A muramidase from Acremonium alcalophilum hydrolyse peptidoglycan found in the gastrointestinal tract of broiler chickens

This study evaluates peptidoglycan hydrolysis by a microbial muramidase from the fungus Acremonium alcalophilum in vitro and in the gastrointestinal tract of broiler chickens. Peptidoglycan used for in vitro studies was derived from 5 gram-positive chicken gut isolate type strains. In vitro peptidoglycan hydrolysis was studied by three approaches: a) helium ion microscopy to identify visual phenotypes of hydrolysis b) reducing end assay to quantify solubilization of peptidoglycan fragments and c) mass spectroscopy to estimate relative abundances of soluble substrates and reaction products. Visual effects of peptidoglycan hydrolysis could be observed by helium ion microscopy and the increase in abundance of soluble peptidoglycan due to hydrolysis was quantified by a reducing end assay. Mass spectroscopy confirmed the release of hydrolysis products and identified muropeptides from the five different peptidoglycan sources. Peptidoglycan hydrolysis in chicken crop, jejunum and caecum samples was measured by quantifying the total and soluble muramic acid content. A significant increase in the proportion of the soluble muramic acid was observed in all three segments upon inclusion of the microbial muramidase in the diet.

Engineered Lysins With Customized Lytic Activities Against Enterococci and Staphylococci

The emergence of multidrug-resistant bacteria has made minor bacterial infections incurable with many existing antibiotics. Lysins are phage-encoded peptidoglycan hydrolases that have demonstrated therapeutic potential as a novel class of antimicrobials. The modular architecture of lysins enables the functional domains – catalytic domain (CD) and cell wall binding domain (CBD) – to be shuffled to create novel lysins. The CD is classically thought to be only involved in peptidoglycan hydrolysis whereas the CBD dictates the lytic spectrum of a lysin. While there are many studies that extended the lytic spectrum of a lysin by domain swapping, few have managed to introduce species specificity in a chimeric lysin. In this work, we constructed two chimeric lysins by swapping the CBDs of two parent lysins with different lytic spectra against enterococci and staphylococci. We showed that these chimeric lysins exhibited customized lytic spectra distinct from the parent lysins. Notably, the chimeric lysin P10N-V12C, which comprises a narrow-spectrum CD fused with a broad-spectrum CBD, displayed species specificity not lysing Enterococcus faecium while targeting Enterococcus faecalis and staphylococci. Such species specificity can be attributed to the narrow-spectrum CD of the chimeric lysin. Using flow cytometry and confocal microscopy, we found that the E. faecium cells that were treated with P10N-V12C are less viable with compromised membranes yet remained morphologically intact. Our results suggest that while the CBD is a major determinant of the lytic spectrum of a lysin, the CD is also responsible in the composition of the final lytic spectrum, especially when it pertains to species-specificity.

New insights in the coordinated amidase and glucosaminidase activity of the major autolysin (Atl) in Staphylococcus aureus

After bacterial cell division, the daughter cells are still covalently interlinked by the peptidoglycan network which is resolved by specific hydrolases (autolysins) to release the daughter cells. In staphylococci, the major autolysin (Atl) with its two domain enzymes, N-acetylmuramyl-L-alanine amidase (AmiA) and β-N-acetylglucosaminidase (GlcA), resolves the peptidoglycan to release the daughter cells. Internal deletions in each of the enzyme domains revealed defined morphological alterations such as cell cluster formation in ΔamiA, ΔglcA and Δatl, and asymmetric cell division in the ΔglcA. A most important finding was that GlcA activity requires the prior removal of the stem peptide by AmiA for its activity thus the naked glycan strand is its substrate. Furthermore, GlcA is not an endo-β-N-acetylglucosaminidase but an exo-enzyme that cuts the glycan backbone to disaccharides independent of its O-acetylation modification. Our results shed new light into the sequential peptidoglycan hydrolysis by AmiA and GlcA during cell division in staphylococci.

Teichoic Acid Polymers Affect Expression and Localization of dl-Endopeptidase LytE Required for Lateral Cell Wall Hydrolysis in Bacillus subtilis

ABSTRACTInBacillus subtilis, thedl-endopeptidase LytE is responsible for lateral peptidoglycan hydrolysis during cell elongation. We found that σI-dependent transcription oflytEis considerably enhanced in a strain with a mutation inltaS, which encodes a major lipoteichoic acid (LTA) synthase. Similar enhancements were observed in mutants that affect the glycolipid anchor and wall teichoic acid (WTA) synthetic pathways. Immunofluorescence microscopy revealed that the LytE foci were considerably increased in these mutants. The localization patterns of LytE on the sidewalls appeared to be helix-like in LTA-defective or WTA-reduced cells and evenly distributed on WTA-depleted or -defective cell surfaces. These results strongly suggested that LTA and WTA affect both σI-dependent expression and localization of LytE. Interestingly, increased LytE localization along the sidewall in theltaSmutant largely occurred in an MreBH-independent manner. Moreover, we found that cell surface decorations with LTA and WTA are gradually reduced at increased culture temperatures and that LTA rather than WTA on the cell surface is reduced at high temperatures. In contrast, the amount of LytE on the cell surface gradually increased under heat stress conditions. Taken together, these results indicated that reductions in these anionic polymers at high temperatures might give rise to increases in SigI-dependent expression and cell surface localization of LytE at high temperatures.IMPORTANCEThe bacterial cell wall is required for maintaining cell shape and bearing environmental stresses. The Gram-positive cell wall consists of mesh-like peptidoglycan and covalently linked wall teichoic acid and lipoteichoic acid polymers. It is important to determine if these anionic polymers are required for proliferation and environmental adaptation. Here, we demonstrated that these polymers affect the expression and localization of a peptidoglycan hydrolase LytE required for lateral cell wall elongation. Moreover, we found that cell surface decorations with teichoic acid polymers are substantially decreased at high temperatures and that the peptidoglycan hydrolase is consequently increased. These findings suggest that teichoic acid polymers control lateral peptidoglycan hydrolysis by LytE, and bacteria drastically change their cell wall content to adapt to their environment.

β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus USA300 Is Increased by Inactivation of the ClpXP Protease

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) has acquired themecAgene encoding a peptidoglycan transpeptidase, penicillin binding protein 2a (PBP2a), which has decreased affinity for β-lactams. Quickly spreading and highly virulent community-acquired (CA) MRSA strains recently emerged as a frequent cause of infection in individuals without exposure to the health care system. In this study, we found that the inactivation of the components of the ClpXP protease substantially increased the β-lactam resistance level of a CA-MRSA USA300 strain, suggesting that the proteolytic activity of ClpXP controls one or more pathways modulating β-lactam resistance. These pathways do not involve the control ofmecAexpression, as the cellular levels of PBP2a were unaltered in theclpmutants. An analysis of the cell envelope properties of theclpXandclpPmutants revealed a number of distinct phenotypes that may contribute to the enhanced β-lactam tolerance. Both mutants displayed significantly thicker cell walls, increased peptidoglycan cross-linking, and altered composition of monomeric muropeptide species compared to those of the wild types. Moreover, changes in Sle1-mediated peptidoglycan hydrolysis and altered processing of the major autolysin Atl were observed in theclpmutants. In conclusion, the results presented here point to an important role for the ClpXP protease in controlling cell wall metabolism and add novel insights into the molecular factors that determine strain-dependent β-lactam resistance.