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MBio Jun 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.
Topics: Staphylococcus aureus; Peptidoglycan; Cell Division; Staphylococcus Phages; Viral Proteins; Bacterial Proteins; Cell Wall; Membrane Proteins
PubMed: 38752726
DOI: 10.1128/mbio.00679-24 -
New Biotechnology Sep 2024Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial...
Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD)) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD) complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 10 cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms.
Topics: Staphylococcus aureus; Bacillus anthracis; Cell Wall; Luminescent Proteins; Protein Multimerization; Protein Domains; Surface Plasmon Resonance; Biosensing Techniques; Peptidoglycan
PubMed: 38750815
DOI: 10.1016/j.nbt.2024.05.004 -
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 -
Archives of Microbiology May 2024Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase...
Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase chain reaction (qPCR) analyses were performed to elucidate the genes related to the induction of the VBNC state. The C. jejuni NCTC11168 strain was cultured under low-temperature or high-osmotic stress conditions to induce the VBNC state. mRNA expression in the VBNC state was investigated using microarray analysis, and the gene encoding peptidoglycan-associated lipoprotein, Pal, was selected as the internal control gene using qPCR analysis and software. The three genes showing particularly large increases in mRNA expression, cj1500, cj1254, and cj1040, were involved in respiration, DNA repair, and transporters, respectively. However, formate dehydrogenase encoded by cj1500 showed decreased activity in the VBNC state. Taken together, C. jejuni actively changed its mRNA expression during induction of the VBNC state, and protein activities did not always match the mRNA expression levels.
Topics: Campylobacter jejuni; Bacterial Proteins; Gene Expression Regulation, Bacterial; Microbial Viability; Osmotic Pressure; Stress, Physiological; RNA, Messenger; Real-Time Polymerase Chain Reaction; Gene Expression Profiling
PubMed: 38744718
DOI: 10.1007/s00203-024-03980-y -
MBio Jun 2024Loss of the inner membrane protein YhcB results in pleomorphic cell morphology and clear growth defects. Prior work suggested that YhcB was directly involved in cell...
Loss of the inner membrane protein YhcB results in pleomorphic cell morphology and clear growth defects. Prior work suggested that YhcB was directly involved in cell division or peptidoglycan assembly. We found that loss of YhcB is detrimental in genetic backgrounds in which lipopolysaccharide (LPS) or glycerophospholipid (GPL) synthesis is altered. The growth defect of Δ could be rescued through inactivation of the Mla pathway, a system responsible for the retrograde transport of GPLs that are mislocalized to the outer leaflet of the outer membrane. Interestingly, this rescue was dependent upon the outer membrane phospholipase PldA that cleaves GPLs at the bacterial surface. Since the freed fatty acids resulting from PldA activity serve as a signal to the cell to increase LPS synthesis, this result suggested that outer membrane lipids are imbalanced in Δ. Mutations that arose in Δ populations during two independent suppressor screens were in genes encoding subunits of the acetyl coenzyme A carboxylase complex, which initiates fatty acid biosynthesis (FAB). These mutations fully restored cell morphology and reduced GPL levels, which were increased compared to wild-type bacteria. Growth of Δ with the FAB-targeting antibiotic cerulenin also increased cellular fitness. Furthermore, genetic manipulation of FAB and lipid biosynthesis showed that decreasing FAB rescued Δ filamentation, whereas increasing LPS alone could not. Altogether, these results suggest that YhcB may play a pivotal role in regulating FAB and, in turn, impact cell envelope assembly and cell division.IMPORTANCESynthesis of the Gram-negative cell envelope is a dynamic and complex process that entails careful coordination of many biosynthetic pathways. The inner and outer membranes are composed of molecules that are energy intensive to synthesize, and, accordingly, these synthetic pathways are under tight regulation. The robust nature of the Gram-negative outer membrane renders it naturally impermeable to many antibiotics and therefore a target of interest for antimicrobial design. Our data indicate that when the inner membrane protein YhcB is absent in , the pathway for generating fatty acid substrates needed for all membrane lipid synthesis is dysregulated which leads to increased membrane material. These findings suggest a potentially novel regulatory mechanism for controlling the rate of fatty acid biosynthesis.
Topics: Escherichia coli; Escherichia coli Proteins; Fatty Acids; Glycerophospholipids; Lipopolysaccharides; Membrane Proteins
PubMed: 38742872
DOI: 10.1128/mbio.00790-24 -
MBio Jun 2024() affects patients with immunosuppression or underlying structural lung diseases such as cystic fibrosis (CF). Additionally, poses clinical challenges due to its...
UNLABELLED
() affects patients with immunosuppression or underlying structural lung diseases such as cystic fibrosis (CF). Additionally, poses clinical challenges due to its resistance to multiple antibiotics. Herein, we investigated the synergistic effect of dual β-lactams [sulopenem and cefuroxime (CXM)] or the combination of sulopenem and CXM with β-lactamase inhibitors [BLIs-avibactam (AVI) or durlobactam (DUR)]. The sulopenem-CXM combination yielded low minimum inhibitory concentration (MIC) values for 54 clinical isolates and ATCC19977 (MIC and MIC ≤0.25 µg/mL). Similar synergistic effects were observed in time-kill studies conducted at concentrations achievable in clinical settings. Sulopenem-CXM outperformed monotherapy, yielding ~1.5 Log CFU/mL reduction during 10 days. Addition of BLIs enhanced this antibacterial effect, resulting in an additional reduction of CFUs (~3 Log for sulopenem-CXM and AVI and ~4 Log for sulopenem-DUR). Exploration of the potential mechanisms of the synergy focused on their interactions with L,D-transpeptidases (Ldts; Ldt-Ldt), penicillin-binding-protein B (PBP B), and D,D-carboxypeptidase (DDC). Acyl complexes, identified via mass spectrometry analysis, demonstrated the binding of sulopenem with Ldt-Ldt, DDC, and PBP B and CXM with Ldt and PBP B. Molecular docking and mass spectrometry data suggest the formation of a covalent adduct between sulopenem and Ldt after the nucleophilic attack of the cysteine residue at the β-lactam carbonyl carbon, leading to the cleavage of the β-lactam ring and the establishment of a thioester bond linking the Ldt with sulopenem. In conclusion, we demonstrated the biochemical basis of the synergy of sulopenem-CXM with or without BLIs. These findings potentially broaden the selection of oral therapeutic agents to combat .
IMPORTANCE
Treating infections from (Mab), particularly those resistant to common antibiotics like macrolides, is notoriously difficult, akin to a never-ending struggle for healthcare providers. The rate of treatment failure is even higher than that seen with multidrug-resistant tuberculosis. The role of combination β-lactams in inhibiting L,D-transpeptidation, the major peptidoglycan crosslink reaction in Mab, is an area of intense investigation, and clinicians have utilized this approach in the treatment of macrolide-resistant Mab, with reports showing clinical success. In our study, we found that cefuroxime and sulopenem, when used together, display a significant synergistic effect. If this promising result seen in lab settings, translates well into real-world clinical effectiveness, it could revolutionize current treatment methods. This combination could either replace the need for more complex intravenous medications or serve as a "step down" to an oral medication regimen. Such a shift would be much easier for patients to manage, enhancing their comfort and likelihood of sticking to the treatment plan, which could lead to better outcomes in tackling these tough infections. Our research delved into how these drugs inhibit cell wall synthesis, examined time-kill data and binding studies, and provided a scientific basis for the observed synergy in cell-based assays.
Topics: Mycobacterium abscessus; Anti-Bacterial Agents; Drug Synergism; Microbial Sensitivity Tests; Humans; Cefuroxime; Mycobacterium Infections, Nontuberculous; beta-Lactamase Inhibitors; Molecular Docking Simulation; Prohibitins
PubMed: 38742824
DOI: 10.1128/mbio.00609-24 -
Microbiology (Reading, England) May 2024Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage...
Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested and against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against subsp. serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 μg ml against ATCC 27853, 16.25 μg ml against . Typhimurium ATCC 13311, and 32.50 μg ml against ATCC BAA-2146 and P2224. LysKpV475 showed bactericidal activity only for ATCC 27853 (32.50 μg ml) and P2307 (65.00 μg ml) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and . Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against . Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant S reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.
Topics: Endopeptidases; Polymyxin B; Anti-Bacterial Agents; Salmonella Phages; Glucans; Animals; Microbial Sensitivity Tests; Gram-Negative Bacteria; Mice; Salmonella typhimurium; Bacteriophages; Viral Proteins
PubMed: 38739436
DOI: 10.1099/mic.0.001462 -
International Journal of Molecular... May 2024C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of was identified....
C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of . leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of . Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against . by modulating the TLR signaling pathway.
Topics: Animals; Aeromonas hydrophila; Bass; Cell Adhesion Molecules; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Immunity, Innate; Lectins, C-Type; Pathogen-Associated Molecular Pattern Molecules; Receptors, Cell Surface; Signal Transduction; Toll-Like Receptors
PubMed: 38732232
DOI: 10.3390/ijms25095013 -
International Journal of Molecular... Apr 2024Insects possess an effective immune system, which has been extensively characterized in several model species, revealing a plethora of conserved genes involved in...
Insects possess an effective immune system, which has been extensively characterized in several model species, revealing a plethora of conserved genes involved in recognition, signaling, and responses to pathogens and parasites. However, some taxonomic groups, characterized by peculiar trophic niches, such as plant-sap feeders, which are often important pests of crops and forestry ecosystems, have been largely overlooked regarding their immune gene repertoire. Here we annotated the immune genes of soft scale insects (Hemiptera: Coccidae) for which omics data are publicly available. By using immune genes of aphids and to query the genome of , as well as the transcriptomes of and sp., we highlight the lack of peptidoglycan recognition proteins, galectins, thaumatins, and antimicrobial peptides in Coccidae. This work contributes to expanding our knowledge about the evolutionary trajectories of immune genes and offers a list of promising candidates for developing new control strategies based on the suppression of pests' immunity through RNAi technologies.
Topics: Animals; Hemiptera; Insect Proteins; Transcriptome; Phylogeny; Antimicrobial Peptides; Galectins; Carrier Proteins
PubMed: 38732132
DOI: 10.3390/ijms25094922 -
The Journal of Biological Chemistry May 2024FtsZ, the tubulin homolog essential for bacterial cell division, assembles as the Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. It is...
FtsZ, the tubulin homolog essential for bacterial cell division, assembles as the Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. It is unclear how FtsZ achieves kinetic polarity that drives treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we carried out structural and biochemical characterization of FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). Interestingly the structures of SmFtsZ, bound to GDP and GMPPNP respectively, were captured as domain swapped dimers. SmFtsZ was found to be a slower GTPase with a higher critical concentration (CC) compared to Escherichia coli FtsZ (EcFtsZ). In FtsZs, a conformational switch from R-state (close) to T-state (open) favors polymerization. We identified that Phe224, located at the interdomain cleft of SmFtsZ, is crucial for R- to T-state transition. SmFtsZ exhibited higher GTPase activity and lower CC, whereas the corresponding EcFtsZ resulted in cell division defects in E. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization. Our structural analysis suggests that the rotation is plausibly triggered upon addition of a GTP-bound monomer to the filament through interaction of the preformed N-terminal domain (NTD). Hence, addition of monomers to the NTD-exposed end of filament is slower in comparison to the C-terminal domain (CTD) end, thus explaining kinetic polarity. In summary, the study highlights the importance of interdomain interactions and conformational changes in regulating FtsZ assembly dynamics.
PubMed: 38718863
DOI: 10.1016/j.jbc.2024.107336