Identification of a new family of peptidoglycan transpeptidases reveals atypical crosslinking is essential for viability in Clostridioides difficile.

Clostridioides difficile, the leading cause of antibiotic-associated diarrhea, relies primarily on 3-3 crosslinks created by L,D-transpeptidases (LDTs) to fortify its peptidoglycan (PG) cell wall. This is unusual, as in most bacteria the vast majority of PG crosslinks are 4-3 crosslinks, which are created by penicillin-binding proteins (PBPs). Here we report the unprecedented observation that 3-3 crosslinking is essential for viability in C. difficile. We also report the discovery of a new family of LDTs that use a VanW domain to catalyze 3-3 crosslinking rather than a YkuD domain as in all previously known LDTs. Bioinformatic analyses indicate VanW domain LDTs are less common than YkuD domain LDTs and are largely restricted to Gram-positive bacteria. Our findings suggest that LDTs might be exploited as targets for antibiotics that kill C. difficile without disrupting the intestinal microbiota that is important for keeping C. difficile in check.

Identification of Clostridioides difficile mutants with increased daptomycin resistance.

Daptomycin is a cyclic lipopeptide antibiotic used to treat infections caused by some Gram-positive bacteria. Daptomycin disrupts synthesis of the peptidoglycan (PG) cell wall by inserting into the cytoplasmic membrane and binding multiple forms of the undecaprenyl carrier lipid required for PG synthesis. Membrane insertion requires phosphatidylglycerol, so studies of daptomycin can provide insight into assembly and maintenance of the cytoplasmic membrane. Here, we studied the effects of daptomycin on Clostridioides difficile, the leading cause of healthcare-associated diarrhea. We observed that growth of C. difficile strain R20291 in the presence of sub-MIC levels of daptomycin resulted in a chaining phenotype, minicell formation, and lysis-phenotypes broadly consistent with perturbation of membranes and PG synthesis. We also selected for and characterized eight mutants with elevated daptomycin resistance. The mutations in these mutants were mapped to four genes: cdsA (cdr20291_2041), ftsH2 (cdr20291_3396), esrR (cdr20291_1187), and draS (cdr20291_2456). Of these four genes, only draS has been characterized previously. Follow-up studies indicate these mutations confer daptomycin resistance by two general mechanisms: reducing the amount of phosphatidylglycerol in the cytoplasmic membrane (cdsA) or altering the regulation of membrane processes (ftsH2, esrR, and draS). Thus, the mutants described here provide insights into phospholipid synthesis and identify signal transduction systems involved in cell envelope biogenesis and stress response in C. difficile. IMPORTANCE: C. difficile is the leading cause of healthcare-associated diarrhea and is a threat to public health due to the risk of recurrent infections. Understanding biosynthesis of the atypical cell envelope of C. difficile may provide insight into novel drug targets to selectively inhibit C. difficile. Here, we identified mutations that increased daptomycin resistance and allowed us to better understand phospholipid synthesis, cell envelope biogenesis, and stress response in C. difficile.

The WalRK Two-Component System Is Essential for Proper Cell Envelope Biogenesis in Clostridioides difficile.

The WalR-WalK two-component regulatory system (TCS) is found in all Firmicutes, in which it regulates the expression of multiple genes required for remodeling the cell envelope during growth and division. Unlike most TCSs, WalRK is essential for viability, so it has attracted interest as a potential antibiotic target. In this study, we used overexpression of WalR and CRISPR interference to investigate the Wal system of Clostridioides difficile, a major cause of hospital-associated diarrhea in high-income countries. We confirmed that the wal operon is essential and identified morphological defects and cell lysis as the major terminal phenotypes of altered wal expression. We also used transcriptome sequencing (RNA-seq) to identify over 150 genes whose expression changes in response to WalR levels. This gene set is enriched in cell envelope genes and includes genes encoding several predicted PG hydrolases and proteins that could regulate PG hydrolase activity. A distinct feature of the C. difficile cell envelope is the presence of an S-layer, and we found that WalR affects expression of several genes which encode S-layer proteins. An unexpected finding was that some Wal-associated phenotypic defects were inverted in comparison to what has been reported for other Firmicutes. For example, downregulation of Wal signaling caused C. difficile cells to become longer rather than shorter, as in Bacillus subtilis. Likewise, downregulation of Wal rendered C. difficile more sensitive to vancomycin, whereas reduced Wal activity is linked to increased vancomycin resistance in Staphylococcus aureus. IMPORTANCE The WalRK two-component system (TCS) is essential for coordinating synthesis and turnover of peptidoglycan in Firmicutes. We investigated the WalRK TCS in Clostridioides difficile, an important bacterial pathogen with an atypical cell envelope. We confirmed that WalRK is essential and regulates cell envelope biogenesis, although several of the phenotypic changes we observed were opposite to what has been reported for other Firmicutes. We also identified over 150 genes whose expression is controlled either directly or indirectly by WalR. Overall, our findings provide a foundation for future investigations of an important regulatory system and potential antibiotic target in C. difficile.

DrpB (YedR) Is a Nonessential Cell Division Protein in Escherichia coli.

We report that the small Escherichia coli membrane protein DrpB (formerly YedR) is involved in cell division. We discovered DrpB in a screen for multicopy suppressors of a ΔftsEX mutation that prevents divisome assembly when cells are plated on low ionic strength medium, such as lysogeny broth without NaCl. Characterization of DrpB revealed that (i) translation initiates at an ATG annotated as codon 22 rather than the GTG annotated as codon 1, (ii) DrpB localizes to the septal ring when cells are grown in medium of low ionic strength but localization is greatly reduced in medium of high ionic strength, (iii) overproduction of DrpB in a ΔftsEX mutant background improves recruitment of the septal peptidoglycan synthase FtsI, implying multicopy suppression works by rescuing septal ring assembly, (iv) a ΔdrpB mutant divides quite normally, but a ΔdrpB ΔdedD double mutant has a strong division and viability defect, albeit only in medium of high ionic strength, and (v) DrpB homologs are found in E. coli and a few closely related enteric bacteria, but not outside this group. In sum, DrpB is a poorly conserved nonessential division protein that improves the efficiency of cytokinesis under suboptimal conditions. Proteins like DrpB are likely to be a widespread feature of the bacterial cell division apparatus, but they are easily overlooked because mutants lack obvious shape defects.IMPORTANCE A thorough understanding of bacterial cell division requires identifying and characterizing all of the proteins that participate in this process. Our discovery of DrpB brings us one step closer to this goal in E. coli.

Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases.

Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile, σV is required for lysozyme resistance, and σV is activated in response to lysozyme. Once activated, σV, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here, we analyze the contribution of individual genes in the σV regulon to lysozyme resistance. Using CRISPR-Cas9-mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find that pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, encoded by pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1,000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed that loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but that loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also used CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficileIMPORTANCEClostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV operon is required for lysozyme resistance. Here, we used CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in C. difficile We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

Activation of the Extracytoplasmic Function σ Factor σP by ß-Lactams in Bacillus thuringiensis Requires the Site-2 Protease RasP.

Bacteria can utilize alternative σ factors to regulate sets of genes in response to changes in the environment. The largest and most diverse group of alternative σ factors are the extracytoplasmic function (ECF) σ factors. σP is an ECF σ factor found in Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Previous work showed that σP is induced by ampicillin, a ß-lactam antibiotic, and required for resistance to ampicillin. However, it was not known how activation of σP is controlled or what other antibiotics may activate σP Here, we report that activation of σP is specific to a subset of ß-lactams and that σP is required for resistance to these ß-lactams. We demonstrate that activation of σP is controlled by the proteolytic destruction of the anti-σ factor RsiP and that degradation of RsiP requires multiple proteases. Upon exposure to ß-lactams, the extracellular domain of RsiP is cleaved by an unknown protease, which we predict cleaves at site-1. Following cleavage by the unknown protease, the N terminus of RsiP is further degraded by the site-2 intramembrane protease RasP. Our data indicate that RasP cleavage of RsiP is not the rate-limiting step in σP activation. This proteolytic cascade leads to activation of σP, which induces resistance to ß-lactams likely via increased expression of ß-lactamases.IMPORTANCE The discovery of antibiotics to treat bacterial infections has had a dramatic and positive impact on human health. However, shortly after the introduction of a new antibiotic, bacteria often develop resistance. The bacterial cell envelope is essential for cell viability and is the target of many of the most commonly used antibiotics, including ß-lactam antibiotics. Resistance to ß-lactams is often dependent upon ß-lactamases. In B. cereus, B. thuringiensis, and some B. anthracis strains, the expression of some ß-lactamases is inducible. This inducible ß-lactamase expression is controlled by activation of an alternative σ factor called σP Here, we show that ß-lactam antibiotics induce σP activation by degradation of the anti-σ factor RsiP.

A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile.

Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile The xylose-inducible expression vector allows for ∼100-fold induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing investigators to control the timing and extent of gene silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression, ∼100-fold). To validate the utility of CRISPRi for deciphering gene function in C. difficile, we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA, and the peptidoglycan synthase gene pbp-0712 CRISPRi confirmed known or expected phenotypes associated with the loss of FtsZ and SlpA and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division, and protection against lysis.IMPORTANCEClostridioides difficile has become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treating C. difficile infections. Here we introduce new plasmid vectors that allow for titratable induction (P xyl ) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in C. difficile Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.

Bacterial sensing: A putative amphipathic helix in RsiV is the switch for activating σV in response to lysozyme.

Extra Cytoplasmic Function (ECF) σ factors are a diverse group of alternate σ factors bacteria use to respond to changes in the environment. The Bacillus subtilis ECF σ factor σV responds to lysozyme. In the absence of lysozyme, σV is held inactive by the anti-σ factor, RsiV. In the presence of lysozyme RsiV is degraded via regulated intramembrane proteolysis, which results in the release of σV and thus activation of lysozyme resistance genes. Signal peptidase is required to initiate degradation of RsiV. Previous work indicated that RsiV only becomes sensitive to signal peptidase upon direct binding to lysozyme. We have identified a unique domain of RsiV that is responsible for protecting RsiV from cleavage by signal peptidase in the absence of lysozyme. We provide evidence that this domain contains putative amphipathic helices. Disruption of the hydrophobic surface of these helices by introducing positively charged residues results in constitutive cleavage of RsiV by signal peptidase and thus constitutive σV activation. We provide further evidence that this domain contains amphipathic helices using a membrane-impermeable reagent. Finally, we show that upon lysozyme binding to RsiV, the hydrophobic face of the amphipathic helix becomes accessible to a membrane-impermeable reagent. Thus, we propose the amphipathic helices protect RsiV from cleavage in the absence of lysozyme. Additionally, we propose the amphipathic helices rearrange to form a suitable signal peptidase substrate upon binding of RsiV to lysozyme leading to the activation of σV.

Mtb PKNA/PKNB Dual Inhibition Provides Selectivity Advantages for Inhibitor Design To Minimize Host Kinase Interactions.

Drug resistant tuberculosis (TB) infections are on the rise and antibiotics that inhibit Mycobacterium tuberculosis through a novel mechanism could be an important component of evolving TB therapy. Protein kinase A (PknA) and protein kinase B (PknB) are both essential serine-threonine kinases in M. tuberculosis. Given the extensive knowledge base in kinase inhibition, these enzymes present an interesting opportunity for antimycobacterial drug discovery. This study focused on targeting both PknA and PknB while improving the selectivity window over related mammalian kinases. Compounds achieved potent inhibition (Ki ≈ 5 nM) of both PknA and PknB. A binding pocket unique to mycobacterial kinases was identified. Substitutions that filled this pocket resulted in a 100-fold differential against a broad selection of mammalian kinases. Reducing lipophilicity improved antimycobacterial activity with the most potent compounds achieving minimum inhibitory concentrations ranging from 3 to 5 µM (1-2 µg/mL) against the H37Ra isolate of M. tuberculosis.

In vitro phenotypic characterization of hepatitis C virus NS3 protease variants observed in clinical studies of telaprevir.

Telaprevir is a linear, peptidomimetic small molecule that inhibits hepatitis C virus (HCV) replication by specifically inhibiting the NS3·4A protease. In phase 3 clinical studies, telaprevir in combination with peginterferon and ribavirin (PR) significantly improved sustained virologic response (SVR) rates in genotype 1 chronic HCV-infected patients compared with PR alone. In patients who do not achieve SVR after treatment with telaprevir-based regimens, variants with mutations in the NS3·4A protease region have been observed. Such variants can contribute to drug resistance and limit the efficacy of treatment. To gain a better understanding of the viral resistance profile, we conducted phenotypic characterization of the variants using HCV replicons carrying site-directed mutations. The most frequently observed (significantly enriched) telaprevir-resistant variants, V36A/M, T54A/S, R155K/T, and A156S, conferred lower-level resistance (3- to 25-fold), whereas A156T and V36M+R155K conferred higher-level resistance (>25-fold) to telaprevir. Rarely observed (not significantly enriched) variants included V36I/L and I132V, which did not confer resistance to telaprevir; V36C/G, R155G/I/M/S, V36A+T54A, V36L+R155K, T54S+R155K, and R155T+D168N, which conferred lower-level resistance to telaprevir; and A156F/N/V, V36A+R155K/T, V36M+R155T, V36A/M+A156T, T54A+A156S, T54S+A156S/T, and V36M+T54S+R155K, which conferred higher-level resistance to telaprevir. All telaprevir-resistant variants remained fully sensitive to alpha interferon, ribavirin, and HCV NS5B nucleoside and nonnucleoside polymerase inhibitors. In general, the replication capacity of telaprevir-resistant variants was lower than that of the wild-type replicon.

Phenotypic characterization of resistant Val36 variants of hepatitis C virus NS3-4A serine protease.

In patients chronically infected with hepatitis C virus (HCV) strains of genotype 1, rapid and dramatic antiviral activity has been observed with telaprevir (VX-950), a highly selective and potent inhibitor of the HCV NS3-4A serine protease. HCV variants with substitutions in the NS3 protease domain were observed in some patients during telaprevir dosing. In this study, purified protease domain proteins and reconstituted HCV subgenomic replicons were used for phenotypic characterization of many of these substitutions. V36A/M or T54A substitutions conferred less than eightfold resistance to telaprevir. Variants with double substitutions at Val36 plus Thr54 had approximately 20-fold resistance to telaprevir, and variants with double substitutions at Val36 plus Arg155 or Ala156 had >40-fold resistance to telaprevir. An X-ray structure of the HCV strain H protease domain containing the V36M substitution in a cocomplex with an NS4A cofactor peptide was solved at a 2.4-A resolution. Except for the side chain of Met36, the V36M variant structure is identical to that of the wild-type apoenzyme. The in vitro replication capacity of most variants was significantly lower than that of the wild-type replicon in cells, which is consistent with the impaired in vivo fitness estimated from telaprevir-dosed patients. Finally, the sensitivity of these replicon variants to alpha interferon or ribavirin remained unchanged compared to that of the wild-type.

Phenotypic and structural analyses of hepatitis C virus NS3 protease Arg155 variants: sensitivity to telaprevir (VX-950) and interferon alpha.

Telaprevir (VX-950) is a highly selective, potent inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease. It has demonstrated strong antiviral activity in patients chronically infected with genotype 1 HCV when dosed alone or in combination with peginterferon alfa-2a. Substitutions of Arg(155) of the HCV NS3 protease domain have been previously detected in HCV isolates from some patients during telaprevir dosing. In this study, Arg(155) was replaced with various residues in genotype 1a protease domain proteins and in genotype 1b HCV subgenomic replicons. Characterization of both the purified enzymes and reconstituted replicon cells demonstrated that substitutions of Arg(155) with these residues conferred low level resistance to telaprevir (<25-fold). An x-ray structure of genotype 1a HCV protease domain with the R155K mutation, in a complex with an NS4A co-factor peptide, was determined at a resolution of 2.5A. The crystal structure of the R155K protease is essentially identical to that of the wild-type apoenzyme (Protein Data Bank code 1A1R) except for the side chain of mutated residue 155. Telaprevir was docked into the x-ray structure of the R155K protease, and modeling analysis suggests that the P2 group of telaprevir loses several hydrophobic contacts with the Lys(155) side chain. It was demonstrated that replicon cells containing substitutions at NS3 protease residue 155 remain fully sensitive to interferon alpha or ribavirin. Finally, these variant replicons were shown to have reduced replication capacity compared with the wild-type HCV replicon in cells.

Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir.

BACKGROUND & AIMS: Telaprevir (VX-950), a hepatitis C virus (HCV) NS3.4A protease inhibitor, has shown strong antiviral activity in phase 1 clinical studies. Because of high levels of HCV replication and the low fidelity of HCV polymerase, selection of resistant isolates during therapy may occur. METHODS: A highly sensitive sequencing method was developed in which approximately 80 clones/sample were analyzed to identify mutations in the NS3 protease catalytic domain in HCV genotype-1-infected patients dosed with 450 mg every 8 hours, 750 mg every 8 hours, or 1250 mg every 12 hours of telaprevir for 14 days. RESULTS: Mutations that confer low-level resistance (V36A/M, T54A, R155K/T, and A156S) and high-level resistance (A156V/T, 36+155, 36+156) to telaprevir were detected and correlated with telaprevir exposure and virologic response. Changes in the frequency of mutations after the end of dosing showed an inverse relationship between in vivo viral fitness and resistance. In the absence of telaprevir selective pressure the majority of resistant variants were replaced by wild-type virus within 3-7 months. CONCLUSIONS: Resistant HCV isolates are selected rapidly during therapy with the highly active protease inhibitor telaprevir. Combination therapy with pegylated interferon-alfa or other direct antiviral drugs seem mandatory to avoid developing resistance.

A structurally unrelated mimic of a Pseudomonas aeruginosa acyl-homoserine lactone quorum-sensing signal.

The pathogenic bacterium Pseudomonas aeruginosa uses acyl-homoserine lactone quorum-sensing signals to coordinate the expression of a battery of virulence genes in a cascade of regulatory events. The quorum-sensing signal that triggers the cascade is N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL), which interacts with two signal receptor-transcription factors, LasR and QscR. This signal is base labile, and it is degraded by mammalian PON lactonases. We have identified a structurally unrelated triphenyl mimic of 3OC12-HSL that is base-insensitive and PON-resistant. The triphenyl mimic seems to interact specifically with LasR but not with QscR. In silico analysis suggests that the mimic fits into the 3OC12-HSL-binding site of LasR and makes key contacts with LasR. The triphenyl mimic is an excellent scaffold for developing quorum-sensing inhibitors, and its stability and potency make it ideal for biotechnology uses such as heterologous gene expression.

Novel Pseudomonas aeruginosa quorum-sensing inhibitors identified in an ultra-high-throughput screen.

The opportunistic pathogen Pseudomonas aeruginosa has two complete acyl-homoserine lactone (acyl-HSL) signaling systems, LasR-LasI and RhlR-RhlI. LasI catalyzes the synthesis of N-3-oxododecanoyl homoserine lactone (3OC12-HSL), and LasR is a transcription factor that requires 3OC12-HSL as a ligand. RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (C4), and RhlR is a transcription factor that responds to C4. LasR and RhlR control the transcription of hundreds of P. aeruginosa genes, many of which are critical virulence determinants, and LasR is required for RhlR function. We developed an ultra-high-throughput cell-based assay to screen a library of approximately 200,000 compounds for inhibitors of LasR-dependent gene expression. Although the library contained a large variety of chemical structures, the two best inhibitors resembled the acyl-homoserine lactone molecule that normally binds to LasR. One compound, a tetrazole with a 12-carbon alkyl tail designated PD12, had a 50% inhibitory concentration (IC50) of 30 nM. The second compound, V-06-018, had an IC50 of 10 microM and is a phenyl ring with a 12-carbon alkyl tail. A microarray analysis showed that both compounds were general inhibitors of quorum sensing, i.e., the expression levels of most LasR-dependent genes were affected. Both compounds also inhibited the production of two quorum-sensing-dependent virulence factors, elastase and pyocyanin. These compounds should be useful for studies of LasR-dependent gene regulation and might serve as scaffolds for the identification of new quorum-sensing modulators.

In vitro characterization of the antibacterial spectrum of novel bacterial type II topoisomerase inhibitors of the aminobenzimidazole class.

Antibiotics with novel mechanisms of action are becoming increasingly important in the battle against bacterial resistance to all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV (topoIV) are the familiar targets of fluoroquinolone and coumarin antibiotics. Here we present the characterization of two members of a new class of synthetic bacterial topoII ATPase inhibitors: VRT-125853 and VRT-752586. These aminobenzimidazole compounds were potent inhibitors of both DNA gyrase and topoIV and had excellent antibacterial activities against a wide spectrum of problematic pathogens responsible for both nosocomial and community-acquired infections, including staphylococci, streptococci, enterococci, and mycobacteria. Consistent with the novelty of their structures and mechanisms of action, antibacterial potency was unaffected by commonly encountered resistance phenotypes, including fluoroquinolone resistance. In time-kill assays, VRT-125853 and VRT-752586 were bactericidal against Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, and Haemophilus influenzae, causing 3-log reductions in viable cells within 24 h. Finally, similar to the fluoroquinolones, relatively low frequencies of spontaneous resistance to VRT-125853 and VRT-752586 were found, a property consistent with their in vitro dual-targeting activities.