell wall precursor. In addition, a combination of antagonization assays, thin layer chromatography, and surface plasmon resonance measurements showed that oyster defensins bind almost irreversibly to the lipid II peptidoglycan precursor, thereby inhibiting the cell wall biosynthesis. To our knowledge, this is the first detailed analysis of the mechanism of action of antibacterial defensins produced by invertebrates. Interestingly, the three defensins, which were chosen as representative of the oyster defensin molecular diversity, bound differentially to lipid II. This correlated with their differential antibacterial activities. From our experimental data and the analysis of oyster defensin sequence diversity, we propose that oyster defensin activity results from selective forces that have conserved residues involved in lipid II binding and diversified residues at the surface of oyster defensins that could improve electrostatic interactions with the bacterial membranes.     

Copsin,a Novel Peptide-based Fungal Antibiotic Interfering with the Peptidoglycan Synthesis

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/β-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.     

The antibacterial properties of secreted phospholipases A2: a major physiological role for the group IIA enzyme that depends on the very high pI of the enzyme to allow penetration of the bacterial cell wall.

The antibacterial properties of human group IIA secreted phospholipase A(2) against Gram-positive bacteria as a result of membrane hydrolysis have been reported. Using Micrococcus luteus as a model system, we demonstrate the very high specificity of this human enzyme for such hydrolysis compared with the group IB, IIE, IIF, V, and X human secreted phospholipase A(2)s. A unique feature of the group IIA enzyme is its very high pI due to a large excess of cationic residues on the enzyme surface. The importance of this global positive charge in bacterial cell membrane hydrolysis and bacterial killing has been examined using charge reversal mutagenesis. The global positive charge on the enzyme surface allows penetration through the bacterial cell wall, thus allowing access of this enzyme to the cell membrane. Reduced bacterial killing was associated with the loss of positive charge and reduced cell membrane hydrolysis. All mutants were highly effective in hydrolyzing the bacterial membrane of cells in which the cell wall was permeabilized with lysozyme. These same overall characteristics were also seen with suspensions of Staphylococcus aureus and Listeria innocua, where cell membrane hydrolysis and antibacterial activity of human group IIA enzyme was also lost as a result of charge reversal mutagenesis.     

Antibacterial activities of synthetic peptides corresponding to the carboxy-terminal region of human beta-defensins 1-3

The antibacterial activities of synthetic human beta-defensin analogs, constrained by a single disulfide bridge and in the reduced form, have been investigated. The peptides span the carboxy-terminal region of human beta-defensins (HBD-1-3), which have a majority of cationic residues present in the native defensins. The disulfide constrained peptides exhibited activity against Escherichia coli and Staphylococcus aureus whereas the reduced forms were active only against E. coli. The antibacterial activities were attenuated in the presence of increasing concentrations of NaCl and divalent cations such as Ca(2+) and Mg(2+). The site of action was the bacterial membrane. Peptides spanning the carboxy-terminal region of human beta-defensins could be of help in understanding facets of antimicrobial activity of beta-defensins such as salt sensitivity and mechanisms of bacterial membrane damage.     

Eurocin,a New Fungal Defensin: STRUCTURE,LIPID BINDING,AND ITS MODE OF ACTION*

Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.     

Antimicrobial Activities of Novel Peptides Derived from Defensin Genes of <Emphasis Type="Italic">Brassica hybrid</Emphasis> cv Pule

Plant defensins are small and basic antimicrobial peptides characterized by conserved cysteine stabilizing structure with α-helix and triple strand antiparallel β-sheet. In the present study, two novel defensin genes, designated as BhDef1 and BhDef2, was isolated from Brassica hybrid cv Pule, a native unexplored Brassicaceae species found in Thailand. The full-length cDNA of BhDef1 and BhDef2 were 240 and 258 bp encoding a 79 and 85 amino acid residues with 29 and 25 amino acid signal peptide at N-terminal, respectively. The putative BhDef1 and BhDef2 mature proteins showed significant similarity to other Brassicaceae defensins. Their secondary structure comprises of one α-helix and a triple stranded β-sheet stabilized by four disulphide bridges of eight cysteines. BhDef1 and BhDef2 also contain a highly conserved γ-core and α-core motif exhibiting antifungal activity against Colletotrichum gloeosporioides causing anthracnose disease. Six out of eight synthetic BhDef peptide derivatives showed antibacterial activity against both gram-positive bacteria and gram-negative bacteria used in this study. BhDef14, the derivative of BhDef1, showed the highest activity against two test pathogenic bacteria. This activity could probably due to a net positively charge and alpha-helical conformation which are known as the key determinant for the bacterial membrane disruption. To our knowledge, this is the first report on defensin genes isolated from B. hybrid cv Pule. The synthetic peptides designed from their sequences showed antifungal and antibacterial activity.     

The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis

Defensins are a class of ubiquitously expressed cationic antimicrobial peptides (CAPs) that play an important role in innate defense. Plant defensins are active against a broad range of microbial pathogens and act via multiple mechanisms, including cell membrane permeabilization. The cytolytic activity of defensins has been proposed to involve interaction with specific lipid components in the target cell wall or membrane and defensin oligomerization. Indeed, the defensin Nicotiana alata defensin 1 (NaD1) binds to a broad range of membrane phosphatidylinositol phosphates and forms an oligomeric complex with phosphatidylinositol (4,5)-bisphosphate (PIP2) that facilitates membrane lysis of both mammalian tumor and fungal cells. Here, we report that the tomato defensin TPP3 has a unique lipid binding profile that is specific for PIP2 with which it forms an oligomeric complex that is critical for cytolytic activity. Structural characterization of TPP3 by X-ray crystallography and site-directed mutagenesis demonstrated that it forms a dimer in a “cationic grip” conformation that specifically accommodates the head group of PIP2 to mediate cooperative higher-order oligomerization and subsequent membrane permeabilization. These findings suggest that certain plant defensins are innate immune receptors for phospholipids and adopt conserved dimeric configurations to mediate PIP2 binding and membrane permeabilization. This mechanism of innate defense may be conserved across defensins from different species.     

Single disulfide and linear analogues corresponding to the carboxy-terminal segment of bovine beta-defensin-2: effects of introducing the beta-hairpin nucleating sequence d-pro-gly on antibacterial activity and Biophysical properties

Mammalian defensins (alpha as well as beta forms) have a beta-hairpin structural motif spanning approximately 20 residues at the carboxy-terminal end. We have investigated the antibacterial activity and biophysical properties of synthetic peptides corresponding to the carboxy-terminal segment of bovine beta-defensin-2 (BNBD-2): VRNHVTC(1)RINRGFC(2)VPIRC(3)PGRTRQIGTC(4)FGPRIKC(5)C(6)RSW (positions of disulfide bonds are C(1)[bond]C(5), C(2)[bond]C(4), and C(3)[bond]C(6)). The parent sequence chosen was RCPGRTRQIGTIFGPRIKCRSW (P1), which spans the carboxy-terminal region of BNBD-2. Since the dipeptide sequence D-Pro-Gly favors nucleation of beta-hairpin structures even in acyclic peptides, analogues of P1 with one D-Pro-Gly at the central portion and two D-Pro-Gly segments near the N- and C-terminal ends were generated. An analogue in which GP (residues 14 and 15) in P1 was switched to PG was also synthesized. It was observed that the cyclic form as well as their linear forms exhibited antibacterial activity. Circular dichroism and theoretical studies indicated that while the beta-hairpin conformation is populated, there is conformational plasticity in the cyclic and linear peptides. The mode of bacterial killing was by membrane permeabilization. The entire mammalian defensin sequence does not appear to be essential for manifestation of antibacterial activity. Hence, short peptides corresponding to the C-terminal segments of mammalian defensins could have potential as therapeutic agents.     

Membrane Lipids Determine the Antibiotic Activity of the Lantibiotic Gallidermin

Lantibiotics, a group of lanthionine-containing peptides, display their antibiotic activity by combining different killing mechanisms within one molecule. The prototype lantibiotic nisin was shown to possess both inhibition of peptidoglycan synthesis and pore formation in bacterial membranes by interacting with lipid II. Gallidermin, which shares the lipid II binding motif with nisin but has a shorter molecular length, differed from nisin in pore formation in several strains of bacteria. To simulate the mode of action, we applied cyclic voltammetry and quartz crystal microbalance to correlate pore formation with lipid II binding kinetics of gallidermin in model membranes. The inability of gallidermin to form pores in DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) (C18/1) and DPoPC (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine) (C16/1) membranes was related to the membrane thickness. For a better simulation of bacterial membrane characteristics, two different phospholipids with branched fatty acids were incorporated into the DPoPC matrix. Phospholipids with methyl branches in the middle of the fatty acid chains favored a lipid II–independent DPoPC permeabilization by gallidermin, while long-branched phospholipids in which the branch is placed near the hydrophilic region induced an identical lipid II–dependent pore formation of gallidermin and nisin. Obviously, the branched lipids altered lipid packing and reduced the membrane thickness. Therefore, the duality of gallidermin activity (pore formation and inhibition of the cell wall synthesis) seems to be balanced by the bacterial membrane composition.     

The mode of action of the lantibiotic lacticin 3147--a complex mechanism involving specific interaction of two peptides and the cell wall precursor lipid II

Lacticin 3147 is a two-peptide lantibiotic produced by Lactococcus lactis in which both peptides, LtnA1 and LtnA2, interact synergistically to produce antibiotic activities in the nanomolar concentration range; the individual peptides possess marginal (LtnA1) or no activity (LtnA2). We analysed the molecular basis for the synergism and found the cell wall precursor lipid II to play a crucial role as a target molecule. Tryptophan fluorescence measurements identified LtnA1, which is structurally similar to the lantibiotic mersacidin, as the lipid II binding component. However, LtnA1 on its own was not able to substantially inhibit cell wall biosynthesis in vitro; for full inhibition, LtnA2 was necessary. Both peptides together caused rapid K(+) leakage from intact cells; in model membranes supplemented with lipid II, the formation of defined pores with a diameter of 0.6 nm was observed. We propose a mode of action model in which LtnA1 first interacts specifically with lipid II in the outer leaflet of the bacterial cytoplasmic membrane. The resulting lipid II:LtnA1 complex is then able to recruit LtnA2 which leads to a high-affinity, three-component complex and subsequently inhibition of cell wall biosynthesis combined with pore formation.     

Analysis of membrane interactions of antibiotic peptides using ITC and biosensor measurements

The interaction of the lantibiotic gallidermin and the glycopeptide antibiotic vancomycin with bacterial membranes was simulated using mass sensitive biosensors and isothermal titration calorimetry (ITC). Both peptides interfere with cell wall biosynthesis by targeting the cell wall precursor lipid II, but differ clearly in their antibiotic activity against individual bacterial strains. We determined the binding affinities of vancomycin and gallidermin to model membranes±lipid II in detail. Both peptides bind to DOPC/lipid II membranes with high affinity (K(D) 0.30 μM and 0.27 μM). Gallidermin displayed also strong affinity to pure DOPC membranes (0.53 μM) an effect that was supported by ITC measurements. A surface acoustic wave (SAW) sensor allowed measurements in the picomolar concentration range and revealed that gallidermin targets lipid II at an equimolar ratio and simultaneously inserts into the bilayer. These results indicate that gallidermin, in contrast to vancomycin, combines cell wall inhibition and interference with the bacterial membrane integrity for potent antimicrobial activity.     

Antibacterial activity and mechanism of action of tick defensin against Gram-positive bacteria

Defensins are a major group of antimicrobial peptides and are found widely in vertebrates, invertebrates and plants. Invertebrate defensins have been identified from insects, scorpions, mussels and ticks. In this study, chemically synthesized tick defensin was used to further investigate the activity spectrum and mode of action of natural tick defensin. Synthetic tick defensin showed antibacterial activity against many Gram-positive bacteria but not Gram-negative bacteria and low hemolytic activity, characteristic of invertebrate defensins. Furthermore, bactericidal activity against pathogenic Gram-positive bacteria including Bacillus cereus, Enterococcus faecalis and methicillin-resistant Staphylococcus aureus was observed. However, more than 30 min was necessary for tick defensin to completely kill bacteria. The interaction of tick defensin with the bacterial cytoplasmic membrane and its ability to disrupt the membrane potential was analyzed. Tick defensin was able to disrupt the membrane potential over a period of 30-60 min consistent with its relatively slow killing. Transmission electron microscopy of Micrococcus luteus treated with tick defensin showed lysis of the cytoplasmic membrane and leakage of cellular cytoplasmic contents. These findings suggest that the primary mechanism of action of tick defensin is bacterial cytoplasmic membrane lysis. In addition, incomplete cell division with multiple cross-wall formation was occasionally seen in tick defensin-treated bacteria showing pleiotropic secondary effects of tick defensin.     

Lipodepsipeptide empedopeptin inhibits cell wall biosynthesis through Ca2+-dependent complex formation with peptidoglycan precursors

Empedopeptin is a natural lipodepsipeptide antibiotic with potent antibacterial activity against multiresistant Gram-positive bacteria including methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae in vitro and in animal models of bacterial infection. Here, we describe its so far elusive mechanism of antibacterial action. Empedopeptin selectively interferes with late stages of cell wall biosynthesis in intact bacterial cells as demonstrated by inhibition of N-acetylglucosamine incorporation into polymeric cell wall and the accumulation of the ultimate soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide in the cytoplasm. Using membrane preparations and the complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes and their respective purified substrates, we show that empedopeptin forms complexes with undecaprenyl pyrophosphate containing peptidoglycan precursors. The primary physiological target of empedopeptin is undecaprenyl pyrophosphate-N-acetylmuramic acid(pentapeptide)-N-acetylglucosamine (lipid II), which is readily accessible at the outside of the cell and which forms a complex with the antibiotic in a 1:2 molar stoichiometry. Lipid II is bound in a region that involves at least the pyrophosphate group, the first sugar, and the proximal parts of stem peptide and undecaprenyl chain. Undecaprenyl pyrophosphate and also teichoic acid precursors are bound with lower affinity and constitute additional targets. Calcium ions are crucial for the antibacterial activity of empedopeptin as they promote stronger interaction with its targets and with negatively charged phospholipids in the membrane. Based on the high structural similarity of empedopeptin to the tripropeptins and plusbacins, we propose this mechanism of action for the whole compound class.     

Bacterial species selective toxicity of two isomeric α/β-peptides: Role of membrane lipids

We have studied how membrane interactions of two synthetic cationic antimicrobial peptides with alternating α- and β-amino acid residues (“α/β-peptides”) impact toxicity to different prokaryotes. Electron microscopic examination of thin sections of Escherichia coli and of Bacillus subtilis exposed to these two α/β-peptides reveals different structural changes in the membranes of these bacteria. These two peptides also have very different effects on the morphology of liposomes composed of phosphatidylethanolamine and phosphatidylglycerol in a 2:1 molar ratio. Freeze fracture electron microscopy indicates that with this lipid mixture, α/β-peptide I induces the formation of a sponge phase. ³¹P NMR and X-ray diffraction are consistent with this conclusion. In contrast, with α/β-peptide II and this same lipid mixture, a lamellar phase is maintained, but with a drastically reduced d-spacing. α/β-Peptide II is more lytic to liposomes composed of these lipids than is I. These findings are consistent with the greater toxicity of α/β-peptide II, relative to α/β-peptide I, to E. coli, a bacterium having a high content of phosphatidylethanolamine. In contrast, both α/β-peptides display similar toxicity toward B. subtilis, in accord with the greater anionic lipid composition in its membrane. This work shows that variations in the selectivity of these peptidic antimicrobial peptides toward different strains of bacteria can be partly determined by the lipid composition of the bacterial cell membrane.     

Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria

We investigated the mechanisms of two tryptophan-rich antibacterial peptides (KT2 and RT2) obtained in a previous optimization screen for increased killing of both Gram-negative and Gram-positive bacteria pathogens. At their minimal inhibitory concentrations (MICs), these peptides completely killed cells of multidrug-resistant, enterohemorrhagic pathogen Escherichia coli O157:H7 within 1–5 min. In addition, both peptides exhibited anti-biofilm activity at sub-MIC levels. Indeed, these peptides prevented biofilm formation and triggered killing of cells in mature E. coli O157:H7 biofilms at 1 μM. Both peptides bound to bacterial surface LPS as assessed using the dansyl-polymyxin displacement assay, and were able to interact with the lipids of liposomes as determined by observing a tryptophan blue shift. Interestingly, even though these peptides were highly antimicrobial, they did not induce pore formation or aggregates in bacterial cell membranes. Instead these peptides readily penetrated into bacterial cells as determined by confocal microscopy of labeled peptides. DNA binding assays indicated that both peptides bound to DNA with higher affinity than the positive control peptide buforin II. We propose that cationic peptides KT2 and RT2 bind to negatively-charged LPS to enable self-promoted uptake and, subsequently interact with cytoplasmic membrane phospholipids through their hydrophobic domains enabling translocation across the bacterial membrane and entry into cells within minutes and binding to DNA and other cytoplasmic membrane. Due to their dual antimicrobial and anti-biofilm activities, these peptides may find use as an alternative to (or in conjunction with) conventional antibiotics to treat acute infections caused by planktonic bacteria and chronic, biofilm-related infections.     

Inhibition of SARS-CoV-2 Infection by Human Defensin HNP1 and Retrocyclin RC-101

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is an enveloped virus responsible for the COVID-19 pandemic. The emergence of new potentially more transmissible and vaccine-resistant variants of SARS-CoV-2 is an ever-present threat. Thus, it remains essential to better understand innate immune mechanisms that can inhibit the virus. One component of the innate immune system with broad antipathogen, including antiviral, activity is a group of cationic immune peptides termed defensins. The ability of defensins to neutralize enveloped and non-enveloped viruses and to inactivate numerous bacterial toxins correlate with their ability to promote the unfolding of proteins with high conformational plasticity. We found that human neutrophil α-defensin HNP1 binds to SARS-CoV-2 Spike protein with submicromolar affinity that is more than 20 fold stronger than its binding to serum albumin. As such, HNP1, as well as a θ-defensin retrocyclin RC-101, both interfere with Spike-mediated membrane fusion, Spike-pseudotyped lentivirus infection, and authentic SARS-CoV-2 infection in cell culture. These effects correlate with the abilities of the defensins to destabilize and precipitate Spike protein and inhibit the interaction of Spike with the ACE2 receptor. Serum reduces the anti-SARS-CoV-2 activity of HNP1, though at high concentrations, HNP1 was able to inactivate the virus even in the presence of serum. Overall, our results suggest that defensins can negatively affect the native conformation of SARS-CoV-2 Spike, and that α- and θ-defensins may be valuable tools in developing SARS-CoV-2 infection prevention strategies.     

Proteinase 3 carries small unusual carbohydrates and associates with αlpha-defensins

The neutrophil granulocyte is an important first line of defense against intruding pathogens and it contains a range of granules armed with antibacterial peptides and proteins. Proteinase 3 (PR3) is one among several serine proteases of the azurophilic granules in neutrophil granulocytes. Here, we characterize the glycosylation of PR3 and its association with antimicrobial human neutrophil peptides (HNPs, α-defensins) and the effect of these on the mechanism of inhibition of the major plasma inhibitor of PR3, α1-antitrypsin. The glycosylation of purified, mature PR3 showed some heterogeneity with carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with α-defensins and oligomers hereof. Irreversible inhibition of PR3 by α1-antitrypsin did not affect its association with defensins. Other proteins from neutrophil granules were also found to be associated with defensins, whereas purified plasma proteins did not carry defensins. These results point to a role of defensins in controlling and targeting the activity of neutrophil granule proteins.     

The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes.

Peptides composed of leucyl and lysyl residues ('LK peptides') with different compositions and sequences were compared for their antibacterial activities using cell wall-less bacteria of the class Mollicutes (acholeplasmas, mycoplasmas and spiroplasmas) as targets. The antibacterial activity of the amphipathic alpha-helical peptides varied with their size, 15 residues being the optimal length, independent of the membrane hydrophobic core thickness and the amount of cholesterol. The 15-residue ideally amphipathic alpha helix with a +5 positive net charge (KLLKLLLKLLLKLLK) had the strongest antibacterial activity, similar to that of melittin. In contrast, scrambled peptides devoid of amphipathy and the less hydrophobic beta-sheeted peptides [(LK)nK], even those 15-residue long, were far less potent than the helical ones. Furthermore, the growth inhibitory activity of the peptides was correlated with their ability to abolish membrane potential. These data are fully consistent with a predominantly flat orientation of LK peptides at the lipid/water interface and strongly supports that these peptides and probably the linear polycationic amphipathic defence peptides act on bacterial membranes in four main steps according to the 'carpet' model: (a) interfacial partitioning with accumulation of monomers on the target membrane (limiting step); (b) peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer (as already shown with liposomes and erythrocytes); (c) plasma membrane permeabilization/depolarization via a detergent-like effect; and (d) rapid bacterial cell death if the extent of depolarization is maintained above a critical threshold.     

How do bacteria resist human antimicrobial peptides?

Cationic antimicrobial peptides (CAMPs), such as defensins, cathelicidins and thrombocidins, are an important human defense mechanism, protecting skin and epithelia against invading microorganisms and assisting neutrophils and platelets. Staphylococcus aureus, Salmonella enterica and other bacterial pathogens have evolved countermeasures to limit the effectiveness of CAMPs, including the repulsion of CAMPs by reducing the net negative charge of the bacterial cell envelope through covalent modification of anionic molecules (e.g. teichoic acids, phospholipids and lipid A); expelling CAMPs through energy-dependent pumps; altering membrane fluidity; and cleaving CAMPs with proteases. Mutants susceptible to CAMPs are more efficiently inactivated by phagocytes and are virulence-attenuated, indicating that CAMP resistance plays a key role in bacterial infections.     

Characterization of cell membrane parameters of clinical isolates of Staphylococcus aureus with varied susceptibility to alpha-melanocyte stimulating hormone

Staphylococcus aureus (S. aureus), a major human pathogen of hospital and community acquired infections, is becoming resistant to almost all commercially available antibiotics. This has prompted development of antimicrobial peptides as therapeutic options. Alpha melanocyte stimulating hormone (α-MSH) is one such peptide known to possess antimicrobial properties. In the present study, we analyzed the antimicrobial activity of α-MSH against 75 clinical strains of S. aureus including both methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) strains. Results of our previous study showed that membrane damage is the major mechanism of staphylocidal activity of α-MSH. In this context, we compared the various bacterial membrane parameters, viz., membrane fluidity, lipid composition, and surface charge of a few selected MSSA and MRSA strains that showed variable susceptibility to the melanocortin peptide. Our results showed that α-MSH killed both type of strains efficiently (≥70% killing in 84% clinical strains after exposure with 6μM of α-MSH for 1h). It was observed that compared to the α-MSH-susceptible strains, the α-MSH-non-susceptible strains had a different membrane order and phospholipid pattern. There was no consistent pattern of cell surface charge to distinguish α-MSH-susceptible strain from a non-susceptible strain. In conclusion, α-MSH possessed potential staphylocidal activity for both against MSSA and MRSA strains. S. aureus strains not susceptible to the peptide exhibited a rigid membrane and a higher amount of the cationic phospholipid as compared to the α-MSH-susceptible strains.