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 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 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 Molecular Characterization of Zinc (Zn) Resistant Bacteria in Banger River, Pekalongan, Indonesia

2018 ◽  
Vol 10 (3) ◽  
pp. 622-628
Author(s):  
Fitri Arum Sasi ◽  
Hermin Pancasakti Kusumaningrum ◽  
Anto Budiharjo
Keyword(s):  
Bacterial Species ◽  
Selective Medium ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative ◽  
Indigenous Bacteria ◽  
Gram Positive Bacteria ◽  
Sequences Analysis

Indigenous bacteria are able to remove the metals contamination in environment. This study aimed to assess the resistance of bacterial species to Zinc (Zn) in Banger River, Pekalongan City. The bacteria from three different parts of Banger River were isolated and inoculated in Zn-selective medium. Then, molecular identification to determine the bacteria species was conducted using polymerase chain reaction (PCR) by applying forward-reverse 16SrRNA gene primers. The sequences analysis was conducted using MUSCLE and MEGA6. There were seven dominant species that possibly resistant to Zn. Approximately, every isolate could reach more than 95 % from 2000 ppm of Zn in the medium. The higher absorption of Zn was found in Z5 isolate. The seven bacteria species were clustered into nine genera i.e. Klebsiela, Xenorhabdus, Cronobacter, Enterobacter, Escherichia, Shigella and Sporomusa known as Gram Negative bacteria and Clostridium and Bacillus as Gram Positive bacteria. In Gram Positive bacteria, especially Bacillus sp, carboxyl group in peptidoglycan play a role as metal binder. In Gram-negative bacteria, lipopolysaccharide (LPS) which is highly anionic component on the outer membrane, able to catch the Zn. Besides that, Enterobacter activates endogen antioxidants such as glutathione peroxidase (GSHPx), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD). The research found there was possible seven novel indigenous bacteria species in Banger that able to remove Zn from the sediment extremely. This finding can be developed as an eco-friendly approach to reduce metals pollution using local microorganisms.

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 Identification and characterization of a family of toxin–antitoxin systems related to the Enterococcus faecalis plasmid pAD1 par addiction module

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 2930-2940 ◽  
Author(s):  
Keith E. Weaver ◽  
Shirisha G. Reddy ◽  
Cassandra L. Brinkman ◽  
Smita Patel ◽  
Kenneth W. Bayles ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Biological Function ◽  
Bacterial Species ◽  
Saturation Mutagenesis ◽  
Regulatory Rna ◽  
Gram Positive ◽  
Peptide Toxin ◽  
Identification And Characterization ◽  
Genetic Context

The par locus of the Enterococcus faecalis plasmid pAD1 is an RNA-regulated addiction module encoding the peptide toxin Fst. Homology searches revealed that Fst belongs to a family of at least nine related peptides encoded on the chromosomes and plasmids of six different Gram-positive bacterial species. Comparison of an alignment of these peptides with the results of a saturation mutagenesis analysis indicated regions of the peptides important for biological function. Examination of the genetic context of the fst genes revealed that all of these peptides are encoded within par-like loci with conserved features similar to pAD1 par. All four Ent. faecalis family members were demonstrated to produce the expected toxin-encoding and regulatory RNA products. The locus from the Ent. faecalis plasmid pAMS1 was demonstrated to function as an addiction module and Fst was shown to be toxic to Staphylococcus aureus, suggesting that a plasmid-encoded module in that species is performing the same function. Thus, the pAD1-encoded par locus appears to be the prototype of a family of related loci found in several Gram-positive species.

scholarly journals Characterization of exosporium layer variability of Clostridioides difficile spores in the epidemically relevant strain R20291

2020 ◽  
Author(s):  
Marjorie Pizarro-Guajardo ◽  
Paulina Calderón ◽  
Alba Romero-Rodriguez ◽  
Daniel Paredes-Sabja
Keyword(s):  
Spore Coat ◽  
Coat Proteins ◽  
Anaerobic Conditions ◽  
Host Interactions ◽  
Clostridioides Difficile ◽  
Transmission Electron ◽  
Antibiotic Associated Diarrhea ◽  
Biological Aspects ◽  
Spore Coat Proteins

AbstractClostridioides difficile is a Gram-positive anaerobic intestinal pathogenic bacterium and the causative agent of antibiotic-associated diarrhea and spores are the transmission vehicle of the disease. In C. difficile spores, the outermost exosporium layer is the first barrier of interaction with the host and should carry spore ligands involved in spore-host interactions. C. difficile forms two types of spores (i.e., thin and thick exosporium layers). In this communication, we contribute to understand several biological aspects of these two exosporium morphotypes. By transmission electron microscopy, we demonstrate that both exosporium morphotypes appear simultaneously during sporulation and that the laminations of the spore-coat are formed under anaerobic conditions. Nycodenz density-gradient allows enrichment of spores with a thick-exosporium layer morphotype and presence of polar appendage. Using translational fluorescent fusions with exosporium proteins BclA3, CdeA, CdeC and CdeM as well as with several spore coat proteins, we observed that expression intensity and distribution of SNAP-translational fusions in R20291 strain is highly heterogeneous. Electron micrographs demonstrate that multicopy expression of CdeC, but not CdeM, SNAP translational fusion, increases the abundance of the thick exosporium morphotype. Collectively, these results raise further questions on how these distinctive exosporium morphotypes are made during spore formation.

scholarly journals Characterization of Enterococcus faecalis Alkaline Phosphatase and Use in IdentifyingStreptococcus agalactiae Secreted Proteins

1999 ◽  
Vol 181 (18) ◽  
pp. 5790-5799 ◽  
Author(s):  
Martin H. Lee ◽  
Aphakorn Nittayajarn ◽  
R. Paul Ross ◽  
Cynthia B. Rothschild ◽  
Derek Parsonage ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Membrane Protein ◽  
Enterococcus Faecalis ◽  
Signal Sequence ◽  
Subcellular Location ◽  
Lactose Permease ◽  
Wild Type ◽  
Alkaline Phosphatases ◽  
Gram Positive ◽  
Gram Positive Bacteria

ABSTRACT We have identified and characterized an Enterococcus faecalis alkaline phosphatase (AP, encoded by phoZ). The predicted gene product shows homology with alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases from Bacillus subtilis. Expression ofphoZ in Escherichia coli, E. faecalis, Streptococcus agalactiae (group B streptococcus [GBS]), or Streptococcus pyogenes (group A streptococcus [GAS]) produces a blue-colony phenotype on plates containing a chromogenic substrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP). Two tests were made to determine if the activity of the enzyme is dependent upon the enzyme’s subcellular location. First, elimination of the signal sequence reduced AP activity to 3% of the wild-type activity (or less) in three species of gram-positive bacteria. Restoration of export, using the signal sequence from C5a peptidase, restored AP activity to at least 50% of that of the wild type. Second, we engineered two chimeric proteins in which AP was fused to either a periplasmic domain or a cytoplasmic domain of lactose permease (a membrane protein). In E. coli, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion. We concluded that AP activity is export dependent. The signal sequence deletion mutant, phoZΔss, was used to identify random genomic fragments from GBS that encode exported proteins or integral membrane proteins. Included in this set of fragments were genes that exhibited homology with the Rib protein (a cell wall protein from GBS) or with DppB (an integral membrane protein from GAS). AP acts as a reporter enzyme in GBS, GAS, and E. faecalis and is expected to be useful in a variety of gram-positive bacteria.

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 The selenophosphate synthetase, selD, is important for Clostridioides difficile physiology

2021 ◽  
Author(s):  
Kathleen N. McAllister ◽  
Andrea Martinez Aguirre ◽  
Joseph A. Sorg
Keyword(s):  
Treatment Options ◽  
Metabolic Process ◽  
Carbon And Nitrogen ◽  
Clostridioides Difficile ◽  
Selenophosphate Synthetase ◽  
Antibiotic Associated Diarrhea ◽  
Initial Characterization ◽  
Significant Burden ◽  
Gut Pathogen

The endospore-forming pathogen, Clostridioides difficile, is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and healthcare. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using RNAseq, we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD+ can be regenerated by other pathways. Importance C. difficile is a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop the C. difficile lifecycle, other non-antibiotic treatment options are in urgent need of development. Towards this goal, we find that a metabolic process used by only a small fraction of the microbiota is important for C. difficile physiology – Stickland metabolism. Here, we use our CRISPR-Cas9 system to ‘knock in’ a copy of the selD gene into the deletion strain to restore selD at its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology – from vegetative growth to spore formation and outgrowth post-germination.

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