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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 -
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 -
Marine Drugs May 2024The deep-sea bacterium CSC3.9 has significant inhibitory effects on agricultural pathogenic fungi and human pathogenic bacteria, especially , the notorious...
The deep-sea bacterium CSC3.9 has significant inhibitory effects on agricultural pathogenic fungi and human pathogenic bacteria, especially , the notorious multidrug-resistant pathogen affecting human public health. We demonstrate that the corresponding antibacterial agents against PAO1 are volatile organic compounds (VOCs, namely VOC-3.9). Our findings show that VOC-3.9 leads to the abnormal cell division of PAO1 by disordering the expression of several essential division proteins associated with septal peptidoglycan synthesis. VOC-3.9 hinders the biofilm formation process and promotes the biofilm dispersion process of PAO1 by affecting its quorum sensing systems. VOC-3.9 also weakens the iron uptake capability of PAO1, leading to reduced enzymatic activity associated with key metabolic processes, such as reactive oxygen species (ROS) scavenging. Overall, our study paves the way to developing antimicrobial compounds against drug-resistant bacteria by using volatile organic compounds.
Topics: Pseudomonas aeruginosa; Volatile Organic Compounds; Anti-Bacterial Agents; Biofilms; Quorum Sensing; Microbial Sensitivity Tests; Reactive Oxygen Species; Humans
PubMed: 38786624
DOI: 10.3390/md22050233 -
Antibiotics (Basel, Switzerland) May 2024Chlamydial infections and diseases caused by filarial nematodes are global health concerns. However, treatment presents challenges due to treatment failures potentially...
Chlamydial infections and diseases caused by filarial nematodes are global health concerns. However, treatment presents challenges due to treatment failures potentially caused by persisting and long regimens against filarial infections accompanied by low compliance. A new treatment strategy could be the targeting of the reduced peptidoglycan structures involved in cell division in the obligate intracellular bacteria and , the latter being obligate endosymbionts supporting filarial development, growth, and survival. Here, cell culture experiments with and showed that the nucleoside antibiotics muraymycin and carbacaprazamycin interfere with bacterial cell division and induce enlarged, aberrant cells resembling the penicillin-induced persistence phenotype in Enzymatic inhibition experiments with purified MraY revealed that muraymycin derivatives abolish the synthesis of the peptidoglycan precursor lipid I. Comparative in silico analyses of chlamydial and wolbachial MraY with the corresponding well-characterized enzyme in revealed a high degree of conservation, providing evidence for a similar mode of inhibition. Muraymycin D2 treatment eradicated persisting non-dividing cells from an established penicillin-induced persistent infection. This finding indicates that nucleoside antibiotics may have additional properties that can break bacterial persistence.
PubMed: 38786149
DOI: 10.3390/antibiotics13050421 -
Frontiers in Microbiology 2024
PubMed: 38784805
DOI: 10.3389/fmicb.2024.1329047 -
Acta Veterinaria Scandinavica May 2024Bacteriophage-encoded endolysins, peptidoglycan hydrolases breaking down the Gram-positive bacterial cell wall, represent a groundbreaking class of novel antimicrobials... (Review)
Review
Bacteriophage-encoded endolysins, peptidoglycan hydrolases breaking down the Gram-positive bacterial cell wall, represent a groundbreaking class of novel antimicrobials to revolutionize the veterinary medicine field. Wild-type endolysins exhibit a modular structure, consisting of enzymatically active and cell wall-binding domains, that enable genetic engineering strategies for the creation of chimeric fusion proteins or so-called 'engineered endolysins'. This biotechnological approach has yielded variants with modified lytic spectrums, introducing new possibilities in antimicrobial development. However, the discovery of highly similar endolysins by different groups has occasionally resulted in the assignment of different names that complicate a straightforward comparison. The aim of this review was to perform a homology-based comparison of the wild-type and engineered endolysins that have been characterized in the context of bovine mastitis-causing streptococci and staphylococci, grouping homologous endolysins with ≥ 95.0% protein sequence similarity. Literature is explored by homologous groups for the wild-type endolysins, followed by a chronological examination of engineered endolysins according to their year of publication. This review concludes that the wild-type endolysins encountered persistent challenges in raw milk and in vivo settings, causing a notable shift in the field towards the engineering of endolysins. Lead candidates that display robust lytic activity are nowadays selected from screening assays that are performed under these challenging conditions, often utilizing advanced high-throughput protein engineering methods. Overall, these recent advancements suggest that endolysins will integrate into the antibiotic arsenal over the next decade, thereby innovating antimicrobial treatment against bovine mastitis-causing streptococci and staphylococci.
Topics: Animals; Mastitis, Bovine; Cattle; Endopeptidases; Staphylococcus; Bacteriophages; Staphylococcal Infections; Streptococcus; Female; Streptococcal Infections; Anti-Bacterial Agents
PubMed: 38769566
DOI: 10.1186/s13028-024-00740-2 -
Systematic and Applied Microbiology May 2024The genus Natronospira is represented by a single species of extremely salt-tolerant aerobic alkaliphilic proteolytic bacterium, isolated from hypersaline soda lakes....
Natronospira bacteriovora sp. nov., and Natronospira elongata sp. nov., extremely salt-tolerant predatory proteolytic bacteria from soda lakes and proposal to classify the genus Natronospira into Natronospiraceae fam. nov., and Natronospirales ord. nov., within the class Gammaproteobacteria.
The genus Natronospira is represented by a single species of extremely salt-tolerant aerobic alkaliphilic proteolytic bacterium, isolated from hypersaline soda lakes. When cells of Gram-positive cocci were used as a substrate instead of proteins at extremely haloalkaline conditions, two new members of this genus were enriched and isolated in pure culture from the same sites. Strains AB-CW1 and AB-CW4 are obligate aerobic heterotrophic proteolytic bacteria able to feed on both live and dead cells of staphylococci and a range of proteins and peptides. Similar to the type species, N. proteinivora, the isolates are extremely salt-tolerant obligate alkaliphiles. However, N. proteinivora was unable to use bacterial cells as a substrate. Electron microscopy showed direct contact between the prey and predator cells. Functional analysis of the AB-CW1 and AB-CW4 genomes identified two sets of genes coding for extracellular enzymes potentially involved in the predation and proteolysis, respectively. The first set includes several copies of lysozyme-like GH23 peptidoglycan-lyase and murein-specific M23 [Zn]-di-peptidase enabling the cell wall degradation. The second set features multiple copies of secreted serine and metallopeptidases apparently allowing for the strong proteolytic phenotype. Phylogenomic analysis placed the isolates into the genus Natronospira as two novel species members, and furthermore indicated that this genus forms a deep-branching lineage of a new family (Natronospiraceae) and order (Natronospirales) within the class Gammaproteobacteria. On the basis of distinct phenotypic and genomic properties, strain AB-CW1 (JCM 335396 = UQM 41579) is proposed to be classified as Natronospira elongata sp. nov., and AB-CW4 (JCM 335397 = UQM 41580) as Natronospira bacteriovora sp. nov.
PubMed: 38759530
DOI: 10.1016/j.syapm.2024.126519 -
Scientific Reports May 2024Alterations in the vaginal microbiota, including both species composition and functional pathways, have been associated with HPV infection and progression of dysplasia...
Alterations in the vaginal microbiota, including both species composition and functional pathways, have been associated with HPV infection and progression of dysplasia to cervical cancer. To further explore this, shotgun metagenomic sequencing was used to taxonomically and functionally characterize the vaginal microbiota of women with and without cervical dysplasia. Women with histologically verified dysplasia (n = 177; low grade dysplasia (LSIL) n = 81, high-grade dysplasia (HSIL) n = 94, cancer n = 2) were compared with healthy controls recruited from the cervical screening programme (n = 177). Women with dysplasia had a higher vaginal microbial diversity, and higher abundances of Gardnerella vaginalis, Aerococcus christensenii, Peptoniphilus lacrimalis and Fannyhessea vaginae, while healthy controls had higher relative abundance of Lactobacillus crispatus. Genes involved in e.g. nucleotide biosynthesis and peptidoglycan biosynthesis were more abundant in women with dysplasia. Healthy controls showed higher abundance of genes important for e.g. amino acid biosynthesis, (especially L-lysine) and sugar degradation. These findings suggest that the microbiota may have a role in creating a pro-oncogenic environment in women with dysplasia. Its role and potential interactions with other components in the microenvironment deserve further exploration.
Topics: Humans; Female; Vagina; Microbiota; Adult; Uterine Cervical Dysplasia; Middle Aged; Case-Control Studies; Metagenomics; Bacteria
PubMed: 38755259
DOI: 10.1038/s41598-024-61942-2 -
Gut May 2024Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known...
OBJECTIVE
Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending how this subset of cells evades the immune system is crucial for advancing novel therapies.
DESIGN
We used the KPC mouse model () and primary tumour cell lines to investigate putative CSC populations. Transcriptomic analyses were conducted to pinpoint new genes involved in immune evasion. Overexpressing and knockout cell lines were established with lentiviral vectors. Subsequent coculture assays, mouse and zebrafish tumorigenesis studies, and database approaches were performed.
RESULTS
Using the KPC mouse model, we functionally confirmed a population of cells marked by EpCAM, Sca-1 and CD133 as authentic CSCs and investigated their transcriptional profile. Immune evasion signatures/genes, notably the gene peptidoglycan recognition protein 1 (PGLYRP1), were significantly overexpressed in these CSCs. Modulating PGLYRP1 impacted CSC immune evasion, affecting their resistance to macrophage-mediated and T-cell-mediated killing and their tumourigenesis in immunocompetent mice. Mechanistically, tumour necrosis factor alpha (TNFα)-regulated PGLYRP1 expression interferes with the immune tumour microenvironment (TME) landscape, promoting myeloid cell-derived immunosuppression and activated T-cell death. Importantly, these findings were not only replicated in human models, but clinically, secreted PGLYRP1 levels were significantly elevated in patients with PDAC.
CONCLUSIONS
This study establishes PGLYRP1 as a novel CSC-associated marker crucial for immune evasion, particularly against macrophage phagocytosis and T-cell killing, presenting it as a promising target for PDAC immunotherapy.
PubMed: 38754953
DOI: 10.1136/gutjnl-2023-330995 -
MBio May 2024Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors...
UNLABELLED
Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors that remodel host metabolism will provide valuable information for target development for antimicrobials. Here, we perform a comprehensive screen for early-gene products of ΦNM1 that inhibit cell growth in . A small membrane protein, Gp11, with inhibitory effects on cell division was identified. A bacterial two-hybrid library containing 345 essential genes was constructed to screen for targets of Gp11, and Gp11 was found to interact with MurG and DivIC. Defects in cell growth and division caused by Gp11 were dependent on MurG and DivIC, which was further confirmed using CRISPRi hypersensitivity assay. Gp11 interacts with MurG, the protein essential for cell wall formation, by inhibiting the production of lipid II to regulate peptidoglycan (PG) biosynthesis on the cell membrane. Gp11 also interacts with cell division protein DivIC, an essential part of the division machinery necessary for septal cell wall assembly, to disrupt the recruitment of division protein FtsW. Mutations in Gp11 result in loss of its ability to cause growth defects, whereas infection with phage in which the gene has been deleted showed a significant increase in lipid II production in . Together, our findings reveal that a phage early-gene product interacts with essential host proteins to disrupt PG biosynthesis and block cell division, suggesting a potential pathway for the development of therapeutic approaches to treat pathogenic bacterial infections.
IMPORTANCE
Understanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in , and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in . Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.
PubMed: 38752726
DOI: 10.1128/mbio.00679-24