Biology subjects

Ellermeier, C. D.

Publications and source records attributed to Ellermeier, C. D..

10 recordsLinked to original sources

bioRxiv2026

Carboxypeptidase activity drives L,D-transpeptidase essentiality during vegetative growth and sporulation in Clostridioides difficile

In most bacteria, peptidoglycan contains mainly 4-3 crosslinks formed by penicillin-binding proteins (PBPs). But in the opportunistic pathogen Clostridioides difficile, 70% of the crosslinks are 3-3 crosslinks formed by L,D-transpeptidases (LDTs), and LDTs are essential for viability. PBPs and LDTs use different acyl donors for crosslinking; PBPs require a pentapeptide, while LDTs require a tetrapeptide. Here, we determined the source of the tetrapeptides in C. difficile and investigated the consequences of reengineering PG crosslinking from predominantly 3-3 to exclusively 4-3. We found that two D-alanyl-D-alanine carboxypeptidases (DD-CPase), DacA and DacC, supply LDTs with tetrapeptides during vegetative growth. Deleting these enzymes was sufficient to bypass the normal requirement for LDTs. The resulting mutant ({Delta}dacAC {Delta}ldt) was remarkably healthy despite the absence of 3-3 crosslinks. Its only major phenotypic defect was a 3- to 4-log decrease in sporulation, which could, however, be overcome by deleting a third DD-CPase, dacB. These findings fill gaps in our understanding of the pathway for LD-transpeptidation in C. difficile and imply that LDTs are not essential components of the elongasome or divisome, both of which function well in the complete absence of LDTs, provided there is sufficient pentapeptide to sustain crosslinking by PBPs. Thus, LDTs are essential for viability because C. difficile has intrinsically high levels of DD-CPase activity. Finally, we propose a model for how PBPs and LDTs work together during PG synthesis. In this model, PBPs construct a sparsely crosslinked PG sacculus that is subsequently strengthened with crosslinks introduced by LDTs. ImportanceSynthesis of peptidoglycan (PG) in the opportunistic gut pathogen Clostridioides difficile depends mostly on 3-3 crosslinks created by L,D-transpeptidases (LDTs). Here we converted C. difficile from an organism that relies primarily on 3-3 crosslinks made by LDTs to one that relies exclusively on 4-3 crosslinks made by an alternative family of crosslinking enzymes, the penicillin-binding proteins (PBPs). Our findings explain why 3-3 crosslinking predominates over 4-3 crosslinking, why C. difficile does not compensate for loss of LDTs by increasing PBP activity, and suggest a model for how PBPs and LDTs work together in this problematic pathogen.

microbiology↗

bioRxiv2026

CenIR, an essential BlaIR-family regulatory system in C. difficile

The CenIR regulatory system of Clostridioides difficile comprises a predicted transcriptional repressor, CenI, and a predicted membrane metalloprotease, CenR. The physiological role of CenIR and activating signal(s) are not known. CenIR belongs to the BlaIR family of regulators that mediate resistance to {beta}-lactam antibiotics. In canonical BlaIR systems, binding of a {beta}-lactam to the extracellular transpeptidase domain of BlaR triggers proteolysis of BlaI and thus induction of a closely linked {beta}-lactamase gene. However, CenR lacks a {beta}-lactam-binding domain and transposon mutagenesis indicated CenI is essential for viability even when {beta}-lactams are not present. Here we confirmed essentiality of CenIR and determined its regulon contains [~]12 genes, including an exported protein of unknown function (CDR_0474) that is induced about 500-fold and a peptidoglycan hydrolase (Cwp6) that is induced about 7-fold when cells are depleted of CenIR. There are no essential genes or {beta}-lactamases in the regulon. Phenotypic characterization of CenIR-depletion strains revealed slower growth, mild elongation and cell lysis. Deletion of cdr_0474 corrected all three defects, while deletion of cwp6 only rescued the lysis phenotype. It was possible to delete cenIR in either a {Delta}cdr_0474 or {Delta}cwp6 background. We propose that CenIR is essential because its absence leads to lysis due to Cwp6 overproduction. Bioinformatic analyses revealed the predicted extracellular sensing domains in annotated "BlaR" proteins are diverse. Thus, BlaIR systems are not dedicated to defense against {beta}-lactams but probably enable bacteria to adapt to a variety of environmental stimuli. ImportanceMany of the regulatory systems for controlling cell envelope biogenesis and stress responses have yet to be studied. Here we characterize a Clostridioides difficile BlaIR-like regulatory system that we have named CenIR for cell envelope. Unlike canonical BlaIR systems, which bind {beta}-lactams and induce a {beta}-lactamase, CenIR lacks a {beta}-lactam binding domain and is essential for viability even in the absence of antibiotics. We identified the genes in the regulon and found that CenIR is essential because its absence leads to overproduction of the Cwp6 peptidoglycan hydrolase. We also show that most annotated BlaIR-like systems lack a {beta}-lactam-binding domain, from which we infer that these systems have much broader physiological roles than generally appreciated.

microbiology↗

bioRxiv2025

Analysis of Essential Genes in Clostridioides difficile by CRISPRi and Tn-seq

Essential genes are interesting in their own right and as potential antibiotic targets. To date, only one report has identified essential genes on a genome-wide scale in Clostridioides difficile, a problematic pathogen for which treatment options are limited. That foundational study used large-scale transposon mutagenesis to identify 404 protein-encoding genes as likely to be essential for vegetative growth of the epidemic strain R20291. Here, we revisit the essential genes of strain R20291 using a combination of CRISPR interference (CRISPRi) and transposon-sequencing (Tn-seq). First, we targeted 181 of the 404 putatively essential genes with CRISPRi. We confirmed essentiality for >90% of the targeted genes and observed morphological defects for >80% of them. Second, we conducted a new Tn-seq analysis, which identified 346 genes as essential, of which 283 are in common with the previous report and might be considered a provisional essential gene set that minimizes false positives. We compare the list of essential genes to those of other bacteria, especially Bacillus subtilis, highlighting some noteworthy differences. Finally, we used fusions to red fluorescent protein (RFP) to identify 18 putative new cell division proteins, three of which are conserved in Bacillota but of largely unknown function. Collectively, our findings provide new tools and insights that advance our understanding of C. difficile. IMPORTANCEClostridioides difficile is an opportunistic pathogen for which better antibiotics are sorely needed. Most antibiotics target pathways that are essential for viability. Here we use saturation transposon mutagenesis and gene silencing with CRISPR interference to identify and characterize genes required for growth on laboratory media. Comparison to the model organism B. subtilis reveals many similarities and a few striking differences that warrant further study and may include opportunities for developing antibiotics that kill C. difficile without decimating the healthy microbiota needed to keep C. difficile in check.

microbiology↗

bioRxiv2025

Identification of two glycosyltransferases required for synthesis of membrane glycolipids in Clostridioides difficile

Clostridioides difficile infections cause over 12,000 deaths and an estimated one billion dollars in healthcare costs annually in the United States. The cell membrane is an essential structure that is important for protection from the extracellular environment, signal transduction, and transport of nutrients. The polar membrane lipids of C. difficile are [~]50% glycolipids, a higher percentage than most other organisms. The glycolipids of C. difficile consist of monohexosyldiradylglycerol (MHDRG) ([~]14%), dihexosyldiradylglycerol (DHDRG) ([~]15%), trihexosyldiradylglycerol (THDRG) ([~]5%), and a unique glycolipid aminohexosyl-hexosyldiradylglycerol (HNHDRG) ([~]16%). Previously, we found HexSDF are required for synthesis of HNHDRG. The enzymes required for synthesis of MHDRG, DHDRG, and THDRG are not known. In this study, we identified the glycosyltransferases UgtA (CDR20291_0008), which is required for synthesis of all glycolipids, and UgtB (CDR20291_1186), which is required for synthesis of DHDRG and THDRG. We propose a model where UgtA synthesizes only MHDRG, HexSDF synthesize HNHDRG from MHDRG, and UgtB synthesizes DHDRG and potentially THDRG from MHDRG. We also report that glycolipids are important for critical cell functions, including sporulation, cell size and morphology, maintaining membrane fluidity, colony morphology, and resistance to some membrane targeting antimicrobials. ImportanceClostridioides difficile infections are the leading cause of healthcare associated diarrhea. C. difficile poses a risk to public health due to its ability to form spores and cause recurrent infections. Glycolipids make up [~]50% of the polar lipids in the C. difficile membrane, a higher percentage than other common pathogens and include a unique glycolipid not present in other organisms. Here, we identify glycosyltransferases required for synthesis of glycolipids in C. difficile and demonstrate the important role glycolipids play in C. difficile physiology.

microbiology↗

bioRxiv2024

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.

microbiology↗

bioRxiv2022

HexSDF are Required for Synthesis of a Novel Glycolipid that Mediates Daptomycin and Bacitracin Resistance in C. difficile

Clostridioides difficile is a Gram-positive opportunistic pathogen that results in 250,000 infections, 12,000 deaths, and $1 billion in medical costs in the US each year. There has been recent interest in using a daptomycin analog, Surotomycin, to treat C. difficile infections. Daptomycin interacts with both phosphatidylglycerol and Lipid II to disrupt the membrane and halt peptidoglycan synthesis. C. difficile has an unusual lipid membrane composition as it has no phosphatidylserine or phosphatidylethanolamine, and [~]50% of its membrane is composed of glycolipids, including the unique C. difficile lipid aminohexosyl-hexosyldiradylglycerol (HNHDRG). We identified a two-component system (TCS) HexRK that is required for C. difficile resistance to daptomycin. Using RNAseq we found that HexRK regulates a three gene operon of unknown function hexSDF. Based on bioinformatic predictions, hexS encodes a monogalactosyldiacylglycerol synthase, hexD encodes a polysaccharide deacetylase, and hexF encodes an MprF-like flippase. We find that deletion of hexRK leads to a 4-fold decrease in daptomycin MIC, and that deletion of hexSDF leads to an 8-16-fold decrease in daptomycin MIC. The {Delta}hexSDF mutant is also 4-fold less resistant to bacitracin but no other cell wall active antibiotics. Our data indicate that in the absence of HexSDF the phospholipid membrane composition is altered. In WT C. difficile the unique glycolipid, HNHDRG makes up [~]17% of the lipids in the membrane. However, in a {Delta}hexSDF mutant, HNHDRG is completely absent. While it is unclear how HNHDRG contributes daptomycin resistance, the requirement for bacitracin resistance suggests it has a general role in cell membrane biogenesis. ImportanceClostridioides difficile is a major cause of hospital acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. Little is understood about C. difficile membrane lipids, but a unique glycolipid, HNHDRG, has been previously identified in C. difficile and, currently, has not been identified in other organisms. Here we show that HexSDF and HexRK are required for synthesis of HNHDRG, and that production of HNHDRG impacts resistance to daptomycin and bacitracin.

microbiology↗

bioRxiv2022

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, where 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. Here 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 the wal operon is essential and identified morphological defects and cell lysis as the major terminal phenotypes of altered wal expression. We also used 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 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 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 in other Firmicutes. For example, down-regulation of Wal signaling caused C. difficile cells to become longer rather than shorter, as in Bacillus subtilis. Likewise, down-regulation of Wal rendered C. difficile more sensitive to vancomycin, whereas reduced Wal activity is linked to increased vancomycin resistance in Staphylococcus aureus. ImportanceThe WalRK two-component system (TCS) is essential for coordinating synthesis and turnover of peptidoglycan in Firmicutes. Here we investigate 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 in 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.

microbiology↗

bioRxiv2020

Lysozyme resistance in C. 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 {sigma}V is required for lysozyme resistance and {sigma}V is activated in response to lysozyme. Once activated {sigma}V, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here we analyze the contribution of individual genes in the csfV regulon to lysozyme resistance. Using CRISPR-Cas9 mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, 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 1000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but 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 use CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile. ImportanceClostridioides 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 use 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 as part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

microbiology↗

bioRxiv2019

Activation of the Extracytoplasmic Function σ factor σP by β-lactams in Bacillus thuringiensis requires the site-2 protease RasP

Bacteria can utilize alternative {sigma} factors to regulate sets of genes in response to changes in the environment. The largest and most diverse group of alternative {sigma} factors are the Extracytoplasmic Function (ECF) {sigma} factors. {sigma}P is an ECF {sigma} factor found in Bacillus anthracis, B. cereus, and B. thuringiensis. Previous work showed {sigma}P is induced by ampicillin, a {beta}-lactam antibiotic, and required for resistance to ampicillin. However, it was not known how activation of {sigma}P is controlled or what other antibiotics may activate {sigma}P. Here we report that activation of {sigma}P is specific to a subset of {beta}-lactams and {sigma}P is required for resistance to these {beta}-lactams. We demonstrate that activation of {sigma}P is controlled by the proteolytic destruction of the anti-{sigma} factor, RsiP, and that degradation of RsiP requires multiple proteases. Upon exposure to {beta}-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 {sigma}P activation. This proteolytic cascade leads to activation of {sigma}P which induces resistance to {beta}-lactams likely via increased expression of {beta}-lactamases.\n\nImportanceThe 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 {beta}-lactam antibiotics. Resistance to {beta}-lactams is often dependent upon {beta}-lactamases. In B. cereus, B. thuringiensis and some B. anthracis strains the expression of some {beta}-lactamases is inducible. This inducible {beta}-lactamase expression is controlled by activation of an alternative {sigma} factor called {sigma}P. Here we show that {beta}-lactam antibiotics induce {sigma}P activation by degradation of the anti-{sigma} factor RsiP.

microbiology↗

bioRxiv2018

A xylose-inducible expression system and a CRISPRi-plasmid for targeted knock-down 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 expression of three genes involved in 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 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.\n\nImportanceClostridioides 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 (Pxyl) 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.

microbiology↗