Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Bovine Muc1 inhibits binding of enteric bacteria to Caco-2 cells

Inhibition of bacterial adhesion to intestinal epithelial receptors by the consumption of natural food components is an attractive strategy for the prevention of microbial related gastrointestinal illness. We hypothesised that Muc1, a highly glycosylated mucin present in cows’ milk, may be one such food component. Purified bovine Muc1 was tested for its ability to inhibit binding of common enteric bacterial pathogens to Caco-2 cells grown in vitro. Muc1 caused dose-dependent binding inhibition of Escherichia coli, Salmonella enterica serovar Typhimurium (S. Typhimurium), Staphylococcus aureus and Bacillus subtilis. This inhibition was more pronounced for the Gram negative compared with Gram positive bacteria. It was also demonstrated that Muc1, immobilised on a membrane, bound all these bacterial species in a dose-dependent manner, although there was greater interaction with the Gram negative bacteria. A range of monosaccharides, representative of the Muc1 oligosaccharide composition, were tested for their ability to prevent binding of E. coli and S. Typhimurium to Caco-2 cells. Inhibition was structure dependent with sialic acid, L(-) fucose and D(+) mannose significantly inhibiting binding of both Gram negative species. N-acetylglucosamine and N-acetylgalactosamine significantly inhibited binding of E. coli whilst galactose, one of the most abundant Muc1 monosaccharides, showed the strongest inhibition against S. Typhimurium. Treatment with sialidase significantly decreased the inhibitory properties of Muc1, demonstrating the importance of sialic acid in adhesion inhibition. It is concluded that bovine Muc1 prevents binding of bacteria to human intestinal cells and may have a role in preventing the binding of common enteropathogenic bacteria to human intestinal epithelial surfaces.     

A protein critical for cell constriction in the Gram‐negative bacterium Caulobacter crescentus localizes at the division site through its peptidoglycan‐binding LysM domains

During division of Gram‐negative bacteria, invagination of the cytoplasmic membrane and inward growth of the peptidoglycan (PG) are followed by the cleavage of connective septal PG to allow cell separation. This PG splitting process requires temporal and spatial regulation of cell wall hydrolases. In Escherichia coli, LytM factors play an important role in PG splitting. Here we identify and characterize a member of this family (DipM) in Caulobacter crescentus. Unlike its E. coli counterparts, DipM is essential for viability under fast‐growth conditions. Under slow‐growth conditions, the ΔdipM mutant displays severe defects in cell division and FtsZ constriction. Consistent with its function in division, DipM colocalizes with the FtsZ ring during the cell cycle. Mutagenesis suggests that the LytM domain of DipM is essential for protein function, despite being non‐canonical. DipM also carries two tandems of the PG‐binding LysM domain that are sufficient for FtsZ ring localization. Localization and fluorescence recovery after photobleaching microscopy experiments suggest that DipM localization is mediated, at least in part, by the ability of the LysM tandems to distinguish septal, multilayered PG from non‐septal, monolayered PG.     

Identification of YfiH (PgeF) as a factor contributing to the maintenance of bacterial peptidoglycan composition

Peptidoglycan (PG) is an essential, envelope‐fortifying macromolecule of eubacterial cell walls. It is a large polymer with multiple glycan strands interconnected by short peptide chains forming a sac‐like structure around cytoplasmic membrane. In most bacteria, the composition of the peptide chain is well‐conserved and distinctive; in E. coli, the peptide chain length varies from two to five amino acids with a tetrapeptide consisting of L‐alanine – D‐glutamic acid – meso‐diaminopimelic acid – D‐alanine. However, it is not known how bacteria conserve the composition and sequence of peptide chains of PG. Here, we find that a conserved open reading frame of unknown function, YfiH (renamed PgeF) contributes to the maintenance of peptide composition in E. coli. Using genetic, biochemical and mass spectrometrical analyses we demonstrate that absence of yfiH results in incorporation of non‐canonical amino acids, L‐serine or glycine in place of L‐alanine in PG sacculi leading to β‐lactam – sensitivity, lethality in mutants defective in PG remodelling or recycling pathways, altered cell morphology and reduced PG synthesis. yfiH orthologs from other Gram‐positive genera were able to compensate the absence of yfiH in E. coli indicating a conserved pathway in bacterial kingdom. Our results suggest editing/quality control mechanisms exist to maintain composition and integrity of bacterial peptidoglycan.     

Peptidoglycan transformations during Bacillus subtilis sporulation

While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross‐linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high‐resolution electron cryotomographic imaging. Three‐dimensional reconstructions of whole cells in near‐native states revealed a thin PG‐like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram‐negative‐like PG layers as well as its thick, archetypal Gram‐positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.     

The Bacillus subtilis cannibalism toxin SDP collapses the proton motive force and induces autolysis

Bacillus subtilis SDP is a peptide toxin that kills cells outside the biofilm to support continued growth. We show that purified SDP acts like endogenously produced SDP; it delays sporulation, and the SdpI immunity protein confers SDP resistance. SDP kills a variety of Gram‐positive bacteria in the phylum Firmicutes, as well as Escherichia coli with a compromised outer membrane, suggesting it participates in defence of the B. subtilis biofilm against Gram‐positive bacteria as well as cannibalism. Fluorescence microscopy reveals that the effect of SDP on cells differs from that of nisin, nigericin, valinomycin and vancomycin‐KCl, but resembles that of CCCP, DNP and azide. Indeed, SDP rapidly collapses the PMF as measured by fluorometry and flow cytometry, which triggers the slower process of autolysis. This secondary consequence of SDP treatment is not required for cell death since the autolysin‐defective lytC, lytD, lytE, lytF strain fails to be lysed but is nevertheless killed by SDP. Collapsing the PMF is an ideal mechanism for a toxin involved in cannibalism and biofilm defence, since this would incapacitate neighbouring cells by inhibiting motility and secretion of proteins and toxins. It would also induce autolysis in many Gram‐positive species, thereby releasing nutrients that promote biofilm growth.     

Water Soluble Coumarin Quaternary Ammonium Chlorides: Synthesis and Biological Evaluation

In the present study, coumarin‐bearing three pyridinium and three tetra‐alkyl ammonium salts were synthesized. The compounds were fully characterized by ¹H‐ and ¹³C‐NMR, LC/MS and IR spectroscopic methods and elemental analyses. The cytotoxic properties of all compounds were tested against human liver cancer (HepG2), human colorectal cancer (Caco‐2) and non‐cancer mouse fibroblast (L‐929) cell lines. Some compounds performed comparable cytotoxicity with standard drug cisplatin. Antibacterial properties of the compounds were tested against Gram‐negative Escherichia coli and Gram‐positive Bacillus subtilis bacteria, but the compounds did not have any antibacterial effect against both bacteria. Enzyme inhibitory properties of all compounds were tested on the activities of human carbonic anhydrase I and II, and xanthine oxidase. All compounds inhibited both enzymes more effectively than standard drugs, acetazolamide and allopurinol, respectively. The biological evaluation results showed that ionic and water soluble coumarin derivatives are promising structures for further investigations especially on enzyme inhibition field.     

Distinct localization of the complement C5b‐9 complex on Gram‐positive bacteria

The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo‐attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b‐9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram‐positive bacteria are protected from MAC‐dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram‐positive pathogens secrete small proteins that inhibit C5b‐9 formation. In this study, we found that complement activation on Gram‐positive bacteria in serum results in specific surface deposition of C5b‐9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS‐stable) forms, indicating the presence of ring‐structured C5b‐9. Surprisingly, confocal microscopy demonstrated that C5b‐9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b‐9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b‐9 complex and Gram‐positive bacteria, whichmight ultimately lead to a new model of MAC assembly and functioning.     

Lipopolysaccharide binding to the periplasmic protein LptA

Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram‐negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram‐negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG₂, that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane. It is hypothesized that LPS binds to LptA to cross the periplasm and that the acyl chains of LPS bind to the central pocket of LptA. The studies described here are the first to comprehensively characterize and quantitate the binding of LPS by LptA. Using site‐directed spin‐labeling electron paramagnetic resonance (EPR) spectroscopy, data were collected for 15 spin‐labeled residues in and around the proposed LPS binding pocket on LptA to observe the mobility changes caused by the presence of exogenous LPS and identify the binding location of LPS to LptA. The EPR data obtained suggest a 1:1 ratio for the LPS:LptA complex and allow the first calculation of dissociation constants for the LptA–LPS interaction. The results indicate that the entire protein is affected by LPS binding, the N‐terminus unfolds in the presence of LPS, and a mutant LptA protein unable to form oligomers has an altered affinity for LPS.     

Interspecies Comparison of the Bacterial Response to Allicin Reveals Species‐Specific Defense Strategies

Allicin, a broad‐spectrum antimicrobial agent from garlic, disrupts thiol and redox homeostasis, proteostasis, and cell membrane integrity. Since medicine demands antimicrobials with so far unexploited mechanisms, allicin is a promising lead structure. While progress is being made in unraveling its mode of action, little is known on bacterial adaptation strategies. Some isolates of Pseudomonas aeruginosa and Escherichia coli withstand exposure to high allicin concentrations due to as yet unknown mechanisms. To elucidate resistance and sensitivity‐conferring cellular processes, the acute proteomic responses of a resistant P. aeruginosa strain and the sensitive species Bacillus subtilis are compared to the published proteomic response of E. coli to allicin treatment. The cellular defense strategies share functional features: proteins involved in translation and maintenance of protein quality, redox homeostasis, and cell envelope modification are upregulated. In both Gram‐negative species, protein synthesis of the majority of proteins is downregulated while the Gram‐positive B. subtilis responded by upregulation of multiple regulons. A comparison of the B. subtilis proteomic response to a library of responses to antibiotic treatment reveals 30 proteins specifically upregulated by allicin. Upregulated oxidative stress proteins are shared with nitrofurantoin and diamide. Microscopy‐based assays further indicate that in B. subtilis cell wall integrity is impaired.     

Cyclic antimicrobial R-, W-rich peptides: the role of peptide structure and <Emphasis Type="Italic">E. coli</Emphasis> outer and inner membranes in activity and the mode of action

This study compares the effect of cyclic R-, W-rich peptides with variations in amino acid sequences and sizes from 5 to 12 residues upon Gram negative and Gram positive bacteria as well as outer membrane-deficient and LPS mutant Escherichia coli (E. coli) strains to analyze the structural determinants of peptide activity. Cyclo-RRRWFW (c-WFW) was the most active and E. coli-selective sequence and bactericidal at the minimal inhibitory concentration (MIC). Removal of the outer membrane distinctly reduced peptide activity and the complete smooth LPS was required for maximal activity. c-WFW efficiently permeabilised the outer membrane of E. coli and promoted outer membrane substrate transport. Isothermal titration calorimetric studies with lipid A-, rough-LPS (r-LPS)- and smooth-LPS (s-LPS)-doped POPC liposomes demonstrated the decisive role of O-antigen and outer core polysaccharides for peptide binding and partitioning. Peptide activity against the inner E. coli membrane (IM) was very low. Even at a peptide to lipid ratio of 8/1, c-WFW was not able to permeabilise a phosphatidylglycerol/phosphatidylethanolamine (POPG/POPE) bilayer. Low influx of propidium iodide (PI) into bacteria confirmed a low permeabilising ability of c-WFW against PE-rich membranes at the MIC. Whilst the peptide effect upon eukaryotic cells correlated with the amphipathicity and permeabilisation of neutral phosphatidylcholine bilayers, suggesting a membrane disturbing mode of action, membrane permeabilisation does not seem to be the dominating antimicrobial mechanism of c-WFW. Peptide interactions with the LPS sugar moieties certainly modulate the transport across the outer membrane and are the basis of the E. coli selectivity of this type of peptides.     

Cyclic diguanylate regulation of Bacillus cereus group biofilm formation

Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram‐negatives, increased levels of the second messenger cyclic diguanylate (c‐di‐GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c‐di‐GMP include cell division, differentiation and virulence. Among Gram‐positive bacteria, where the function of c‐di‐GMP signalling is less well characterized, c‐di‐GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c‐di‐GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c‐di‐GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c‐di‐GMP signalling between B. subtilis and B. cereus group bacteria.     

Biocontrol of Rhizoctonia Damping-off of Tomato by Bacillus subtilis Combined with Burkholderia cepacia

Bacillus subtilis strain RB14‐C and Burkholderia cepacia strain BY were used in combination to control damping‐off of tomato plants caused by Rhizoctonia solani. Microcosm tests showed complete inhibition of R. solani growth on filter disks buried in soil added with the mixture of both bacteria. Single BY inhibited the fungus, but not completely, and RB14‐C had only slight inhibitory effect on pathogen growth. The efficacy of this combining treatment was checked in pot experiments, where bacteria were applied to the soil in several combinations: RB14‐C and BY together 4 days before seed planting, RB14‐C 4 days and BY 2 days before seed planting, RB14‐C 4 days and BY immediately before seeds. The effect of these treatments on population of R. solani in soil and infection of plants was compared with the activity of single application of each agent. All bacterial treatments significantly decreased damping‐off of tomato plants. The best control was obtained when BY was added 2 days after RB14‐C. In this treatment plant protection was significantly higher than that obtained in other combined applications and obtained by single strains, except BY added to the soil 4 days before seed planting. The lowest suppression indicated BY introduced to the soil before seed planting. RB14‐C only slightly decreased number of R. solani in the soil. In contrast, BY drastically reduced population of the pathogen. However, there was not a clear relation between decrease of pathogen density in soil and the rate of plant infection. The results show that combination of B. subtilis RB14‐C with B. cepacia BY can lead to greater damping‐off suppression than biocontrol exhibited by these strains used separately, but the effect of combining bacterial agents was clearly related to the order in which both agents were introduced.     

A two‐component Kdo hydrolase in the inner membrane of Francisella novicida

Lipid A coats the outer surface of the outer membrane of Gram‐negative bacteria. In Francisella tularensis subspecies novicida lipid A is present either as the covalently attached anchor of lipopolysaccharide (LPS) or as free lipid A. The lipid A moiety of Francisella LPS is linked to the core domain by a single 2‐keto‐3‐deoxy‐D‐manno‐octulosonic acid (Kdo) residue. F. novicida KdtA is bi‐functional, but F. novicida contains a membrane‐bound Kdo hydrolase that removes the outer Kdo unit. The hydrolase consists of two proteins (KdoH1 and KdoH2), which are expressed from adjacent, co‐transcribed genes. KdoH1 (related to sialidases) has a single predicted N‐terminal transmembrane segment. KdoH2 contains 7 putative transmembrane sequences. Neither protein alone catalyses Kdo cleavage when expressed in E. coli. Activity requires simultaneous expression of both proteins or mixing of membranes from strains expressing the individual proteins under in vitro assay conditions in the presence of non‐ionic detergent. In E. coli expressing KdoH1 and KdoH2, hydrolase activity is localized in the inner membrane. WBB06, a heptose‐deficient E. coli mutant that makes Kdo₂‐lipid A as its sole LPS, accumulates Kdo‐lipid A when expressing the both hydrolase components, and 1‐dephospho‐Kdo‐lipid A when expressing both the hydrolase and the Francisella lipid A 1‐phosphatase (LpxE).     

Determinants that govern the recognition and uptake of Escherichia coli O157 : H7 by Acanthamoeba castellanii

Predation by phagocytic predators is a major source of bacterial mortality. The first steps in protozoan predation are recognition and consumption of their bacterial prey. However, the precise mechanisms governing prey recognition and phagocytosis by protists, and the identities of the molecular and cellular factors involved in these processes are, as yet, ill‐characterized. Here, we show that that the ability of the phagocytic bacterivorous amoebae, Acanthamoeba castellanii, to recognize and internalize Escherichia coli, a bacterial prey, varies with LPS structure and composition. The presence of an O‐antigen carbohydrate is not required for uptake of E. coli by A. castellanii. However, O1‐antigen types, not O157 O‐antigen types, inhibit recognition and uptake of bacteria by amoeba. This finding implies that O‐antigen may function as an antipredator defence molecule. Recognition and uptake of E. coli by A. castellanii is mediated by the interaction of mannose‐binding protein located on amoebae's surface with LPS carbohydrate. Phagocytic mammalian cells also use mannose‐binding lectins to recognize and/or mediate phagocytosis of E. coli. Nonetheless, A. castellanii's mannose binding protein apparently displays no sequence similarity with any known metazoan mannose binding protein. Hence, the similarity in bacterial recognition mechanisms of amoebae and mammalian phagocytes may be a result of convergent evolution.     

De‐O‐acetylation of peptidoglycan regulates glycan chain extension and affects in vivo survival of Neisseria meningitidis

Peptidoglycan O‐acetylation is a modification found in many bacteria. In Gram‐positive pathogens, it contributes to virulence by conferring resistance to host lysozyme. However, in Gram‐negative pathogens, its contribution to physiology and virulence is unknown. We examined the contribution of patA, patB and ape1 to peptidoglycan O‐acetylation in the major human pathogen Neisseria meningitidis (Nm). Using genetic expression of all possible combinations of the three genes in Escherichia coli and Nm, we confirmed that PatA and PatB were required for PG O‐acetylation, while ApeI removed the O‐acetyl group. ApeI was active on all O‐acetylated muropeptides produced by PatA and PatB during heterologous expression in E. coli and was also active on several PG structures in vitro. Interestingly, in Nm, ApeI was found to preferentially de‐O‐acetylate muropeptides with tripeptide stems (GM3), suggesting that its activity is highly regulated. Accordingly, de‐O‐acetylation of GM3 regulated glycan chain elongation and cell size. Additionally, the virulence of Nm lacking ApeI was drastically reduced suggesting that regulation of glycan chain length by O‐acetylation contributes to bacterial fitness in the host. Altogether, our results suggest that ApeI represents an attractive target for new drug development.     

A novel cysteine‐free venom peptide with strong antimicrobial activity against antibiotics‐resistant pathogens from the scorpion Opistophthalmus glabrifrons

Antibiotic‐resistant bacteria, such as methicillin‐resistant Staphylococcus aureus and vancomycin‐resistant Enterococcus, pose serious threat to human health. The outbreak of antibiotic‐resistant pathogens in recent years emphasizes once again the urgent need for the development of new antimicrobial agents. Here, we discovered a novel antimicrobial peptide from the scorpion Opistophthalmus glabrifrons, which was referred to as Opisin. Opisin consists of 19 amino acid residues without disulfide bridges. It is a cationic, amphipathic, and α‐helical molecule. Protein sequence homology search revealed that Opisin shares 42.1–5.3% sequence identities to the 17/18‐mer antimicrobial peptides from scorpions. Antimicrobial assay showed that Opisin is able to potently inhibit the growth of the tested Gram‐positive bacteria with the minimal inhibitory concentration (MIC) values of 4.0–10.0 μM; in contrast, it possesses much lower activity against the tested Gram‐negative bacteria and a fungus. It is interesting to see that Opisin is able to strongly inhibit the growth of methicillin‐ and vancomycin‐resistant pathogens with the MICs ranging from 2.0 to 4.0 μM and from 4.0 to 6.0 μM, respectively. We found that at a concentration of 5 × MIC, Opisin completely killed all the cultured methicillin‐resistant Staphylococcus aureus. These results suggest that Opisin is a promising therapeutic candidate for the treatment of the antibiotic‐resistant bacterial infections. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.