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BioRxiv : the Preprint Server For... Apr 2024Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular...
Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of , we identify a connection between and trehalose monomycolate (TMM) transporter in a suppressor screen, and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 are responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism, but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth, but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation, and in which the connection between Wag31, PlrA and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate synthesis of the different layers of the mycobacterial cell wall in changing conditions.
PubMed: 38746181
DOI: 10.1101/2024.04.29.591792 -
Molecules (Basel, Switzerland) Dec 2023The emergence of Multidrug Resistance (MDR) strains of bacteria has accelerated the search for new antibacterials. The specific bacterial peptidoglycan biosynthetic... (Review)
Review
The emergence of Multidrug Resistance (MDR) strains of bacteria has accelerated the search for new antibacterials. The specific bacterial peptidoglycan biosynthetic pathway represents opportunities for the development of novel antibacterial agents. Among the enzymes involved, Mur ligases, described herein, and especially the amide ligases MurC-F are key targets for the discovery of multi-inhibitors, as they share common active sites and structural features.
Topics: Ligases; Anti-Bacterial Agents; Bacteria; Catalytic Domain; Drug Resistance, Microbial; Peptidoglycan
PubMed: 38138566
DOI: 10.3390/molecules28248076 -
The ISME Journal Sep 2023Membrane vesicles are produced by Gram-negative and Gram-positive bacteria. While membrane vesicles are potent elicitors of eukaryotic cells and involved in cell-cell...
Membrane vesicles are produced by Gram-negative and Gram-positive bacteria. While membrane vesicles are potent elicitors of eukaryotic cells and involved in cell-cell communication, information is scarce about their general biology in the context of community members and the environment. Streptococcus sanguinis, a Gram-positive oral commensal, is prevalent in the oral cavity and well-characterized for its ability to antagonize oral pathobionts. We have found that production and dissemination of membrane vesicles by S. sanguinis is dependent on environmental and community factors. Co-culture with interacting commensal Corynebacterium durum, as well as with the periodontal pathobiont Filifactor alocis had no effect on S. sanguinis vesicle number and size, whereas the periodontal pathobiont Porphyromonas gingivalis abolished S. sanguinis vesicle production. Using both correlation and differential expression analyses to examine the transcriptomic changes underlying vesicle production, we found that differential expression of genes encoding proteins related to the cytoplasmic membrane and peptidoglycan correlate with the abundance of membrane vesicles. Proteomic characterizations of the vesicle cargo identified a variety of proteins, including those predicted to influence host interactions or host immune responses. Cell culture studies of gingival epithelial cells demonstrated that both crude and highly purified membrane vesicles could induce the expression of IL-8, TNF-α, IL-1β, and Gro-α within 6 hours of inoculation at levels comparable to whole cells. Our findings suggest that production of membrane vesicles by S. sanguinis is heavily influenced by community and environmental factors and plays an important role in communication with host cells.
Topics: Streptococcus sanguis; Proteomics; Mouth; Gingiva; Gram-Positive Bacteria
PubMed: 37355741
DOI: 10.1038/s41396-023-01456-3 -
The Journal of Cell Biology Feb 2024To divide, bacteria must synthesize their peptidoglycan (PG) cell wall, a protective meshwork that maintains cell shape. FtsZ, a tubulin homolog, dynamically assembles...
To divide, bacteria must synthesize their peptidoglycan (PG) cell wall, a protective meshwork that maintains cell shape. FtsZ, a tubulin homolog, dynamically assembles into a midcell band, recruiting division proteins, including the PG synthases FtsW and FtsI. FtsWI are activated to synthesize PG and drive constriction at the appropriate time and place. However, their activation pathway remains unresolved. In Caulobacter crescentus, FtsWI activity requires FzlA, an essential FtsZ-binding protein. Through time-lapse imaging and single-molecule tracking of Caulobacter FtsW and FzlA, we demonstrate that FzlA is a limiting constriction activation factor that signals to promote conversion of inactive FtsW to an active, slow-moving state. We find that FzlA interacts with the DNA translocase FtsK and place FtsK genetically in a pathway with FzlA and FtsWI. Misregulation of the FzlA-FtsK-FtsWI pathway leads to heightened DNA damage and cell death. We propose that FzlA integrates the FtsZ ring, chromosome segregation, and PG synthesis to ensure robust and timely constriction during Caulobacter division.
Topics: Caulobacter; Cell Death; Cell Division; Cell Wall; Chromosome Segregation; Bacterial Proteins; Peptidoglycan
PubMed: 38015166
DOI: 10.1083/jcb.202211026 -
Nature Microbiology Jul 2023Bdellovibrio bacteriovorus is a microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens....
Bdellovibrio bacteriovorus is a microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometre-scale resolution. With high-resolution images of predation in a native (hydrated, unstained) state, we discover several surprising features of the process, including macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following growth and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid.
Topics: Humans; Animals; Bdellovibrio; Electron Microscope Tomography; Predatory Behavior; Bdellovibrio bacteriovorus
PubMed: 37349588
DOI: 10.1038/s41564-023-01401-2 -
MBio Oct 2023The rising prevalence of antimicrobial resistance in has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative...
The rising prevalence of antimicrobial resistance in has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative treatment options a high priority. Peptidoglycan hydrolases, a diverse group of bacteriolytic enzymes, show high promise as such alternatives due to their rapid and specific lysis of bacterial cells, independent of antibiotic resistance profiles. However, using these enzymes for the systemic treatment of local infections, such as osteomyelitis foci, needs improvement, as the therapeutic distributes throughout the whole host, resulting in low concentrations at the actual infection site. In addition, the occurrence of intracellularly persisting bacteria can lead to relapsing infections. Here, we describe an approach using tissue-targeting to increase the local concentration of therapeutic enzymes in the infected bone. The enzymes were modified with a short targeting moiety that mediated accumulation of the therapeutic in osteoblasts and additionally enables targeting of intracellularly surviving bacteria.
Topics: Humans; Staphylococcus aureus; Peptidoglycan; N-Acetylmuramoyl-L-alanine Amidase; Staphylococcal Infections; Bacteria; Anti-Bacterial Agents
PubMed: 37768041
DOI: 10.1128/mbio.01830-23 -
Microbiology Spectrum Sep 2023strains of phylogenetic group B2 are often associated with urinary tract infections (UTIs) and several other diseases. Recent genomic and transcriptomic analyses have...
strains of phylogenetic group B2 are often associated with urinary tract infections (UTIs) and several other diseases. Recent genomic and transcriptomic analyses have not suggested or identified specific genes required for virulence, but have instead suggested multiple virulence strategies and complex host-pathogen interactions. Previous analyses have not compared core gene expression between phylogenetic groups or between pathogens and nonpathogens within phylogenetic groups. We compared the core gene expression of 35 strains from three phylogenetic groups that included both pathogens and nonpathogens after growth in a medium that allowed comparable growth of both types of strains. K-means clustering suggested a B2 cluster with 17 group B2 strains and two group A strains; an AD cluster with six group A strains, five group D strains and one B2 strain; and four outliers which included the highly studied model uropathogenic strains UTI89 and CFT073. Half of the core genes were differentially expressed between B2 and AD cluster strains, including transcripts of genes for all aspects of macromolecular synthesis-replication, transcription, translation, and peptidoglycan synthesis-energy metabolism, and environmental-sensing transcriptional regulators. Notably, core gene expression between nonpathogenic and uropathogenic transcriptomes within phylogenetic groups did not differ. If differences between pathogens and nonpathogens exist, then the differences do not require transcriptional reprogramming. In summary, B2 cluster strains have a distinct transcription pattern that involves hundreds of genes. We propose that this transcription pattern is one factor that contributes to virulence. IMPORTANCE is a diverse species and an opportunistic pathogen that is associated with various diseases, such as urinary tract infections. When examined, phylogenetic group B2 strains are more often associated with these diseases, but the specific properties that contribute to their virulence are not known. From a comparative transcriptomic analysis, we found that group B2 strains grown in a nutrient-rich medium had a distinct transcription pattern, which is the first evidence that core gene expression differs between phylogenetic groups. Understanding the consequences of group B2 transcription pattern will provide important information on basic biology, the basis for virulence, and possibly for developing therapies for a majority of urinary tract infections and other group B2-associated diseases.
PubMed: 37724859
DOI: 10.1128/spectrum.02085-23 -
Microbiome Research Reports 2023The peptide MS2-L represents toxins of the ssRNA Leviviridae phage family and consists of a predicted N-terminal soluble domain followed by a transmembrane domain....
The peptide MS2-L represents toxins of the ssRNA Leviviridae phage family and consists of a predicted N-terminal soluble domain followed by a transmembrane domain. MS2-L mediates bacterial cell lysis through the formation of large lesions in the cell envelope, but further details of this mechanism as a prerequisite for applied bioengineering studies are lacking. The chaperone DnaJ is proposed to modulate MS2-L activity, whereas other cellular targets of MS2-L are unknown. Here, we provide a combined and overexpression approach to reveal molecular insights into MS2-L action and its interaction with DnaJ. Full-length MS2-L and truncated derivatives were synthesized cell-free and co-translationally inserted into nanodiscs or solubilized in detergent micelles. By native liquid bead ion desorption mass spectrometry, we demonstrate that MS2-L assembles into high oligomeric states after membrane insertion. Oligomerization is directed by the transmembrane domain and is impaired in detergent environments. Studies with truncated MS2-L derivatives provide evidence that the soluble domain acts as a modulator of oligomer formation. DnaJ strongly interacts with MS2-L in membranes as well as in detergent environments. However, this interaction affects neither the MS2-L membrane insertion efficiency nor its oligomerization in nanodisc membranes. In accordance with the in vitro data, the assembly of MS2-L derivatives into large membrane located clusters was monitored by overexpression of corresponding fusions with fluorescent monitors in cells. Analysis by cryo-electron microscopy indicates that lesion formation is initiated in the outer membrane, followed by disruption of the peptidoglycan layer and disintegration of the inner membrane. MS2-L forms oligomeric complexes similar to the related phage toxin ΦX174-E. The oligomeric interface of both peptides is located within their transmembrane domains. We propose a potential function of the higher-order assembly of small phage toxins in membrane disintegration and cell lysis.
PubMed: 38045926
DOI: 10.20517/mrr.2023.28 -
The Journal of Physical Chemistry. B Dec 2023Unlike typical Gram-positive bacteria, the cell envelope of mycobacteria is unique and composed of a mycobacterial outer membrane, also known as the mycomembrane, a... (Review)
Review
Unlike typical Gram-positive bacteria, the cell envelope of mycobacteria is unique and composed of a mycobacterial outer membrane, also known as the mycomembrane, a peptidoglycan layer, and a mycobacterial inner membrane, which is analogous to that of Gram-negative bacteria. Despite its importance, however, our understanding of this complex cell envelope is rudimentary at best. Thus, molecular modeling and simulation of such an envelope can benefit the scientific community by proposing new hypotheses about the biophysical properties of its different layers. In this Perspective, we present recent advances in molecular modeling and simulation of the mycobacterial cell envelope from individual components to cell envelope assemblies. We also show how modeling other types of cell envelopes, such as that of , may help modeling part of the mycobacterial envelopes. We hope that the studies presented here are just the beginning of the road and more and more new modeling and simulation studies help us to understand crucial questions related to mycobacteria such as antibiotic resistance or bacterial survival in the host.
Topics: Cell Membrane; Cell Wall; Mycobacterium; Models, Molecular; Gram-Negative Bacteria
PubMed: 38091517
DOI: 10.1021/acs.jpcb.3c06136 -
Microbial Genomics Jan 2024Many peptidoglycan-deficient bacteria such as the are known host-associated lineages, lacking the environmental resistance mechanisms and metabolic capabilities...
Many peptidoglycan-deficient bacteria such as the are known host-associated lineages, lacking the environmental resistance mechanisms and metabolic capabilities necessary for a free-living lifestyle. Several peptidoglycan-deficient and non-sporulating orders of interest are thought to be descended from Gram-positive sporulating through reductive evolution. Here we annotate 2650 genomes belonging to the class , according to the Genome Taxonomy Database, to predict the peptidoglycan and sporulation phenotypes of three novel orders, , and , known only through environmental sequence surveys. These lineages are interspersed between peptidoglycan-deficient non-sporulating orders including the and , and more typical Gram-positive orders such as the and . We use the extant genotypes to perform ancestral state reconstructions. The novel orders are predicted to have small genomes with minimal metabolic capabilities and to comprise a mix of peptidoglycan-deficient and/or non-sporulating species. In contrast to expectations based on cultured representatives, the order lacks many of the genes involved in peptidoglycan and endospore formation. The reconstructed evolutionary history of these traits suggests multiple independent whole-genome reductions and loss of phenotype via intermediate transition states that continue into the present. We suggest that the evolutionary history of the reduced-genome lineages within the class is one driven by multiple independent transitions to host-associated lifestyles, with the degree of reduction in environmental resistance and metabolic capabilities correlated with degree of host association.
Topics: Peptidoglycan; Mycoplasmatales; Gram-Positive Bacteria; Firmicutes; Genotype
PubMed: 38189216
DOI: 10.1099/mgen.0.001176