scholarly journals Regulation and Anaerobic Function of the Clostridioides difficile β-Lactamase

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Brindar K. Sandhu ◽  
Adrianne N. Edwards ◽  
Sarah E. Anderson ◽  
Emily C. Woods ◽  
Shonna M. McBride
Keyword(s):  
Antibiotic Resistance ◽  
Redox State ◽  
Signal Sequence ◽  
Bacterial Species ◽  
Resistance Mechanism ◽  
Regulatory System ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Reducing Environment

ABSTRACT Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a β-lactamase, which is a common β-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficile bla operon that encodes a lipoprotein of unknown function and a β-lactamase that was greatly induced in response to several classes of β-lactam antibiotics. An in-frame deletion of the operon abolished β-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the β-lactam ampicillin. We found that the activity of this β-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to β-lactams. These results provide support for the function of a β-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile β-lactamase activity.

scholarly journals Characterization of a β-lactamase that contributes to intrinsic β-lactam resistance inClostridioides difficile

2019 ◽  
Author(s):  
Brindar K. Sandhu ◽  
Adrianne N. Edwards ◽  
Sarah E. Anderson ◽  
Emily C. Woods ◽  
Shonna M. McBride
Keyword(s):  
Membrane Protein ◽  
Unknown Function ◽  
Signal Sequence ◽  
Bacterial Species ◽  
Resistance Mechanism ◽  
Regulatory System ◽  
Gram Positive ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea

ABSTRACTClostrididioides difficilecauses severe antibiotic-associated diarrhea and colitis.C. difficileis an anaerobic, Gram-positive spore former that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance ofC. difficileto β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance inC. difficileare not fully understood. Our data reinforce prior evidence thatC. difficileproduces a β-lactamase, which is a common β-lactam resistance mechanism found in other bacterial species. We identified an operon encoding a lipoprotein of unknown function and a β-lactamase that was greatly induced in response to several classes of β-lactam antibiotics. An in-frame deletion of the operon abolished β-lactamase activity inC. difficilestrain 630Δermand resulted in decreased resistance to the β-lactam ampicillin. We found that the activity of this β-lactamase, herein named BlaD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a co-transcribed lipoprotein, BlaX, aids in BlaD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of theblaXDoperon in response to β-lactams. These results provide support for the function of a β-lactamase inC. difficileantibiotic resistance, and reveal the unique roles of a co-regulated lipoprotein and reducing environment in β-lactamase activity.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal human pathogen. One of the highest risk factors for contractingC. difficileinfection is antibiotic treatment, which causes microbiome dysbiosis.C. difficileis resistant to β-lactam antibiotics, the most commonly prescribed class of antibiotics.C. difficileproduces a recently discovered β-lactamase, which cleaves and inactivates numerous β-lactams. In this study, we report the contribution of this anaerobic β-lactamase to ampicillin resistance inC. difficile, as well as the transcriptional regulation of the gene,blaD, by a BlaRI system. In addition, our data demonstrate co-transcription ofblaDwithblaX, which encodes a membrane protein of previously unknown function. Furthermore, we provide evidence that BlaX enhances β-lactamase activity in a portion ofC. difficilestrains. This study demonstrates a novel interaction between a β-lactamase and a membrane protein in a Gram-positive pathogen, and due to the anaerobic nature of the β-lactamase activity, suggests that more β-lactamases are yet to be identified in other anaerobes.

scholarly journals Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases

2020 ◽  
Vol 202 (22) ◽  
Author(s):  
Gabriela M. Kaus ◽  
Lindsey F. Snyder ◽  
Ute Müh ◽  
Matthew J. Flores ◽  
David L. Popham ◽  
...  
Keyword(s):  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Content Type ◽  
Hospital Acquired Infections ◽  
Major Mechanism ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
High Level ◽  
Hospital Acquired ◽  
Very High

ABSTRACT 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. difficile. IMPORTANCE Clostridioides 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.

scholarly journals Relationship between Glycopeptide Production and Resistance in the Actinomycete Nonomuraea sp. ATCC 39727

2014 ◽  
Vol 58 (9) ◽  
pp. 5191-5201 ◽  
Author(s):  
Giorgia Letizia Marcone ◽  
Elisa Binda ◽  
Lucia Carrano ◽  
Mervyn Bibb ◽  
Flavia Marinelli
Keyword(s):  
Antibiotic Resistance ◽  
Gene Dosage ◽  
Antibiotic Production ◽  
Resistance Mechanism ◽  
Biosynthetic Gene Cluster ◽  
Penicillin G ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
High Level

ABSTRACTGlycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens.Nonomuraeasp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonicalvanHAXgenes. Consequently, we investigated the role of the β-lactam-sensitived,d-peptidase/d,d-carboxypeptidase encoded byvanYn, the onlyvan-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic inNonomuraeasp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we variedvanYngene dosage and expressedvanHatAatXatfrom the teicoplanin producerActinoplanes teichomyceticusinNonomuraeasp. ATCC 39727. Knocking outvanYn, complementing avanYnmutant, or duplicatingvanYnhad no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production.Nonomuraeasp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYnactivity. The heterologous expression ofvanHatAatXatincreased A40926 resistance inNonomuraeasp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. ThevanYn-based self-resistance inNonomuraeasp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants ofEnterococcus faeciumselectedin vitrofor high-level resistance to glycopeptides and penicillins.

scholarly journals Linearmycins Activate a Two-Component Signaling System Involved in Bacterial Competition and Biofilm Morphology

2017 ◽  
Vol 199 (18) ◽  
Author(s):  
Reed M. Stubbendieck ◽  
Paul D. Straight
Keyword(s):  
Antibiotic Resistance ◽  
Abc Transporter ◽  
Bacterial Communities ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Bioactive Metabolites ◽  
Signaling System ◽  
Content Type ◽  
Two Component ◽  
Analytical Approaches

ABSTRACT Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilis. IMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that includes the gastrointestinal tract of animals and the rhizosphere of plants. Interactions between different species in bacterial communities have impacts on our health and industry. However, many approaches currently used to study whole bacterial communities do not resolve mechanistic details of interspecies interactions, including how bacteria sense and respond to their competitors. Using a competition model, we have uncovered dual functions for a previously uncharacterized two-component signaling system involved in specific antibiotic resistance and biofilm morphology. Insights gleaned from signaling within interspecies interaction models build a more complete understanding of gene functions important for bacterial communities and will enhance community-level analytical approaches.

scholarly journals Gram-Negative Taxa and Antimicrobial Susceptibility after Fecal Microbiota Transplantation for Recurrent Clostridioides difficile Infection

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Danielle Barrios Steed ◽  
Tiffany Wang ◽  
Divyanshu Raheja ◽  
Alex D. Waldman ◽  
Ahmed Babiker ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Susceptibility ◽  
Fecal Microbiota Transplantation ◽  
Antibiotic Prescription ◽  
Multidrug Resistant ◽  
Fecal Microbiota ◽  
Gram Negative ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Susceptibility Profiles

ABSTRACT Fecal microbiota transplantation (FMT) has promising applications in reducing multidrug-resistant organism (MDRO) colonization and antibiotic resistance (AR) gene abundance. However, data on clinical microbiology results after FMT are limited. We examined the changes in antimicrobial susceptibility profiles in patients with Gram-negative infections in the year before and the year after treatment with FMT for recurrent Clostridioides difficile infection (RCDI). We also examined whether a history of FMT changed health care provider behavior with respect to culture ordering and antibiotic prescription. Medical records for RCDI patients who underwent FMT at Emory University between July 2012 and March 2017 were reviewed retrospectively. FMT-treated patients with Gram-negative culture data in the 1-year period preceding and the 1-year period following FMT were included. Demographic and clinical data were abstracted, including CDI history, frequency of Gram-negative cultures, microbiological results, and antibiotic prescription in response to positive cultures in the period following FMT. Twelve patients were included in this case series. We pooled data from infections at all body sites and found a decrease in the number of total and Gram-negative cultures post-FMT. We compared susceptibility profiles across taxa given the potential for horizontal transmission of AR elements and observed increased susceptibility to nitrofurantoin, trimethoprim-sulfamethoxazole, and the aminoglycosides. FMT did not drastically influence health care provider ordering of bacterial cultures or antibiotic prescribing practices. We observed a reduction in Gram-negative cultures and a trend toward increased antimicrobial susceptibility. This study supports further investigation of FMT as a means of improving antimicrobial susceptibility. IMPORTANCE Fecal microbiota transplantation (FMT), which is highly efficacious in treating recurrent C. difficile infection (RCDI), has a promising application in decolonization of multidrug-resistant organisms, reduction of antibiotic resistance gene abundance, and restoration of healthy intestinal microbiota. However, data representing clinical microbiology results after FMT are limited. We sought to characterize the differences in culture positivity and antimicrobial susceptibility profiles in patients with Gram-negative infections in the year before and the year after FMT for RCDI. Drawing on prior studies that had demonstrated the success of FMT in eradicating extraintestinal infections and the occurrence of patient-level interspecies transfer of resistance elements, we employed an agnostic analytic approach of reviewing the data irrespective of body site or species. In a small RCDI population, we observed an improvement in the antimicrobial susceptibility profile of Gram-negative bacteria following FMT, which supports further study of FMT as a strategy to combat antibiotic resistance.

scholarly journals The Inactivation of Enzymes Belonging to the Central Carbon Metabolism Is a Novel Mechanism of Developing Antibiotic Resistance

mSystems ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Teresa Gil-Gil ◽  
Fernando Corona ◽  
José Luis Martínez ◽  
Alejandra Bernardini
Keyword(s):  
Antibiotic Resistance ◽  
Stenotrophomonas Maltophilia ◽  
Bacterial Species ◽  
Bacterial Metabolism ◽  
Resistant Bacteria ◽  
Uridine Diphosphate ◽  
Biosynthesis Pathway ◽  
Content Type ◽  
Peptidoglycan Biosynthesis ◽  
Central Carbon

ABSTRACT Fosfomycin is a bactericidal antibiotic, analogous to phosphoenolpyruvate, that exerts its activity by inhibiting the activity of MurA. This enzyme catalyzes the first step of peptidoglycan biosynthesis, the transfer of enolpyruvate from phosphoenolpyruvate to uridine-diphosphate-N-acetylglucosamine. Fosfomycin is increasingly being used, mainly for treating infections caused by Gram-negative multidrug-resistant bacteria. The mechanisms of mutational resistance to fosfomycin in Stenotrophomonas maltophilia, an opportunistic pathogen characterized by its low susceptibility to commonly used antibiotics, were studied in the current work. None of the mechanisms reported so far for other organisms, which include the production of fosfomycin-inactivating enzymes, target modification, induction of an alternative peptidoglycan biosynthesis pathway, and the impaired entry of the antibiotic, are involved in the acquisition of such resistance by this bacterial species. Instead, the unique cause of resistance in the mutants studied is the mutational inactivation of different enzymes belonging to the Embden-Meyerhof-Parnas central metabolism pathway. The amount of intracellular fosfomycin accumulation did not change in any of these mutants, showing that neither inactivation nor transport of the antibiotic is involved. Transcriptomic analysis also showed that the mutants did not present changes in the expression level of putative alternative peptidoglycan biosynthesis pathway genes or any related enzyme. Finally, the mutants did not present an increased phosphoenolpyruvate concentration that might compete with fosfomycin for its binding to MurA. On the basis of these results, we describe a completely novel mechanism of antibiotic resistance based on mutations of genes encoding metabolic enzymes. IMPORTANCE Antibiotic resistance has been largely considered a specific bacterial response to an antibiotic challenge. Indeed, its study has been mainly concentrated on mechanisms that affect the antibiotics (mutations in transporters, efflux pumps, and antibiotic-modifying enzymes, or their regulators) or their targets (i.e., target mutations, protection, or bypass). Usually, antibiotic resistance-associated metabolic changes were considered a consequence (fitness costs) and not a cause of antibiotic resistance. Herein, we show that alterations in the central carbon bacterial metabolism can also be the cause of antibiotic resistance. In the study presented here, Stenotrophomonas maltophilia acquires fosfomycin resistance through the inactivation of glycolytic enzymes belonging to the Embden-Meyerhof-Parnas pathway. Besides resistance to fosfomycin, this inactivation also impairs the bacterial gluconeogenic pathway. Together with previous work showing that antibiotic resistance can be under metabolic control, our results provide evidence that antibiotic resistance is intertwined with the bacterial metabolism.

scholarly journals The Formation of Streptococcus mutans Persisters Induced by the Quorum-Sensing Peptide Pheromone Is Affected by the LexA Regulator

2015 ◽  
Vol 197 (6) ◽  
pp. 1083-1094 ◽  
Author(s):  
Vincent Leung ◽  
Dragana Ajdic ◽  
Stephanie Koyanagi ◽  
Céline M. Lévesque
Keyword(s):  
Quorum Sensing ◽  
Streptococcus Mutans ◽  
Bacterial Species ◽  
Regulatory System ◽  
Bacterial Survival ◽  
Chronic Infections ◽  
Persister Cells ◽  
Gene Encoding ◽  
Content Type ◽  
Peptide Pheromone

The presence of multidrug-tolerant persister cells within microbial populations has been implicated in the resiliency of bacterial survival against antibiotic treatments and is a major contributing factor in chronic infections. The mechanisms by which these phenotypic variants are formed have been linked to stress response pathways in various bacterial species, but many of these mechanisms remain unclear. We have previously shown that in the cariogenic organismStreptococcus mutans, the quorum-sensing peptide CSP (competence-stimulating peptide) pheromone was a stress-inducible alarmone that triggered an increased formation of multidrug-tolerant persisters. In this study, we characterized SMU.2027, a CSP-inducible gene encoding a LexA ortholog. We showed that in addition to exogenous CSP exposure, stressors, including heat shock, oxidative stress, and ofloxacin antibiotic, were capable of triggering expression oflexAin an autoregulatory manner akin to that of LexA-like transcriptional regulators. We demonstrated the role of LexA and its importance in regulating tolerance toward DNA damage in a noncanonical SOS mechanism. We showed its involvement and regulatory role in the formation of persisters induced by the CSP-ComDE quorum-sensing regulatory system. We further identified key genes involved in sugar and amino acid metabolism, the clustered regularly interspaced short palindromic repeat (CRISPR) system, and autolysin from transcriptomic analyses that contribute to the formation of quorum-sensing-induced persister cells.

scholarly journals Clostridium difficileLipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Oscar R. Diaz ◽  
Cameron V. Sayer ◽  
David L. Popham ◽  
Aimee Shen
Keyword(s):  
Clostridium Difficile ◽  
Spore Germination ◽  
Protective Layer ◽  
Thick Layer ◽  
Lytic Enzyme ◽  
Gram Positive ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Biochemical Analyses

ABSTRACTClostridium difficile, also known asClostridioides difficile, is a Gram-positive, spore-forming bacterium that is a leading cause of antibiotic-associated diarrhea.C. difficileinfections begin when its metabolically dormant spores germinate to form toxin-producing vegetative cells. Successful spore germination depends on the degradation of the cortex, a thick layer of modified peptidoglycan that maintains dormancy. Cortex degradation is mediated by the SleC cortex lytic enzyme, which is thought to recognize the cortex-specific modification muramic-δ-lactam.C. difficilecortex degradation also depends on thePeptostreptococcaceae-specific lipoprotein GerS for unknown reasons. In this study, we tested whether GerS regulates production of muramic-δ-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of ΔgerSspores to those of spores lacking either CwlD or PdaA, both of which mediate cortex modification inBacillus subtilis, we determined thatC. difficileGerS, CwlD, and PdaA are all required to generate muramic-δ-lactam. Both GerS and CwlD were needed to cleave the peptide side chains from N-acetylmuramic acid, suggesting that these two factors act in concert. Consistent with this hypothesis, biochemical analyses revealed that GerS and CwlD directly interact and that CwlD modulates GerS incorporation into mature spores. Since ΔgerS, ΔcwlD, and ΔpdaAspores exhibited equivalent germination defects, our results indicate thatC. difficilespore germination depends on cortex-specific modifications, reveal GerS as a novel regulator of these processes, and highlight additional differences in the regulation of spore germination inC. difficilerelative toB. subtilisand other spore-forming organisms.IMPORTANCEThe Gram-positive, spore-forming bacteriumClostridium difficileis a leading cause of antibiotic-associated diarrhea. BecauseC. difficileis an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of ΔgerSspores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwlD and PdaA. These analyses revealed that GerS, CwlD, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in ΔgerS, ΔcwlD, and ΔpdaAmutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwlD are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight intoC. difficilespore germination.

scholarly journals Production of p-cresol by Decarboxylation of p-HPA by All Five Lineages of Clostridioides difficile Provides a Growth Advantage

2021 ◽  
Vol 11 ◽  
Author(s):  
Mark A. Harrison ◽  
Harparkash Kaur ◽  
Brendan W. Wren ◽  
Lisa F. Dawson
Keyword(s):  
Pseudomembranous Colitis ◽  
Bacterial Species ◽  
Host Cells ◽  
Gram Negative ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Tyrosine Metabolism ◽  
Hydroxyphenylacetic Acid ◽  
Intracellular Phosphate ◽  
Differential Efficiency

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is capable of causing severe symptoms, such as pseudomembranous colitis and toxic megacolon. An unusual feature of C. difficile is the distinctive production of high levels of the antimicrobial compound para-cresol. p-Cresol production provides C. difficile with a competitive colonization advantage over gut commensal species, in particular, Gram-negative species. p-Cresol is produced by the conversion of para-hydroxyphenylacetic acid (p-HPA) via the actions of HpdBCA decarboxylase coded by the hpdBCA operon. Host cells and certain bacterial species produce p-HPA; however, the effects of p-HPA on the viability of C. difficile and other gut microbiota are unknown. Here we show that representative strains from all five C. difficile clades are able to produce p-cresol by two distinct mechanisms: (i) via fermentation of p-tyrosine and (ii) via uptake and turnover of exogenous p-HPA. We observed strain-specific differences in p-cresol production, resulting from differential efficiency of p-tyrosine fermentation; representatives of clade 3 (CD305) and clade 5 (M120) produced the highest levels of p-cresol via tyrosine metabolism, whereas the toxin A-/B+ isolate from clade 4 (M68) produced the lowest level of p-cresol. All five lineages share at least 97.3% homology across the hpdBCA operon, responsible for decarboxylation of p-HPA to p-cresol, suggesting that the limiting step in p-cresol production may result from tyrosine to p-HPA conversion. We identified that elevated intracellular p-HPA, modulated indirectly via CodY, controls p-cresol production via inducing the expression of HpdBCA decarboxylase ubiquitously in C. difficile populations. Efficient turnover of p-HPA is advantageous to C. difficile as p-HPA has a deleterious effect on the growth of C. difficile and other representative Gram-negative gut bacteria, transduced potentially by the disruption of membrane permeability and release of intracellular phosphate. This study provides insights into the importance of HpdBCA decarboxylase in C. difficile pathogenesis, both in terms of p-cresol production and detoxification of p-HPA, highlighting its importance to cell survival and as a highly specific therapeutic target for the inhibition of p-cresol production across C. difficile species.

scholarly journals Sulfur Assimilation Alters Flagellar Function and Modulates the Gene Expression Landscape of Serratia marcescens

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Mark T. Anderson ◽  
Lindsay A. Mitchell ◽  
Anna Sintsova ◽  
Katherine A. Rice ◽  
Harry L. T. Mobley
Keyword(s):  
Serratia Marcescens ◽  
Bacterial Species ◽  
Regulatory System ◽  
Nutrient Utilization ◽  
Cytolytic Activity ◽  
Large Set ◽  
Cysteine Biosynthesis ◽  
Sulfur Assimilation ◽  
Major Pathway ◽  
Content Type

ABSTRACT Sulfur is an essential nutrient that contributes to cellular redox homeostasis, transcriptional regulation, and translation initiation when incorporated into different biomolecules. Transport and reduction of extracellular sulfate followed by cysteine biosynthesis is a major pathway of bacterial sulfur assimilation. For the opportunistic pathogen Serratia marcescens, function of the cysteine biosynthesis pathway is required for extracellular phospholipase activity and flagellum-mediated surface motility, but little else is known about the influence of sulfur assimilation on the physiology of this organism. In this work, it was determined that an S. marcescens cysteine auxotroph fails to differentiate into hyperflagellated and elongated swarmer cells and that cysteine, but not other organic sulfur molecules, restores swarming motility to these bacteria. The S. marcescens cysteine auxotroph further exhibits reduced transcription of phospholipase, hemolysin, and flagellin genes, each of which is subject to transcriptional control by the flagellar regulatory system. Based on these data and the central role of cysteine in sulfur assimilation, it was reasoned that environmental sulfur availability may contribute to the regulation of these functions in S. marcescens. Indeed, bacteria that are starved for sulfate exhibit substantially reduced transcription of the genes for hemolysin, phospholipase, and the FlhD flagellar master regulator. A global transcriptomic analysis further defined a large set of S. marcescens genes that are responsive to extracellular sulfate availability, including genes that encode membrane transport, nutrient utilization, and metabolism functions. Finally, sulfate availability was demonstrated to alter S. marcescens cytolytic activity, suggesting that sulfate assimilation may impact the virulence of this organism. IMPORTANCE Serratia marcescens is a versatile bacterial species that inhabits diverse environmental niches and is capable of pathogenic interactions with host organisms ranging from insects to humans. This report demonstrates for the first time the extensive impacts that environmental sulfate availability and cysteine biosynthesis have on the transcriptome of S. marcescens. The finding that greater than 1,000 S. marcescens genes are differentially expressed depending on sulfate availability suggests that sulfur abundance is a crucial factor that controls the physiology of this organism. Furthermore, the high relative expression levels for the putative virulence factors flagella, phospholipase, and hemolysin in the presence of sulfate suggests that a sulfur-rich host environment could contribute to the transcription of these genes during infection.

Sign in / Sign up

Export Citation Format

Share Document