Peptidoglycan Biosynthesis Research Articles

Unlocking the mystery of lysine toxicity on Microcystis aeruginosa

Lysine toxicity on certain groups of bacterial cells has been recognized for many years, but the detailed molecular mechanisms that drive this phenomenon have not been elucidated. Many cyanobacteria including Microcystis aeruginosa cannot efficiently export and degrade lysine, although they have evolved to maintain a single copy of the lysine uptake system through which arginine or ornithine can also be transported into the cytoplasm. Autoradiographic analysis using 14C-l-lysine confirmed that lysine was competitively uptaken into cells with arginine or ornithine, which explained the arginine or ornithine-mediated alleviation of lysine toxicity in M. aeruginosa. A relatively non-specific MurE amino acid ligase could incorporate l-lysine into the 3rd position of UDP-N-acetylmuramyl-tripeptide by replacing meso-diaminopimelic acid during the stepwise addition of amino acids on peptidoglycan (PG) biosynthesis. However, further transpeptidation was blocked because lysine substitution at the pentapeptide of the cell wall inhibited the activity of transpeptidases. The leaky PG structure caused irreversible damage to the photosynthetic system and membrane integrity. Collectively, our results suggest that a lysine-mediated coarse-grained PG network and the absence of concrete septal PG lead to the death of slow-growing cyanobacteria.

Structural basis for phage single gene lysis protein mechanisms.

The rapid emergence of antibiotic resistant bacterial strains represents a significant threat to public health, making the development of novel therapeutic strategies critical. Bacterial cell wall biosynthesis has been exploited as a major target for antibiotics and historically been quite successful in treating bacterial infections. A major component of the bacterial cell wall is the peptidoglycan (PG) layer, providing structural support to maintain cell shape and protect bacteria from osmotic lysis. One promising strategy to inhibit PG biosynthesis is to leverage the naturally occurring bacteriophage-derived lysis proteins. Small lytic phages of the families Microviridae and Leviviridae disrupt the formation of the PG matrix through a single gene lysis protein (Sgl). Sgls lack enzymatic activity to degrade cell wall directly and instead induce the host cell to undergo autolysis by inhibiting cell wall synthesis. Three Sgls have been identified that target PG biosynthesis enzymes and act as protein antibiotics. The A2 protein of levivirus Qβ, protein E of ΦX174 (EΦX174) and lysis protein of levivirus M (LysM) were shown to inhibit MurA, MraY and MurJ steps of the PG synthesis pathway respectively. To date, the molecular details of how Sgls inhibit PG biosynthesis enzymes are not well understood. Here we present the cryo-EM structure of the EΦX174-MraY inhibition complex which reveals that E inhibits MraY activity by blocking the substrate binding sites. Furthermore, we are currently exploring other peptidoglycan targeting Sgls from various bacteriophages to expand our understanding of cell wall synthesis inhibition by protein antibiotics. Structure elucidation of these Sgl-PG biosynthetic enzyme complexes helps identify structural properties of the inhibitor binding sites, providing insights into rational design strategies for developing novel bacteriophage Sgl-based inhibitors targeting cell wall biosynthesis.

Molecular characterisation of the transglycosylases involved in lipopolysaccharide maturation and peptidoglycan biogenesis.

Lipopolysaccharide (LPS) O-antigens and the Peptidoglycan (PG) cell wall are core components of the cell envelope of Gram-negative bacteria. The assembly of both polysaccharides requires glycosyltransferases (GT) to generate the glycan polymer. In both cases this requires a homologous TM-embedded GT enzyme, with PG biosynthesis catalysed by RodA, a member of the Shape, Elongation, Division and Sporulation (SEDS) GT family, and the final stage of O-antigen maturation of LPS being controlled by WaaL. Structures of both C. metallidurans WaaL and E. coli RodA have been solved by using single particle cryo-electron microscopy, the latter in complex with the transpeptidase PBP2. We have used molecular modelling and dynamics simulations to understand how both enzymes engage with their substrates. In doing so we have identified key residues involved in their coordination and catalysis. For RodA, we identify two binding sites for Lipid II and propose a mechanism for Lipid II polymerization into peptidoglycan. We also identify two equivalent sites in WaaL for the binding of undecaprenyl-linked O-antigen and LPS. The active sites for both enzymes sit on the periplasmic face of the transmembrane bundle, with the lipidic substrates anchored within the cell membrane to allow catalysis to proceed. As part of this study we compare and contrast the two critical enzymes.

P922 Gut microbiome and mycobiome in inflammatory bowel disease patients with Clostridium difficile infection

Abstract Background Clostridium difficile infection (CDI) is common in patients with inflammatory bowel disease (IBD) and has been reported as a risk factor for poor outcome. However, gut microbiome and mycobiome of IBD patients with CDI have been barely investigated. This study aimed to assess the gut microbiome and mycobiome in IBD patients with CDI. Methods We collected fecal samples from patients with IBD and concomitant CDI (IBD-CDI group, n=38), patients with IBD and no CDI (IBD-only group, n=91), and healthy subjects (HC, n=40). Patients characteristics including demographic data, disease severity, and medication history were collected. 16S rRNA sequencing, metagenomic sequencing, and untargeted metabolomic analysis were carried out in the samples. Results We found that the bacterial alpha diversity of the IBD-CDI group was lower than the IBD-only group and HC. And the bacterial and fungal beta diversity variations between IBD patients and HC were significant, regardless of CDI status. But the IBD-CDI group did not significantly cluster separately from the IBD-only group. Several bacterial taxa, including Ruminococcus gnavus, Clostridium innocuum, and Veillonella were overrepresented in the IBD-CDI group compared with IBD-only group and HC. Furthermore, IBD patients with CDI were distinguished from the IBD-only group and HC by several fungal taxa, including underrepresentation of Candida albicans, and overrepresentation of Saccharomyces cerevisiae, Eremothecium sinecaudum, Torulaspora delbrueckii, Grosmannia clavigera, and Eremothecium gossypii. The fungal-bacterial transkingdom network was altered in the IBD patients with CDI with a decreased network density. Notably, the relative abundance of Grosmannia clavigera was markedly higher in severe ulcerative colitis patients (defined by Truelove and Witts criteria and Mayo score). Besides, prior antibiotic exposure was associated with decreased biodiversity and increased dysbiosis. Functional differences in IBD patients with CDI include enrichment of branched-chain fatty acid biosynthesis, succinate fermentation to butanoate, peptidoglycan biosynthesis, and tetrahydrofolate biosynthesis. The metabolomic data indicated that the differential metabolites were predominantly involved in amino sugar and nucleotide sugar metabolism, fatty acid metabolism, porphyrin and chlorophyll metabolism, and neomycin, kanamycin and gentamicin biosynthesis. Conclusion IBD patients with CDI had pronounced microbial dysbiosis. Gut micro-ecological changes and interkingdom crosstalk in IBD patients with CDI might provide insight into the pathological process and potential strategies for diagnosis and treatment in this subset of patients.

Bacteroid Development, Transcriptome, and Symbiotic Nitrogen-Fixing Comparison of Bradyrhizobium arachidis in Nodules of Peanut (Arachis hypogaea) and Medicinal Legume Sophora flavescens.

Bradyrhizobium arachidis strain CCBAU 051107 could differentiate into swollen and nonswollen bacteroids in determinate root nodules of peanut (Arachis hypogaea) and indeterminate nodules of Sophora flavescens, respectively, with different N2 fixation efficiencies. To reveal the mechanism of bacteroid differentiation and symbiosis efficiency in association with different hosts, morphologies, transcriptomes, and nitrogen fixation efficiencies of the root nodules induced by strain CCBAU 051107 on these two plants were compared. Our results indicated that the nitrogenase activity of peanut nodules was 3 times higher than that of S. flavescens nodules, demonstrating the effects of rhizobium-host interaction on symbiotic effectiveness. With transcriptome comparisons, genes involved in biological nitrogen fixation (BNF) and energy metabolism were upregulated, while those involved in DNA replication, bacterial chemotaxis, and flagellar assembly were significantly downregulated in both types of bacteroids compared with those in free-living cells. However, expression levels of genes involved in BNF, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, hydrogenase synthesis, poly-β-hydroxybutyrate (PHB) degradation, and peptidoglycan biosynthesis were significantly greater in the swollen bacteroids of peanut than those in the nonswollen bacteroids of S. flavescens, while contrasting situations were found in expression of genes involved in urea degradation, PHB synthesis, and nitrogen assimilation. Especially higher expression of ureABEF and aspB genes in bacteroids of S. flavescens might imply that the BNF product and nitrogen transport pathway were different from those in peanut. Our study revealed the first differences in bacteroid differentiation and metabolism of these two hosts and will be helpful for us to explore higher-efficiency symbiosis between rhizobia and legumes. IMPORTANCE Rhizobial differentiation into bacteroids in leguminous nodules attracts scientists to investigate its different aspects. The development of bacteroids in the nodule of the important oil crop peanut was first investigated and compared to the status in the nodule of the extremely promiscuous medicinal legume Sophora flavescens by using just a single rhizobial strain of Bradyrhizobium arachidis, CCBAU 051107. This strain differentiates into swollen bacteroids in peanut nodules and nonswollen bacteroids in S. flavescens nodules. The N2-fixing efficiency of the peanut nodules is three times higher than that of S. flavescens. By comparing the transcriptomes of their bacteroids, we found that they have similar gene expression spectra, such as nitrogen fixation and motivity, but different spectra in terms of urease activity and peptidoglycan biosynthesis. Those altered levels of gene expression might be related to their functions and differentiation in respective nodules. Our studies provided novel insight into the rhizobial differentiation and metabolic alteration in different hosts.

Comparative transcriptomics and genomics from continuous axenic media growth identifies Coxiella burnetii intracellular survival strategies.

Coxiella burnetii (Cb) is an obligate intracellular pathogen in nature and the causative agent of acute Q fever as well as chronic diseases. In an effort to identify genes and proteins crucial to their normal intracellular growth lifestyle, we applied a 'reverse evolution' approach where the avirulent Nine Mile Phase II strain of Cb was grown for 67 passages in chemically defined ACCM-D media and gene expression patterns and genome integrity from various passages was compared to passage number one following intracellular growth. Transcriptomic analysis identified a marked downregulation of the structural components of the type 4B secretion system (T4BSS), the general secretory (Sec) pathway, as well as 14 out of 118 previously identified genes encoding effector proteins. Additional downregulated pathogenicity determinants genes included several chaperones, LPS, and peptidoglycan biosynthesis. A general marked downregulation of central metabolic pathways was also observed, which was balanced by a marked upregulation of genes encoding transporters. This pattern reflected the richness of the media and diminishing anabolic, and ATP-generation needs. Finally, genomic sequencing and comparative genomic analysis demonstrated an extremely low level of mutation across passages, despite the observed Cb gene expression changes following acclimation to axenic media.

Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA–MurG interaction

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.

Comparative Proteomics of Outer Membrane Vesicles from Polymyxin-Susceptible and Extremely Drug-Resistant Klebsiella pneumoniae.

Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria serve as transporters for the delivery of cargo such as virulence and antibiotic resistance factors. OMVs play a key role in the defense against membrane-targeting antibiotics such as the polymyxin B. Herein, we conducted comparative proteomics of OMVs from paired Klebsiella pneumoniae ATCC 700721 polymyxin-susceptible (polymyxin B MIC = 0.5 mg/L) and an extremely resistant (polymyxin B MIC ≥128 mg/L), following exposure to 2 mg/L of polymyxin B. Comparative profiling of the OMV subproteome of each strain revealed proteins from multiple perturbed pathways, particularly in the polymyxin-susceptible strain, including outer membrane assembly (lipopolysaccharide, O-antigen, and peptidoglycan biosynthesis), cationic antimicrobial peptide resistance, β-lactam resistance, and quorum sensing. In the polymyxin-susceptible strain, polymyxin B treatment reduced the expression of OMV proteins in the pathways related to adhesion, virulence, and the cell envelope stress responses, whereas, in the polymyxin-resistant strain, the proteins involved in LPS biosynthesis, RNA degradation, and nucleotide excision repair were significantly overexpressed in response to polymyxin B treatment. Intriguingly, the key polymyxin resistance enzymes 4-amino-4-deoxy-l-arabinose transferase and the PhoPQ two-component protein kinase were significantly downregulated in the OMVs of the polymyxin-susceptible strain. Additionally, a significant reduction in class A β-lactamase proteins was observed following polymyxin B treatment in the OMVs of both strains, particularly the OMVs of the polymyxin-susceptible strain. These findings shed new light on the OMV subproteome of extremely polymyxin resistant K. pneumoniae, which putatively may serve as active decoys to make the outer membrane more impervious to polymyxin attack. IMPORTANCE OMVs can help bacteria to fight antibiotics not only by spreading antibiotic resistance genes but also by acting as protective armor against antibiotics. By employing proteomics, we found that OMVs have a potential role in shielding K. pneumoniae and acting as decoys to polymyxin attack, through declining the export of proteins (e.g., 4-amino-4-deoxy-l-arabinose transferase) involved in polymyxin resistance. Furthermore, polymyxin B treatment of both strains leads to shedding of the OMVs with perturbed proteins involved in outer membrane remodeling (e.g., LPS biosynthesis) as well as pathogenic potential of K. pneumoniae (e.g., quorum sensing). The problematic extended spectrum beta-lactamases SHV and TEM were significantly reduced in both strains, suggesting that polymyxin B may act as a potentiator to sensitize the bacterium to β-lactam antibiotics. This study highlights the importance of OMVs as "molecular mules" for the intercellular transmission and delivery of resistance and cellular repair factors in the bacterial response to polymyxins.

Bioinformatics Analysis to Uncover the Potential Drug Targets Responsible for Mycobacterium tuberculosis Peptidoglycan and Lysine Biosynthesis.

Drug-resistant tuberculosis (TB), which results mainly from the selection of naturally resistant strains of Mycobacterium tuberculosis (MTB) due to mismanaged treatment, poses a severe challenge to the global control of TB. Therefore, screening novel and unique drug targets against this pathogen is urgently needed. The metabolic pathways of Homo sapiens and MTB were compared using the Kyoto Encyclopedia of Genes and Genomes tool, and further, the proteins that are involved in the metabolic pathways of MTB were subtracted and proceeded to protein-protein interaction network analysis, subcellular localization, drug ability testing, and gene ontology. The study aims to identify enzymes for the unique pathways for further screening to determine the feasibility of the therapeutic targets. The qualitative characteristics of 28 proteins identified as drug target candidates were studied. The results showed that 12 were cytoplasmic, 2 were extracellular, 12 were transmembrane, and 3 were unknown. Furthermore, druggability analysis revealed 14 druggable proteins, of which 12 were novel and responsible for MTB peptidoglycan and lysine biosynthesis. The novel targets obtained in this study are used to develop antimicrobial treatments against pathogenic bacteria. Future studies should further shed light on the clinical implementation to identify antimicrobial therapies against MTB.

Lonicera japonica Thunb. as a promising antibacterial agent for Bacillus cereus ATCC14579 based on network pharmacology, metabolomics, and in vitro experiments.

Lonicera japonica Thunb. has attracted much attention for its treatment of bacterial and viral infectious diseases, while its active ingredients and potential mechanisms of action have not been fully elucidated. Here, we combined metabolomics, and network pharmacology to explore the molecular mechanism of Bacillus cereus ATCC14579 inhibition by Lonicera japonica Thunb. In vitro inhibition experiments showed that the Lonicera japonica Thunb.'s water extracts, ethanolic extract, luteolin, quercetin, and kaempferol strongly inhibited Bacillus cereus ATCC14579. In contrast, chlorogenic acid and macranthoidin B had no inhibitory effect on Bacillus cereus ATCC14579. Meanwhile, the minimum inhibitory concentrations of luteolin, quercetin, and kaempferol against Bacillus cereus ATCC14579 were 15.625 μg mL-1, 31.25 μg mL-1, and 15.625 μg mL-1. Based on the previous experimental basis, the metabolomic analysis showed the presence of 16 active ingredients in Lonicera japonica Thunb.'s water extracts and ethanol extracts, with differences in the luteolin, quercetin, and kaempferol contents between the water extracts and ethanol extracts. Network pharmacology studies indicated that fabZ, tig, glmU, secA, deoD, nagB, pgi, rpmB, recA, and upp were potential key targets. Active ingredients of Lonicera japonica Thunb. may exert their inhibitory effects by inhibiting ribosome assembly, the peptidoglycan biosynthesis process, and the phospholipid biosynthesis process of Bacillus cereus ATCC14579. An alkaline phosphatase activity assay, peptidoglycan concentration assay, and protein concentration assay showed that luteolin, quercetin, and kaempferol disrupted the Bacillus cereus ATCC14579 cell wall and cell membrane integrity. Transmission electron microscopy results showed significant changes in the morphology and ultrastructure of the cell wall and cell membrane of Bacillus cereus ATCC14579, further confirming the disruption of the cell wall and cell membrane integrity of Bacillus cereus ATCC14579 by luteolin, quercetin, and kaempferol. In conclusion, Lonicera japonica Thunb. can be used as a potential antibacterial agent for Bacillus cereus ATCC14579, which may exert its antibacterial activity by destroying the integrity of the cell wall and membrane.

Altered gut microbiomes are associated with the symptomatic status of unruptured intracranial aneurysms.

Gut microbiome has recently been recognized as an important environmental factor affecting the occurrence and development of unruptured intracranial aneurysms (UIA). This study aimed to investigate the relationship between gut microbiome and symptomatic UIA, which is a predictor of instability and a high propensity to rupture. A total of 132 patients including 86 asymptomatic UIA and 46 symptomatic UIA were recruited in the study. The composition of gut bacterial communities was determined by 16S ribosomal RNA gene sequencing. In addition, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was used to predict the functional composition of the gut microbiome. There is no difference in the fecal microbial alpha diversity between symptomatic and asymptomatic UIA, but gut microbiome composition changed significantly. At the order level, the relative abundance of Clostridiales was significantly enriched in the symptomatic compared with asymptomatic UIA (p = 0.043). In addition, similar alterations were observed at the family levels of Ruminococcaceae. The Linear discriminant analysis (LEfSe) revealed Fournierella, Ruthenibacterium, and Anaerotruncus as discriminative features in the symptomatic group. Notably, functional differences in gut microbiome of patients with symptomatic UIA included decreased propionate metabolism pathway and enrichment of peptidoglycan biosynthesis pathways. The present study comprehensively characterizes gut microbiome in a large cohort of different risk statuses of UIA patients and demonstrates the potential biological function of gut microbiome involved in the development of UIA. It may provide additional benefits in guiding UIA management and improving patient outcomes.

LdtC Is a Key l,d-Transpeptidase for Peptidoglycan Assembly in Mycobacterium smegmatis.

The peptidoglycan of mycobacteria has two types of direct cross-links, classical 4-3 cross-links that occur between diaminopimelate (DAP) and alanine residues, and nonclassical 3-3 cross-links that occur between DAP residues on adjacent peptides. The 3-3 cross-links are synthesized by the concerted action of d,d-carboxypeptidases and l,d-transpeptidases (Ldts). Mycobacterial genomes encode several Ldt proteins that can be classified into six classes based upon sequence identity. As a group, the Ldt enzymes are resistant to most β-lactam antibiotics but are susceptible to carbapenem antibiotics, with the exception of LdtC, a class 5 enzyme. In previous work, we showed that loss of LdtC has the greatest effect on the carbapenem susceptibility phenotype of Mycobacterium smegmatis (also known as Mycolicibacterium smegmatis) compared to other ldt deletion mutants. In this work, we show that a M. smegmatis mutant lacking the five ldt genes other than ldtC has a wild-type phenotype with the exception of increased susceptibility to rifampin. In contrast, a mutant lacking all six ldt genes has pleiotropic cell envelope defects, is temperature sensitive, and has increased susceptibility to a variety of antibiotics. These results indicate that LdtC is capable of functioning as the sole l,d-transpeptidase in M. smegmatis and suggest that it may represent a carbapenem-resistant pathway for peptidoglycan biosynthesis. IMPORTANCE Mycobacteria have several enzymes to catalyze nonclassical 3-3 linkages in the cell wall peptidoglycan. Understanding the biology of these cross-links is important for the development of antibiotic therapies to target peptidoglycan biosynthesis. Our work provides evidence that LdtC can function as the sole enzyme for 3-3 cross-link formation in M. smegmatis and suggests that LdtC may be part of a carbapenem-resistant l,d-transpeptidase pathway.

New Insights into the Lactic Acid Resistance Determinants of Listeria monocytogenes Based on Transposon Sequencing and Transcriptome Sequencing Analyses.

Listeria monocytogenes is a foodborne pathogen that can tolerate a variety of extreme environments. In particular, its acid resistance (AR) capability is considered one of the key factors threating food safety. Here, we employed a microbial functional genomic technology termed transposon sequencing (Tn-seq), leading to the identification of two genes involved in cell wall peptidoglycan biosynthesis (murF) and phosphate transport (lmo2248) that play key roles in lactic acid resistance (LAR) of L. monocytogenes. Deletion of lmo2248 significantly impaired the ability of LAR in L. monocytogenes, demonstrating the accuracy of the Tn-seq results. Transcriptome analysis revealed that 31.7% of the L. monocytogenes genes on the genome were differentially expressed under lactic acid (LA) treatment, in which genes involved in phosphate transport were influenced most significantly. These findings shed light on the LAR mechanisms of L. monocytogenes, which may contribute to the development of novel strategies against foodborne pathogens. IMPORTANCE Listeria monocytogenes is a Gram-positive foodborne pathogen with high lethality and strong stress resistance, and its strong acid tolerance leads to many foodborne illnesses occurring in low-pH foods. Lactic acid is a generally recognized as safe (GRAS) food additive approved for use by the FDA. However, the genetic determinants of lactic acid resistance in L. monocytogenes have not been fully identified. In this study, the lactic acid resistance determinants of L. monocytogenes were comprehensively identified by Tn-seq on a genome-wide scale. Two genes, murF (cell wall peptidoglycan biosynthesis) and lmo2248 (phosphate transport), were identified to play an important role in the lactic acid resistance. Moreover, genome-wide transcriptomic analysis showed that phosphotransferase system (PTS)-related genes play a key role at the transcriptional level. These findings contribute to a better understanding of the lactic acid resistance mechanism of L. monocytogenes and may provide unique targets for the development of other novel antimicrobial agents.

Methylarsenite is a broad-spectrum antibiotic disrupting cell wall biosynthesis and cell membrane potential.

Methylarsenite (MAs(III)), a product of arsenic biomethylation or bioreduction of methylarsenate (MAs(V)), has been proposed as a primitive antibiotic. However, the antibacterial property and the bactericidal mechanism of MAs(III) remain largely unclear. In this study, we found that MAs(III) is highly toxic to 14 strains of bacteria, especially against 9 strains of Gram-positive bacteria with half maximal inhibitory concentration (IC50) in the sub micromolar range for Staphyloccocus aureus, Microbacterium sp., Pseudarthrobacter siccitolerans and several Bacillus species. In a co-culture of B. subtilis 168 and MAs(III)-producer Enterobacter sp. CZ-1, the later reduced non-toxic MAs(V) to highly toxic MAs(III) to kill the former and gain a competitive advantage. MAs(III) induced autolysis of B. subtilis 168. Deletion of the autolysins LytC, LytD, LytE, and LytF suppressed MAs(III)-induced autolysis in B. subtilis 168. Transcriptomic analysis showed that MAs(III) downregulated the expression of the major genes involved in the biosynthesis of the cell wall peptidoglycan. Overexpression of an UDP-N-acetylglucosamine enolpyruvyl transferase gene murAA alleviated MAs(III)-induced autolysis in B. subtilis 168. MAs(III) disrupted the membrane potential of B. subtilis 168 and caused severe membrane damage. The results suggest that MAs(III) is a broad-spectrum antibiotic preferentially against Gram-positive bacteria by disrupting the cell wall biosynthesis pathway and cell membrane potential.

676. Synergistic Bacteriostatic and Bactericidal Activity of Fosfomycin Plus Trimethoprim-Sulfamethoxazole Combination Against Carbapenem-Resistant Enterobacteriales, Multidrug-Resistant P. aeruginosa, Carbapenem-Colistin-Resistant A. baumannii, MRSA, and Enterococcus spp.

Abstract Background Fosfomycin (F) inhibits the first committed step of peptidoglycan biosynthesis catalyzed by the MurA enzyme which uses phosphoenolpyruvate as a substrate. Trimethoprim and sulfamethoxazole (TS) inhibit successive steps in folate biosynthesis and are commonly used as a fixed combination (co-trimoxazole). In this study we explored the combination (FTS) of F with TS in several gram-negative and gram-positive bacteria of increasing concern. Methods We used the synergy-checkerboard variant of the agar minimal inhibitory concentration (MIC) assay with Mueller-Hinton Agar (glucose-6-phosphate supplemented; CLSI) against a selection of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and Enterococcus spp. clinical isolates to characterize an FTS combination. Component antibiotics, F and TS, and a variety of other antibacterial comparators were evaluated by agar method or broth microdilution. Bactericidal assays of FTS were conducted in broth. Results Antibacterial synergy with FTS was exhibited against all E. coli (20/20) at antibacterial concentrations below the individual clinical breakpoints for F and TS. Against K. pneumoniae isolates, all (12/12) that were resistant to F, and many (10/13) resistant to both F and TS, were susceptible to FTS. All P. aeruginosa isolates (18/18) were dually resistant to F and TS. Extrapolating breakpoints for Enterobacteriales, clinically-relevant synergy of FTS was exerted against 11/18 isolates, and susceptibility was achievable for one component in combination against 7/18 isolates. Against A. baumannii, susceptibility values below extrapolated breakpoints for FTS were achieved for most isolates (17/19), and for a few (2/19), susceptibility was observed for one component in combination. FTS was active against all MRSA and Enterococcus spp. regardless of F or TS susceptibility. Conclusion The unique synergistic and bactericidal activity of the FTS combination was not impacted by resistance of the tested species to any other antibacterial agent including carbapenem-resistant Enterobacteriales, P. aeruginosa, carbapenem-colistin-resistant A. baumannii, MRSA, or Enterococcus spp. Disclosures John L. Pace, PhD, FleurirABX, LLC: Board Member|FleurirABX, LLC: Inventor of company patents|FleurirABX, LLC: Ownership Interest.

Synthesis of an Antimicrobial Enterobactin-Muraymycin Conjugate for Improved Activity Against Gram-Negative Bacteria.

Overcoming increasing antibiotic resistance requires the development of novel antibacterial agents that address new targets in bacterial cells. Naturally occurring nucleoside antibiotics (such as muraymycins) inhibit the bacterial membrane protein MraY, a clinically unexploited essential enzyme in peptidoglycan (cell wall) biosynthesis. Even though a range of synthetic muraymycin analogues has already been reported, they generally suffer from limited cellular uptake and a lack of activity against Gram-negative bacteria. We herein report an approach to overcome these hurdles: a synthetic muraymycin analogue has been conjugated to a siderophore, i. e. the enterobactin derivative EntKL , to increase the cellular uptake into Gram-negative bacteria. The resultant conjugate showed significantly improved antibacterial activity against an efflux-deficient E. coli strain, thus providing a proof-of-concept of this novel approach and a starting point for the future optimisation of such conjugates towards potent agents against Gram-negative pathogens.

Specific Features of the Proteomic Response of Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation

Studying the effects of terahertz (THz) radiation on the proteome of temperature-sensitive organisms is limited by a number of significant technical difficulties, one of which is maintaining an optimal temperature range to avoid thermal shock as much as possible. In the case of extremophilic species with an increased temperature tolerance, it is easier to isolate the effects of THz radiation directly. We studied the proteomic response to terahertz radiation of the thermophilic Geobacillus icigianus, persisting under wide temperature fluctuations with a 60 °C optimum. The experiments were performed with a terahertz free-electron laser (FEL) from the Siberian Center for Synchrotron and Terahertz Radiation, designed and employed by the Institute of Nuclear Physics of the SB of the RAS. A G. icigianus culture in LB medium was THz-irradiated for 15 min with 0.23 W/cm2 and 130 μm, using a specially designed cuvette. The life cycle of this bacterium proceeds under conditions of wide temperature and osmotic fluctuations, which makes its enzyme systems stress-resistant. The expression of several proteins was shown to change immediately after fifteen minutes of irradiation and after ten minutes of incubation at the end of exposure. The metabolic systems of electron transport, regulation of transcription and translation, cell growth and chemotaxis, synthesis of peptidoglycan, riboflavin, NADH, FAD and pyridoxal phosphate cofactors, Krebs cycle, ATP synthesis, chaperone and protease activity, and DNA repair, including methylated DNA, take part in the fast response to THz radiation. When the response developed after incubation, the systems of the cell's anti-stress defense, chemotaxis, and, partially, cell growth were restored, but the respiration and energy metabolism, biosynthesis of riboflavin, cofactors, peptidoglycan, and translation system components remained affected and the amino acid metabolism system was involved.

The Potential of 24-Propylcholestrol as Antibacterial Oral Bacteria of Enterococcus faecalis ATCC 29212 and Inhibitor Biofilms Formation: in vitro and in silico Study.

Uncontrolled biofilm can cause several diseases such as dental caries, gingivitis, and periodontitis. Dental caries is a disease caused by the accumulation of plaque-containing pathogenic bacteria, including Enterococcus faecalis. These bacteria infect the root canals of teeth and colonize to form biofilms. Biofilm inhibition is carried out by interfering with cell wall formation metabolism. MurA enzyme has a role in peptidoglycan biosynthesis of cell walls. Enterococcal surface protein (Esp) is the main contributor of E. faecalis to form biofilms. In addition, inhibition of biofilms by interfering with the quorum sensing (QS) system, suppressing gelatinase virulence factors by blocking autoinducers gelatinase biosynthesis-activating pheromone (GBAP). Knowing the potential of Piper betel Linn. compounds as antibacterial in vitro and antibiofilm agents against E. faecalis in silico. The compounds were purified by a bioactivity-guided chromatographic method. Antibacterial activity was tested by disc diffusion method, in vitro studies. In silico study, compound P. betel L. was used as the test ligand and compared with positive control fosfomycin, ambuic acid, quercetin, and taxifolin. The proteins used MurA, Esp, GBAP, and gelatinase were docking with the Autodock Vina PyRx 0.8 followed by the PYMOL program and visualized with the Discovery Studio 2020 program. An antibacterial compound was identified 24-propylcholesterol which can inhibit the activity of E. faecalis ATCC 29212 with MIC value of 78.1 µg/mL and MBC value of 156.3 µg/mL. Molecular docking results showed the binding affinity of 24-propylcholesterol with MurA, ESP, GBAP, and gelatinase enzymes was -7.6, -8.7, -5.3, and -7.9 kcal/mol. 24-propylcholesterol has potential as an antibacterial against E. faecalis and as an antibiofilm through in silico inhibition of QS. However, further research is needed in vitro and in vivo to determine the effectiveness of these compounds as antibacterial and antibiofilm.

Undecaprenyl phosphate translocases confer conditional microbial fitness

The microbial cell wall is essential for maintenance of cell shape and resistance to external stressors1. The primary structural component of the cell wall is peptidoglycan, a glycopolymer with peptide crosslinks located outside of the cell membrane1. Peptidoglycan biosynthesis and structure are responsive to shifting environmental conditions such as pH and salinity2–6, but the mechanisms underlying such adaptations are incompletely understood. Precursors of peptidoglycan and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered across the cell membrane bound to the recyclable lipid carrier undecaprenyl phosphate7 (C55-P, also known as UndP). Here we identify the DUF368-containing and DedA transmembrane protein families as candidate C55-P translocases, filling a critical gap in knowledge of the proteins required for the biogenesis of microbial cell surface polymers. Gram-negative and Gram-positive bacteria lacking their cognate DUF368-containing protein exhibited alkaline-dependent cell wall and viability defects, along with increased cell surface C55-P levels. pH-dependent synthetic genetic interactions between DUF368-containing proteins and DedA family members suggest that C55-P transporter usage is dynamic and modulated by environmental inputs. C55-P transporter activity was required by the cholera pathogen for growth and cell shape maintenance in the intestine. We propose that conditional transporter reliance provides resilience in lipid carrier recycling, bolstering microbial fitness both inside and outside the host.

Discovery of a fragment hit compound targeting D-Ala:D-Ala ligase of bacterial peptidoglycan biosynthesis

Bacterial resistance is an increasing threat to healthcare systems, highlighting the need for discovering new antibacterial agents. An established technique, fragment-based drug discovery, was used to target a bacterial enzyme Ddl involved in the biosynthesis of peptidoglycan. We assembled general and focused fragment libraries that were screened in a biochemical inhibition assay. Screening revealed a new fragment-hit inhibitor of DdlB with a Ki value of 20.7 ± 4.5 µM. Binding to the enzyme was confirmed by an orthogonal biophysical method, surface plasmon resonance, making the hit a promising starting point for fragment development.