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Microbiology Spectrum Jun 2024Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread...
Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpA) protein from . Unlike other enzymes within the M23 family, EnpA demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpA fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpA results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.
Topics: Osmolar Concentration; Enterococcus faecalis; Humans; Bacterial Proteins; Protein Engineering; Anti-Bacterial Agents; Animals; N-Acetylmuramoyl-L-alanine Amidase; Cattle; Recombinant Fusion Proteins; Cell Wall; Catalytic Domain; Drug Resistance, Bacterial
PubMed: 38695664
DOI: 10.1128/spectrum.03546-23 -
Antimicrobial Agents and Chemotherapy May 2024The dual β-lactam approach has been successfully applied to overcome target redundancy in nontuberculous mycobacteria. Surprisingly, this approach has not been...
The dual β-lactam approach has been successfully applied to overcome target redundancy in nontuberculous mycobacteria. Surprisingly, this approach has not been leveraged for despite the high conservation of peptidoglycan synthesis. Through a comprehensive screen of oral β-lactam pairs, we have discovered that cefuroxime strongly potentiates the bactericidal activity of tebipenem and sulopenem-advanced clinical candidates-and amoxicillin, at concentrations achieved clinically. β-lactam pairs thus have the potential to reduce TB treatment duration.
PubMed: 38690896
DOI: 10.1128/aac.00034-24 -
PLoS Biology Apr 2024Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These...
Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These lipid-linked precursors are then flipped across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. Despite its essential role in envelope biogenesis, UndP is maintained at low levels in the cytoplasmic membrane. The mechanisms by which bacteria distribute this limited resource among competing pathways is currently unknown. Here, we report that the Bacillus subtilis transcription factor SigM and its membrane-anchored anti-sigma factor respond to UndP levels and prioritize its use for the synthesis of the only essential surface polymer, the cell wall. Antibiotics that target virtually every step in PG synthesis activate SigM-directed gene expression, confounding identification of the signal and the logic of this stress-response pathway. Through systematic analyses, we discovered 2 distinct responses to these antibiotics. Drugs that trap UndP, UndP-linked intermediates, or precursors trigger SigM release from the membrane in <2 min, rapidly activating transcription. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP induce SigM more slowly. We show that activation in the latter case can be explained by the accumulation of UndP-linked wall teichoic acid precursors that cannot be transferred to the PG due to the block in its synthesis. Furthermore, we report that reduction in UndP synthesis rapidly induces SigM, while increasing UndP production can dampen the SigM response. Finally, we show that SigM becomes essential for viability when the availability of UndP is restricted. Altogether, our data support a model in which the SigM pathway functions to homeostatically control UndP usage. When UndP levels are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of UndP are reduced, SigM activates genes that increase flux through the PG synthesis pathway, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways. Analogous homeostatic pathways that prioritize UndP usage are likely to be common in bacteria.
Topics: Cell Wall; Bacillus subtilis; Signal Transduction; Bacterial Proteins; Peptidoglycan; Polyisoprenyl Phosphates; Anti-Bacterial Agents; Gene Expression Regulation, Bacterial; Cell Membrane
PubMed: 38683856
DOI: 10.1371/journal.pbio.3002589 -
The Journal of Biological Chemistry Jun 2024The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall...
The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.
Topics: Cysteine Endopeptidases; Bacterial Proteins; Aminoacyltransferases; Fimbriae, Bacterial; Fimbriae Proteins; Crystallography, X-Ray; Actinomyces; Substrate Specificity; Models, Molecular
PubMed: 38679328
DOI: 10.1016/j.jbc.2024.107329 -
Microorganisms Mar 2024Peptidoglycan hydrolases are enzymes responsible for breaking the peptidoglycan present in the bacterial cell wall, facilitating cell growth, cell division and...
Peptidoglycan hydrolases are enzymes responsible for breaking the peptidoglycan present in the bacterial cell wall, facilitating cell growth, cell division and peptidoglycan turnover. subsp. (), the causal agent of citrus canker, encodes an M23 peptidase EnvC homolog. EnvC is a LytM factor essential for cleaving the septal peptidoglycan, thereby facilitating the separation of daughter cells. In this study, the investigation focused on EnvC contribution to the virulence and cell separation of . It was observed that disruption of the gene (Δ) led to a reduction in virulence. Upon inoculation into leaves of Rangpur lime ( Osbeck), the Δ exhibited a delayed onset of citrus canker symptoms compared with the wild-type . Mutant complementation restored the wild-type phenotype. Sub-cellular localization confirmed that EnvC is a periplasmic protein. Moreover, the Δ mutant exhibited elongated cells, indicating a defect in cell division. These findings support the role of EnvC in the regulation of cell wall organization, cell division, and they clarify the role of this peptidase in virulence.
PubMed: 38674634
DOI: 10.3390/microorganisms12040691 -
International Journal of Molecular... Apr 2024We explored the metabolic integration of and its obligate endosymbiont by the transcriptomic analysis of the fat body of quasi-aposymbiotic cockroaches, where the... (Comparative Study)
Comparative Study
Comparative Transcriptomics of Fat Bodies between Symbiotic and Quasi-Aposymbiotic Adult Females of with Emphasis on the Metabolic Integration with Its Endosymbiont and Its Immune System.
We explored the metabolic integration of and its obligate endosymbiont by the transcriptomic analysis of the fat body of quasi-aposymbiotic cockroaches, where the endosymbionts were almost entirely removed with rifampicin. Fat bodies from quasi-aposymbiotic insects displayed large differences in gene expression compared to controls. In quasi-aposymbionts, the metabolism of phenylalanine and tyrosine involved in cuticle sclerotization and pigmentation increased drastically to compensate for the deficiency in the biosynthesis of these amino acids by the endosymbionts. On the other hand, the uricolytic pathway and the biosynthesis of uric acid were severely decreased, probably because the reduced population of endosymbionts was unable to metabolize urea to ammonia. Metabolite transporters that could be involved in the endosymbiosis process were identified. Immune system and antimicrobial peptide (AMP) gene expression was also reduced in quasi-aposymbionts, genes encoding peptidoglycan-recognition proteins, which may provide clues for the maintenance of the symbiotic relationship, as well as three AMP genes whose involvement in the symbiotic relationship will require additional analysis. Finally, a search for AMP-like factors that could be involved in controlling the endosymbiont identified two orphan genes encoding proteins smaller than 200 amino acids underexpressed in quasi-aposymbionts, suggesting a role in the host-endosymbiont relationship.
Topics: Symbiosis; Animals; Fat Body; Female; Transcriptome; Gene Expression Profiling; Immune System; Bacteroidetes; Antimicrobial Peptides
PubMed: 38673813
DOI: 10.3390/ijms25084228 -
Microbiome Apr 2024The deep sea represents the largest marine ecosystem, driving global-scale biogeochemical cycles. Microorganisms are the most abundant biological entities and play a...
BACKGROUND
The deep sea represents the largest marine ecosystem, driving global-scale biogeochemical cycles. Microorganisms are the most abundant biological entities and play a vital role in the cycling of organic matter in such ecosystems. The primary food source for abyssal biota is the sedimentation of particulate organic polymers. However, our knowledge of the specific biopolymers available to deep-sea microbes remains largely incomplete. One crucial rate-limiting step in organic matter cycling is the depolymerization of particulate organic polymers facilitated by extracellular enzymes (EEs). Therefore, the investigation of active EEs and the microbes responsible for their production is a top priority to better understand the key nutrient sources for deep-sea microbes.
RESULTS
In this study, we conducted analyses of extracellular enzymatic activities (EEAs), metagenomics, and metatranscriptomics from seawater samples of 50-9305 m from the Mariana Trench. While a diverse array of microbial groups was identified throughout the water column, only a few exhibited high levels of transcriptional activities. Notably, microbial populations actively transcribing EE genes involved in biopolymer processing in the abyssopelagic (4700 m) and hadopelagic zones (9305 m) were primarily associated with the class Actinobacteria. These microbes actively transcribed genes coding for enzymes such as cutinase, laccase, and xyloglucanase which are capable of degrading phytoplankton polysaccharides as well as GH23 peptidoglycan lyases and M23 peptidases which have the capacity to break down peptidoglycan. Consequently, corresponding enzyme activities including glycosidases, esterase, and peptidases can be detected in the deep ocean. Furthermore, cell-specific EEAs increased at 9305 m compared to 4700 m, indicating extracellular enzymes play a more significant role in nutrient cycling in the deeper regions of the Mariana Trench.
CONCLUSIONS
Transcriptomic analyses have shed light on the predominant microbial population actively participating in organic matter cycling in the deep-sea environment of the Mariana Trench. The categories of active EEs suggest that the complex phytoplankton polysaccharides (e.g., cutin, lignin, and hemicellulose) and microbial peptidoglycans serve as the primary nutrient sources available to deep-sea microbes. The high cell-specific EEA observed in the hadal zone underscores the robust polymer-degrading capacities of hadal microbes even in the face of the challenging conditions they encounter in this extreme environment. These findings provide valuable new insights into the sources of nutrition, the key microbes, and the EEs crucial for biopolymer degradation in the deep seawater of the Mariana Trench. Video Abstract.
Topics: Polysaccharides; Seawater; Phytoplankton; Nutrients; Peptidoglycan; Bacteria; Metagenomics; Microbiota
PubMed: 38664737
DOI: 10.1186/s40168-024-01789-x -
Frontiers in Plant Science 2024Autolysins are endogenous cell wall degrading enzymes (CWDEs) in bacteria that remodel the peptidoglycan layer of its own cell wall. In the genome, at least 35...
Autolysins are endogenous cell wall degrading enzymes (CWDEs) in bacteria that remodel the peptidoglycan layer of its own cell wall. In the genome, at least 35 autolysin genes have been identified. However, the study of their roles in bacterial physiology has been hampered by their complexity and functional redundancy. GLB191 is an effective biocontrol strain against grape downy mildew disease, the biocontrol effect of which results from both direct effect against the pathogen and stimulation of the plant defense. In this study, we show that the autolysin -acetylglucosaminidase LytD, a major autolysin of vegetative growth in , plays an important role in its biocontrol activity against grape downy mildew. Disruption of resulted in reduced suppression of the pathogen and stimulation of the plant defense. LytD is also shown to affect the biofilm formation and colonization of on grape leaves. This is the first report that demonstrates the role of an endogenous CWDE in suppressing plant disease infection of a biological control microorganism. These findings not only expand our knowledge on the biological function of autolysins but also provide a new target to promote the biocontrol activity of .
PubMed: 38660441
DOI: 10.3389/fpls.2024.1381018 -
Current Opinion in Microbiology Jun 2024Members of the order Mycobacteriales are distinguished by a characteristic diderm cell envelope, setting them apart from other Actinobacteria species. In addition to the... (Review)
Review
Members of the order Mycobacteriales are distinguished by a characteristic diderm cell envelope, setting them apart from other Actinobacteria species. In addition to the conventional peptidoglycan cell wall, these organisms feature an extra polysaccharide polymer composed of arabinose and galactose, termed arabinogalactan. The nonreducing ends of arabinose are covalently linked to mycolic acids (MAs), forming the immobile inner leaflet of the highly hydrophobic MA membrane. The contiguous outer leaflet of the MA membrane comprises trehalose mycolates and various lipid species. Similar to all actinobacteria, Mycobacteriales exhibit apical growth, facilitated by a polar localized elongasome complex. A septal cell envelope synthesis machinery, the divisome, builds instead of the cell wall structures during cytokinesis. In recent years, a growing body of knowledge has emerged regarding the cell wall synthesizing complexes of Mycobacteriales., focusing particularly on three model species: Corynebacterium glutamicum, Mycobacterium smegmatis, and Mycobacterium tuberculosis.
Topics: Cell Wall; Mycolic Acids; Galactans; Peptidoglycan; Mycobacterium tuberculosis; Corynebacterium glutamicum; Mycobacterium smegmatis; Arabinose; Bacterial Proteins
PubMed: 38653035
DOI: 10.1016/j.mib.2024.102478 -
ELife Apr 2024Mechanisms by which (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB)...
Mechanisms by which (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. and experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.
Topics: Animals; Peptidoglycan; Mice; BCG Vaccine; Mycobacterium tuberculosis; Tuberculosis; Humans; Mice, Inbred C57BL; Macrophages; Female; Nod1 Signaling Adaptor Protein; Disease Models, Animal; Bacterial Proteins
PubMed: 38639995
DOI: 10.7554/eLife.89157