Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides.

The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the two-component systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (Moskowitz, S. M.; Antimicrob. Agents Chemother. 2012, 56, 1019-1030; Cheng, H. Y.; J. Biomed. Sci. 2010, 17, 60). Our data suggested that the two systems have opposing effects on the properties of Salmonella enterica. The knockout of PhoPQ made the bacteria more susceptible to AMPs by making the surface less rigid, more polarized, and permeable with a slightly more negatively charged cell wall. In addition, the periplasmic space is thinner. In contrast, the knockout of PmrAB did not affect its susceptibility, while it made the bacterial outer layer very rigid, less polarized, and less permeable than the other two mutants, with a negatively charged cell wall similar to the WT. Overall, the data suggest that the coexistence of systems with opposing effects on the biophysical properties of the bacteria contribute to their membrane flexibility, which, on the one hand, is important to accommodate changing environments and, on the other hand, may inhibit the development of meaningful resistance to AMPs.

Defensin-based anti-infective strategies.

Cationic and amphiphilic peptides are widely distributed in eukaryotic organisms and constitute a first line of host defense against invading pathogens. Some of these host defense peptides (HDPs) combine specific antibiotic activities with modulation of immune responses. Moreover, they are active against bacteria resistant to conventional antibiotics and show only modest resistance development under in vitro selection pressure. Based on these features, HDPs and particularly defensins are considered a promising source of novel anti-infective agents. This review summarizes the current knowledge about defensins from different kingdoms and discusses their potential for therapeutic application.

Determination of Bacterial Membrane Impairment by Antimicrobial Agents.

The bacterial cytoplasmic membrane separates the cell from its environment and acts as a selective permeability barrier. In addition, it functions in energy conservation, transport, signaling, and biosynthesis processes. Antimicrobial agents disrupting these functions may lead to pleiotropic effects, including leakage of low molecular weight compounds such as ions, amino acids, and ATP and subsequent membrane depolarization. This updated chapter describes two techniques to assess antibiotic-induced membrane impairment in vivo.

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.

Antibiotic activities of host defense peptides: more to it than lipid bilayer perturbation.

Defensins are small basic amphiphilic peptides (up to 5 kDa) that have been shown to be important effector molecules of the innate immune system of animals, plants and fungi. In addition to immune modulatory functions, they have potent direct antimicrobial activity against a broad spectrum of bacteria, fungi and/or viruses, which makes them promising lead compounds for the development of next-generation antiinfectives. The mode of antibiotic action of defensins was long thought to result from electrostatic interaction between the positively charged defensins and negatively charged microbial membranes, followed by unspecific membrane permeabilization or pore-formation. Microbial membranes are more negatively charged than human membranes, which may explain to some extent the specificity of defensin action against microbes and associated low toxicity for the host. However, research during the past decade has demonstrated that defensin activities can be much more targeted and that microbe-specific lipid receptors are involved in the killing activity of various defensins. In this respect, human, fungal and invertebrate defensins have been shown to bind to and sequester the bacterial cell wall building block lipid II, thereby specifically inhibiting cell wall biosynthesis. Moreover, plant and insect defensins were found to interact with fungal sphingolipid receptors, resulting in fungal cell death. This review summarizes the current knowledge on the mode of action and structure of defensins from different kingdoms, with specific emphasis on their interaction with microbial lipid receptors.

Human beta-defensin 3 inhibits cell wall biosynthesis in Staphylococci.

Human beta-defensin 3 (hBD3) is a highly charged (+11) cationic host defense peptide, produced by epithelial cells and neutrophils. hBD3 retains antimicrobial activity against a broad range of pathogens, including multiresistant Staphylococcus aureus, even under high-salt conditions. Whereas antimicrobial host defense peptides are assumed to act by permeabilizing cell membranes, the transcriptional response pattern of hBD3-treated staphylococcal cells resembled that of vancomycin-treated cells (V. Sass, U. Pag, A. Tossi, G. Bierbaum, and H. G. Sahl, Int. J. Med. Microbiol. 298:619-633, 2008) and suggested that inhibition of cell wall biosynthesis is a major component of the killing process. hBD3-treated cells, inspected by transmission electron microscopy, showed localized protrusions of cytoplasmic contents, and analysis of the intracellular pool of nucleotide-activated cell wall precursors demonstrated accumulation of the final soluble precursor, UDP-MurNAc-pentapeptide. Accumulation is typically induced by antibiotics that inhibit membrane-bound steps of cell wall biosynthesis and also demonstrates that hBD3 does not impair the biosynthetic capacity of cells and does not cause gross leakage of small cytoplasmic compounds. In in vitro assays of individual membrane-associated cell wall biosynthesis reactions (MraY, MurG, FemX, and penicillin-binding protein 2 [PBP2]), hBD3 inhibited those enzymes which use the bactoprenol-bound cell wall building block lipid II as a substrate; quantitative analysis suggested that hBD3 may stoichiometrically bind to lipid II. We report that binding of hBD3 to defined, lipid II-rich sites of cell wall biosynthesis may lead to perturbation of the biosynthesis machinery, resulting in localized lesions in the cell wall as demonstrated by electron microscopy. The lesions may then allow for osmotic rupture of cells when defensins are tested under low-salt conditions.

Determination of Bacterial Membrane Impairment by Antimicrobial Agents.

The bacterial cytoplasmic membrane separates the cell from its environment and acts as a selective permeability barrier. In addition, it functions in energy conservation, transport, and biosynthesis processes. Antimicrobial agents disrupting these functions may lead to pleiotropic effects, including leakage of low molecular weight compounds such as ions, amino acids and ATP, and subsequent membrane depolarization. This article describes two techniques to assess antibiotic-induced membrane impairment in vivo.

Development of in vitro resistance to chitosan is related to changes in cell envelope structure of Staphylococcus aureus.

The bacterial cell envelope is believed to be a principal target for initiating the staphylocidal pathway of chitosan. The present study was therefore designed to investigate possible changes in cell surface phenotypes related to the in vitro chitosan resistance development in the laboratory strain S. aureus SG511-Berlin. Following a serial passage experiment, a stable chitosan-resistant variant (CRV) was identified, exhibiting >50-fold reduction in its sensitivity towards chitosan. Our analyses of the CRV identified phenotypic and genotypic features that readily distinguished it from its chitosan-susceptible parental strain, including: (i) a lower overall negative cell surface charge; (ii) cross-resistance to a number of antimicrobial agents; (iii) major alterations in cell envelope structure, cellular bioenergetics and metabolism (based on transcriptional profiling); and (iv) a repaired sensor histidine kinase GraS. Our data therefore suggest a close nexus between changes in cell envelope properties with the in vitro chitosan-resistant phenotype in S. aureus SG511-Berlin.

AmiD Is a Novel Peptidoglycan Amidase in Wolbachia Endosymbionts of Drosophila melanogaster.

Wolbachia endobacteria are obligate intracellular bacteria with a highly reduced genome infecting many arthropod and filarial species, in which they manipulate arthropod reproduction to increase their transmission and are essential for nematode development and survival. The Wolbachia genome encodes all enzymes required for the synthesis of the cell wall building block lipid II, although a peptidoglycan-like structure has not been detected. Despite the ability to synthesize lipid II, Wolbachia from arthropods and nematodes have only a subset of genes encoding enzymes involved in the periplasmic processing of lipid II and peptidoglycan recycling, with arthropods having two more than nematodes. We functionally analyzed the activity of the putative cell wall hydrolase AmiD from the Wolbachia endosymbiont of Drosophila melanogaster, an enzyme not encoded by the nematode endobacteria. Wolbachia AmiD has Zn2+-dependent amidase activity and cleaves intact peptidoglycan, monomeric lipid II and anhydromuropeptides, substrates that are generated during bacterial growth. AmiD may have been maintained in arthropod Wolbachia to avoid host immune recognition by degrading cell wall fragments in the periplasm. This is the first description of a wolbachial lipid II processing enzyme putatively expressed in the periplasm.

Killing of staphylococci by θ-defensins involves membrane impairment and activation of autolytic enzymes.

θ-Defensins are cyclic antimicrobial peptides expressed in leukocytes of Old world monkeys. To get insight into their antibacterial mode of action, we studied the activity of RTDs (rhesus macaque θ-defensins) against staphylococci. We found that in contrast to other defensins, RTDs do not interfere with peptidoglycan biosynthesis, but rather induce bacterial lysis in staphylococci by interaction with the bacterial membrane and/or release of cell wall lytic enzymes. Potassium efflux experiments and membrane potential measurements revealed that the membrane impairment by RTDs strongly depends on the energization of the membrane. In addition, RTD treatment caused the release of Atl-derived cell wall lytic enzymes probably by interaction with membrane-bound lipoteichoic acid. Thus, the premature and uncontrolled activity of these enzymes contributes strongly to the overall killing by θ-defensins. Interestingly, a similar mode of action has been described for Pep5, an antimicrobial peptide of bacterial origin.