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ELife Jun 2024is a microbiota species in humans that can modulate host immunity (Griffin and Hang, 2022), but has also acquired antibiotic resistance and is a major cause of...
is a microbiota species in humans that can modulate host immunity (Griffin and Hang, 2022), but has also acquired antibiotic resistance and is a major cause of hospital-associated infections (Van Tyne and Gilmore, 2014). Notably, diverse strains of produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity (Rangan et al., 2016; Pedicord et al., 2016; Kim et al., 2019) and immune checkpoint inhibitor antitumor activity (Griffin et al., 2021). However, the functions of SagA in were unknown. Here, we report that deletion of impaired growth and resulted in bulged and clustered enterococci due to defective peptidoglycan cleavage and cell separation. Moreover, Δ showed increased antibiotic sensitivity, yielded lower levels of active muropeptides, displayed reduced activation of the peptidoglycan pattern-recognition receptor NOD2, and failed to promote cancer immunotherapy. Importantly, the plasmid-based expression of SagA, but not its catalytically inactive mutant, restored Δ growth, production of active muropeptides, and NOD2 activation. SagA is, therefore, essential for growth, stress resistance, and activation of host immunity.
Topics: Enterococcus faecium; N-Acetylmuramoyl-L-alanine Amidase; Immune Checkpoint Inhibitors; Humans; Animals; Bacterial Proteins; Peptidoglycan; Mice
PubMed: 38857064
DOI: 10.7554/eLife.95297 -
Applied and Environmental Microbiology Jun 2024Lytic enzymes, or lysins for short, break down peptidoglycan and interrupt the continuity of the cell wall, which, in turn, causes osmotic lysis of the bacterium. Their... (Review)
Review
Lytic enzymes, or lysins for short, break down peptidoglycan and interrupt the continuity of the cell wall, which, in turn, causes osmotic lysis of the bacterium. Their ability to destroy bacteria from without makes them promising antimicrobial agents that can be used as alternatives or supplements to antibiotics. In this paper, we briefly summarize basic terms and concepts used to describe lysin sequences and delineate major lysin groups. More importantly, we describe the domain repertoire found in lysins and critically review bioinformatic tools or databases which are used in studies of these enzymes (with particular emphasis on the repositories of Hidden Markov models). Finally, we present a novel comprehensive, meticulously curated set of lysin-related family and domain models, sort them into clusters that reflect major families, and demonstrate that the selected models can be used to efficiently search for new lysins.
PubMed: 38842338
DOI: 10.1128/aem.02361-23 -
Frontiers in Microbiology 2024Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant...
Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant effect. It has been widely used in recent years in the development of vaccines. Existing anchoring proteins for BLP surfaces are few in number, so screening and characterization of new anchoring proteins are necessary. In this research, we created the OACD (C-terminal domain of outer membrane protein A) to serve as an anchoring protein on the surface of BLP produced by the immunomodulatory bacteria 23017. We used red fluorescent protein (RFP) to demonstrate the novel surface display system's effectiveness, stability, and ability to be adapted to a wide range of lactic acid bacteria. Furthermore, this study employed this surface display method to develop a novel vaccine (called COB17) by using the multi-epitope antigen of as the model antigen. The vaccine can induce more than 50% protection rate against type A challenge in mice immunized with a single dose and has been tested through three routes. The vaccine yields protection rates of 75% for subcutaneous, 50% for intranasal, and 75% for oral immunization. Additionally, it elicits a strong mucosal immune response, markedly increasing levels of specific IgG, high-affinity IgG, specific IgA, and SIgA antibodies. Additionally, we used protein anchors (PA) and OACD simultaneous to show several antigens on the BLP surface. The discovery of novel BLP anchoring proteins may expand the possibilities for creating mucosal immunity subunit vaccines. Additionally, it may work in concert with PA to provide concepts for the creation of multivalent or multiple vaccines that may be used in clinical practice to treat complex illnesses.
PubMed: 38841059
DOI: 10.3389/fmicb.2024.1395837 -
MBio Jun 2024expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here,...
expresses a type IV protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria and does so partly by secreting the effector TfcB. Here, we report the structure of TfcB, comprising an N-terminal domain similar to the catalytic domain of glycosyl hydrolase (GH-19) chitinases and a C-terminal domain for recognition and translocation by the T4SS. Utilizing a two-hybrid assay to measure effector interactions with the T4SS coupling protein VirD4, we documented the existence of five more T4SS substrates. One of these was protein 20845, an annotated nuclease. A mutant lacking the gene for 20845 was impaired for killing , , and . Moreover, the cloned 20845 gene conferred robust toxicity, with the recombinant being rescued when 20845 was co-expressed with its cognate immunity protein. The 20845 effector was an 899 amino-acid protein, comprised of a GHH-nuclease domain in its N-terminus, a large central region of indeterminant function, and a C-terminus for secretion. Engineered variants of the 20845 gene that had mutations in the predicted catalytic site did not impede , indicating that the antibacterial effect of 20845 involves its nuclease activity. Using flow cytometry with DNA staining, we determined that 20845, but not its mutant variants, confers a loss in DNA content of target bacteria. Database searches revealed that uncharacterized homologs of 20845 occur within a range of bacteria. These data indicate that the T4SS promotes interbacterial competition through the action of multiple toxic effectors, including a potent, novel DNase.IMPORTANCE is a multi-drug-resistant, Gram-negative bacterium that is an emerging pathogen of humans. Patients with cystic fibrosis are particularly susceptible to infection. In hospital water systems and various types of infections, co-exists with other bacteria, including other pathogens such as . We previously demonstrated that has a functional VirB/D4 type VI protein secretion system (T4SS) that promotes contact-dependent killing of other bacteria. Since most work on antibacterial systems involves the type VI secretion system, this observation remains noteworthy. Moreover, currently stands alone as a model for a human pathogen expressing an antibacterial T4SS. Using biochemical, genetic, and cell biological approaches, we now report both the discovery of a novel antibacterial nuclease (TfdA) and the first structural determination of a bactericidal T4SS effector (TfcB).
PubMed: 38832773
DOI: 10.1128/mbio.01198-24 -
Frontiers in Pharmacology 2024Antibiotic-resistant bacteria are rapidly emerging, and the increasing prevalence of multidrug-resistant (MDR) poses a severe threat to humans and healthcare... (Review)
Review
Endolysins: a new antimicrobial agent against antimicrobial resistance. Strategies and opportunities in overcoming the challenges of endolysins against Gram-negative bacteria.
Antibiotic-resistant bacteria are rapidly emerging, and the increasing prevalence of multidrug-resistant (MDR) poses a severe threat to humans and healthcare organizations, due to the lack of innovative antibacterial drugs. Endolysins, which are peptidoglycan hydrolases encoded by a bacteriophage, are a promising new family of antimicrobials. Endolysins have been demonstrated as an effective therapeutic agent against bacterial infections of and many other Gram-positive and Gram-negative bacteria. Endolysin research has progressed from basic characterization to sophisticated protein engineering methodologies, including advanced preclinical and clinical testing. Endolysin are therapeutic agent that shows antimicrobial properties against bacterial infections caused by drug-resistant Gram-negative bacteria, there are still barriers to their implementation in clinical settings, such as safety concerns with outer membrane permeabilizers (OMP) use, low efficiency against stationary phase bacteria, and stability issues. The application of protein engineering and formulation techniques to improve enzyme stability, as well as combination therapy with other types of antibacterial drugs to optimize their medicinal value, have been reviewed as well. In this review, we summarize the clinical development of endolysin and its challenges and approaches for bringing endolysin therapies to the clinic. This review also discusses the different applications of endolysins.
PubMed: 38831886
DOI: 10.3389/fphar.2024.1385261 -
PLoS Genetics Jun 2024The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer...
The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer layer of mycolic acids called the mycomembrane (MM) and a cell wall composed of peptidoglycan and arabinogalactan. This envelope architecture is unique among bacteria and contributes significantly to the virulence of pathogenic Mycobacteriales like Mycobacterium tuberculosis. Characterization of pathways that govern envelope biogenesis in these organisms is therefore critical in understanding their biology and for identifying new antibiotic targets. To better understand MM biogenesis, we developed a cell sorting-based screen for mutants defective in the surface exposure of a porin normally embedded in the MM of the model organism Corynebacterium glutamicum. The results revealed a requirement for the conserved σD envelope stress response in porin export and identified MarP as the site-1 protease, respectively, that activate the response by cleaving the membrane-embedded anti-sigma factor. A reporter system revealed that the σD pathway responds to defects in mycolic acid and arabinogalactan biosynthesis, suggesting that the stress response has the unusual property of being induced by activating signals that arise from defects in the assembly of two distinct envelope layers. Our results thus provide new insights into how C. glutamicum and related bacteria monitor envelope integrity and suggest a potential role for members of the σD regulon in protein export to the MM.
Topics: Cell Wall; Corynebacterium glutamicum; Mycolic Acids; Sigma Factor; Cell Membrane; Stress, Physiological; Porins; Bacterial Proteins; Galactans; Gene Expression Regulation, Bacterial; Peptidoglycan
PubMed: 38829907
DOI: 10.1371/journal.pgen.1011127 -
BMC Microbiology May 2024Urinary tract infections (UTIs) are common bacterial infections, primarily caused by uropathogenic Escherichia coli (UPEC), leading to significant health issues and...
BACKGROUND
Urinary tract infections (UTIs) are common bacterial infections, primarily caused by uropathogenic Escherichia coli (UPEC), leading to significant health issues and economic burden. Although antibiotics have been effective in treating UPEC infections, the rise of antibiotic-resistant strains hinders their efficacy. Hence, identifying novel bacterial targets for new antimicrobial approaches is crucial. Bacterial factors required for maintaining the full virulence of UPEC are the potential target. MepM, an endopeptidase in E. coli, is involved in the biogenesis of peptidoglycan, a major structure of bacterial envelope. Given that the bacterial envelope confronts the hostile host environment during infections, MepM's function could be crucial for UPEC's virulence. This study aims to explore the role of MepM in UPEC pathogenesis.
RESULTS
MepM deficiency significantly impacted UPEC's survival in urine and within macrophages. Moreover, the deficiency hindered the bacillary-to-filamentous shape switch which is known for aiding UPEC in evading phagocytosis during infections. Additionally, UPEC motility was downregulated due to MepM deficiency. As a result, the mepM mutant displayed notably reduced fitness in causing UTIs in the mouse model compared to wild-type UPEC.
CONCLUSIONS
This study provides the first evidence of the vital role of peptidoglycan endopeptidase MepM in UPEC's full virulence for causing UTIs. MepM's contribution to UPEC pathogenesis may stem from its critical role in maintaining the ability to resist urine- and immune cell-mediated killing, facilitating the morphological switch, and sustaining motility. Thus, MepM is a promising candidate target for novel antimicrobial strategies.
Topics: Urinary Tract Infections; Uropathogenic Escherichia coli; Animals; Mice; Escherichia coli Infections; Virulence; Endopeptidases; Escherichia coli Proteins; Female; Peptidoglycan; Macrophages; Humans; Disease Models, Animal
PubMed: 38816687
DOI: 10.1186/s12866-024-03290-9 -
PLoS Biology May 2024The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive...
The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria.
Topics: Peptidoglycan; Cell Division; Hydrolysis; Escherichia coli Proteins; Escherichia coli; Adenosine Triphosphate; Cryoelectron Microscopy; Cell Wall; Protein Conformation; Models, Molecular; N-Acetylmuramoyl-L-alanine Amidase; Bacterial Outer Membrane Proteins; ATP-Binding Cassette Transporters; Cystic Fibrosis Transmembrane Conductance Regulator; Lipoproteins; Cell Cycle Proteins
PubMed: 38814940
DOI: 10.1371/journal.pbio.3002628 -
International Journal of Antimicrobial... May 2024Clostridioides difficile has emerged as a major cause of life-threatening diarrheal disease. Conventional antibiotics used in current standards of care exacerbate the...
OBJECTIVES
Clostridioides difficile has emerged as a major cause of life-threatening diarrheal disease. Conventional antibiotics used in current standards of care exacerbate the emergence of antibiotic-resistant strains and pose a risk of recurrent C. difficile infection (CDI). Thus, there is an urgent need for alternative therapeutics that selectively eliminate C. difficile without disturbing the commensal microbiota. This study aimed to explore the potential of endolysins as an alternative therapeutic agent to antibiotics. Endolysin is a bacteriophage-derived peptidoglycan hydrolase that aids in the release of phage progeny during the final stage of infection.
METHODS
In order to exploit endolysin as a therapeutic agent against CDI, the bactericidal activity of 23 putative endolysins was compared and ΦCD27 endolysin CD27L was selected and modified to CD27L_EAD by cleaving the cell-wall binding domain of CD27L.
RESULTS
CD27L_EAD exhibited greater bacteriolytic activity than CD27L and its activity was stable over a wide range of salt concentrations and pH conditions. CD27L_EAD was added to a co-culture of human gut microbiota with C. difficile and the bacterial community structure was analyzed. CD27L_EAD did not impair the richness and diversity of the bacterial population but remarkably attenuated the abundance of C. difficile. Furthermore, the co-administration of vancomycin exerted synergistic bactericidal activity against C. difficile. β-diversity analysis revealed that CD27L_EAD did not significantly disturb the composition of the microbial community, whereas the abundance of some species belonging to the family Lachnospiraceae decreased after CD27L_EAD treatment.
CONCLUSIONS
This study provides insights into endolysin as a prospective therapeutic agent for the treatment of CDI without damaging the normal gut microbiota.
PubMed: 38810936
DOI: 10.1016/j.ijantimicag.2024.107222 -
Scientific Reports May 2024Recently, Lactobacillus johnsonii N6.2-derived extracellular vesicles (EVs) were shown to reduce apoptosis in human beta cell lines and stimulate insulin secretion in...
Recently, Lactobacillus johnsonii N6.2-derived extracellular vesicles (EVs) were shown to reduce apoptosis in human beta cell lines and stimulate insulin secretion in human islets. Our goal was to identify a physiologically relevant environmental condition that induces a hypervesiculation phenotype in L. johnsonii N6.2 and to evaluate if transcriptional changes are involved in this process. Culturing this strain in the presence of 0.2% bovine bile, which mimics a stressor encountered by the bacterium in the small intestine, resulted in approximately a 100-fold increase in EVs relative to cells grown in media without bile. Whole transcriptome analysis of cells grown with bile revealed upregulation of several peptidoglycan hydrolases as well as several genes involved in fatty acid utilization. These results suggest that the hypervesiculation phenotype may be the result of increased cell wall turnover combined with increased accumulation of phospholipids, in agreement with our previous proteomic and lipidomics results. Additionally, EVs isolated from L. johnsonii N6.2 grown in presence of bile maintained their immunomodulatory properties in host-derived βlox5 pancreatic and THP-1 macrophage cell lines. Our findings suggest that in L. johnsonii N6.2 vesiculogenesis is significantly impacted by the expression of cell wall modifying enzymes and proteins utilized for exogenous fatty acid uptake that are regulated at the transcriptional level. Furthermore, this data suggests that vesiculogenesis could be stimulated in vivo using small molecules thereby maximizing the beneficial interactions between bacteria and their hosts.
Topics: Extracellular Vesicles; Humans; Lactobacillus johnsonii; Bile; Animals; Cell Line; Cattle; THP-1 Cells; Cell Wall; Gene Expression Profiling
PubMed: 38806562
DOI: 10.1038/s41598-024-62843-0