Regulation of Virulence by the RevR Response Regulator in Clostridium perfringens

ABSTRACTClostridium perfringenscauses clostridial myonecrosis or gas gangrene and produces several extracellular hydrolytic enzymes and toxins, many of which are regulated by the VirSR signal transduction system. TherevRgene encodes a putative orphan response regulator that has similarity to the YycF (WalR), VicR, PhoB, and PhoP proteins from other Gram-positive bacteria. RevR appears to be a classical response regulator, with an N-terminal receiver domain and a C-terminal domain with a putative winged helix-turn-helix DNA binding region. To determine its functional role, arevRmutant was constructed by allelic exchange and compared to the wild type by microarray analysis. The results showed that more than 100 genes were differentially expressed in the mutant, including several genes involved in cell wall metabolism. TherevRmutant had an altered cellular morphology; unlike the short rods observed with the wild type, the mutant cells formed long filaments. These changes were reversed upon complementation with a plasmid that carried the wild-typerevRgene. Several genes encoding extracellular hydrolytic enzymes (sialidase, hyaluronidase, and α-clostripain) were differentially expressed in therevRmutant. Quantitative enzyme assays confirmed that these changes led to altered enzyme activity and that complementation restored the wild-type phenotype. Most importantly, therevRmutant was attenuated for virulence in the mouse myonecrosis model compared to the wild type and the complemented strains. These results provide evidence that RevR regulates virulence inC. perfringens; it is the first response regulator other than VirR to be shown to regulate virulence in this important pathogen.

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CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

mBio ◽

10.1128/mbio.00770-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 27

Author(s):

Jihong Li ◽

Menglin Ma ◽

Mahfuzur R. Sarker ◽

Bruce A. McClane

Keyword(s):

Clostridium Perfringens ◽

Null Mutant ◽

Intestinal Infection ◽

Wild Type ◽

Global Regulator ◽

Content Type ◽

Gram Positive Bacteria ◽

Type D ◽

Epsilon Toxin ◽

Virulence Properties

ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. IMPORTANCE Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.

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Salivaricin D, a Novel Intrinsically Trypsin-Resistant Lantibiotic from Streptococcus salivarius 5M6c Isolated from a Healthy Infant

Applied and Environmental Microbiology ◽

10.1128/aem.06588-11 ◽

2011 ◽

Vol 78 (2) ◽

pp. 402-410 ◽

Cited By ~ 39

Author(s):

Dagim Jirata Birri ◽

Dag Anders Brede ◽

Ingolf F. Nes

Keyword(s):

Abc Transporters ◽

Response Regulator ◽

Probiotic Bacterium ◽

Streptococcus Salivarius ◽

Human Pathogens ◽

Gene Encoding ◽

Content Type ◽

Gram Positive Bacteria ◽

Genes Encoding ◽

Immunity Function

ABSTRACTIn this work, we purified and characterized a newly identified lantibiotic (salivaricin D) fromStreptococcus salivarius5M6c. Salivaricin D is a 34-amino-acid-residue peptide (3,467.55 Da); the locus of the gene encoding this peptide is a 16.5-kb DNA segment which contains genes encoding the precursor of two lantibiotics, two modification enzymes (dehydratase and cyclase), an ABC transporter, a serine-like protease, immunity proteins (lipoprotein and ABC transporters), a response regulator, and a sensor histidine kinase. The immunity gene (salI) was heterologously expressed in a sensitive indicator and provided significant protection against salivaricin D, confirming its immunity function. Salivaricin D is a naturally trypsin-resistant lantibiotic that is similar to nisin-like lantibiotics. It is a relatively broad-spectrum bacteriocin that inhibits members of many genera of Gram-positive bacteria, including the important human pathogensStreptococcus pyogenesandStreptococcus pneumoniae. Thus,Streptococcus salivarius5M6c may be a potential biological agent for the control of oronasopharynx-colonizing streptococcal pathogens or may be used as a probiotic bacterium.

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Acquisition of the Ability To Assimilate Mannitol by Saccharomyces cerevisiae through Dysfunction of the General Corepressor Tup1-Cyc8

Applied and Environmental Microbiology ◽

10.1128/aem.02906-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 9-16 ◽

Cited By ~ 17

Author(s):

Moeko Chujo ◽

Shiori Yoshida ◽

Anri Ota ◽

Kousaku Murata ◽

Shigeyuki Kawai

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Basis ◽

Mutant Allele ◽

Wild Type ◽

Growth Data ◽

Content Type ◽

Corepressor Complex ◽

Marine Biomass ◽

Prolonged Culture ◽

Genes Encoding

ABSTRACTSaccharomyces cerevisiaenormally cannot assimilate mannitol, a promising brown macroalgal carbon source for bioethanol production. The molecular basis of this inability remains unknown. We found that cells capable of assimilating mannitol arose spontaneously from wild-typeS. cerevisiaeduring prolonged culture in mannitol-containing medium. Based on microarray data, complementation analysis, and cell growth data, we demonstrated that acquisition of mannitol-assimilating ability was due to spontaneous mutations in the genes encoding Tup1 or Cyc8, which constitute a general corepressor complex that regulates many kinds of genes. We also showed that anS. cerevisiaestrain carrying a mutant allele ofCYC8exhibited superior salt tolerance relative to other ethanologenic microorganisms; this characteristic would be highly beneficial for the production of bioethanol from marine biomass. Thus, we succeeded in conferring the ability to assimilate mannitol onS. cerevisiaethrough dysfunction of Tup1-Cyc8, facilitating production of ethanol from mannitol.

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Nisin Resistance of Listeria monocytogenes Is Increased by Exposure to Salt Stress and Is Mediated via LiaR

Applied and Environmental Microbiology ◽

10.1128/aem.01797-13 ◽

2013 ◽

Vol 79 (18) ◽

pp. 5682-5688 ◽

Cited By ~ 52

Author(s):

Teresa M. Bergholz ◽

Silin Tang ◽

Martin Wiedmann ◽

Kathryn J. Boor

Keyword(s):

Salt Stress ◽

Listeria Monocytogenes ◽

Safety Concern ◽

Response Regulator ◽

Brain Heart Infusion ◽

Control Measures ◽

Protective Effects ◽

Wild Type ◽

Content Type ◽

Type Strains

ABSTRACTGrowth ofListeria monocytogeneson refrigerated, ready-to-eat food is a significant food safety concern. Natural antimicrobials, such as nisin, can be used to control this pathogen on food, but little is known about how other food-related stresses may impact how the pathogen responds to these compounds. Prior work demonstrated that exposure ofL. monocytogenesto salt stress at 7°C led to increased expression of genes involved in nisin resistance, including the response regulatorliaR. We hypothesized that exposure to salt stress would increase subsequent resistance to nisin and that LiaR would contribute to increased nisin resistance. Isogenic deletion mutations inliaRwere constructed in 7 strains ofL. monocytogenes, and strains were exposed to 6% NaCl in brain heart infusion broth and then tested for resistance to nisin (2 mg/ml Nisaplin) at 7°C. For the wild-type strains, exposure to salt significantly increased subsequent nisin resistance (P< 0.0001) over innate levels of resistance. Compared to the salt-induced nisin resistance of wild-type strains, ΔliaRstrains were significantly more sensitive to nisin (P< 0.001), indicating that induction of LiaFSR led to cross-protection ofL. monocytogenesagainst subsequent inactivation by nisin. Transcript levels of LiaR-regulated genes were induced by salt stress, and lmo1746 andtelAwere found to contribute to LiaR-mediated salt-induced nisin resistance. These data suggest that environmental stresses similar to those on foods can influence the resistance ofL. monocytogenesto antimicrobials such as nisin, and potential cross-protective effects should be considered when selecting and applying control measures for this pathogen on ready-to-eat foods.

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An intramembrane sensory circuit monitors sortase A–mediated processing of streptococcal adhesins

Science Signaling ◽

10.1126/scisignal.aas9941 ◽

2019 ◽

Vol 12 (580) ◽

pp. eaas9941 ◽

Cited By ~ 2

Author(s):

Jeffrey W. Hall ◽

Bruno P. Lima ◽

Gaetan G. Herbomel ◽

Tata Gopinath ◽

LeAnna McDonald ◽

...

Keyword(s):

Cell Membrane ◽

Biofilm Formation ◽

Response Regulator ◽

Sortase A ◽

Gram Positive Bacteria ◽

Regulatory Circuit ◽

Bacterial Adhesins ◽

Genes Encoding ◽

Quality Control Mechanism ◽

Cognate Response Regulator

Bacterial adhesins mediate adhesion to substrates and biofilm formation. Adhesins of the LPXTG family are posttranslationally processed by the cell membrane–localized peptidase sortase A, which cleaves the LPXTG motif. This generates a short C-terminal peptide (C-pep) that remains in the cell membrane, whereas the mature adhesin is incorporated into the cell wall. Genes encoding adhesins of the oral bacteriumStreptococcus gordoniiwere differentially expressed depending on whether the bacteria were isolated from saliva or dental plaque and appeared to be coordinately regulated. Deletion ofsspAandsspB (sspAB), both of which encode LPXTG-containing adhesins, unexpectedly enhanced adhesion and biofilm formation. C-peps produced from a model LPXTG-containing adhesin localized to the cell membrane and bound to and inhibited the intramembrane sensor histidine kinase SGO_1180, thus preventing activation of the cognate response regulator SGO_1181. The absence of SspAB C-peps induced the expression of thescaCBAoperon encoding the lipoprotein adhesin ScaA, which was sufficient to preserve and even enhance biofilm formation. This C-pep–driven regulatory circuit also exists in pathogenic streptococci and is likely conserved among Gram-positive bacteria. This quality control mechanism ensures that the bacteria can form biofilms under diverse environmental conditions and may play a role in optimizing adhesion and biofilm formation.

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TheprrF-Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.02707-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 863-875 ◽

Cited By ~ 41

Author(s):

Alexandria A. Reinhart ◽

Daniel A. Powell ◽

Angela T. Nguyen ◽

Maura O'Neill ◽

Louise Djapgne ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Iron Homeostasis ◽

Opportunistic Pathogen ◽

Wild Type ◽

Content Type ◽

Genes Encoding ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

Heme Regulation ◽

Heme Binding Protein

Pseudomonas aeruginosais an opportunistic pathogen that requires iron to cause infection, but it also must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are part ofP. aeruginosa'siron regulatory network and affect the expression of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs are encoded in tandem inP. aeruginosa, allowing for the expression of a distinct, heme-responsive sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of growth, mass spectrometry, and gene expression analysis, we showed that the ΔprrF1,2mutant, which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis. We also identifiedphuS, encoding a heme binding protein involved in heme acquisition, andvreR, encoding a previously identified regulator ofP. aeruginosavirulence genes, as novel targets ofprrF-mediated heme regulation. Finally, we showed that theprrFlocus encoding the PrrF and PrrH sRNAs is required forP. aeruginosavirulence in a murine model of acute lung infection. Moreover, we showed that inoculation with a ΔprrF1,2deletion mutant protects against future challenge with wild-typeP. aeruginosa. Combined, these data demonstrate that theprrF-encoded sRNAs are critical regulators ofP. aeruginosavirulence.

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The Atypical Response Regulator AtvR Is a New Player in Pseudomonas aeruginosa Response to Hypoxia and Virulence

Infection and Immunity ◽

10.1128/iai.00207-17 ◽

2017 ◽

Vol 85 (8) ◽

Cited By ~ 5

Author(s):

Gilberto Hideo Kaihami ◽

Leandro Carvalho Dantas Breda ◽

José Roberto Fogaça de Almeida ◽

Thays de Oliveira Pereira ◽

Gianlucca Gonçalves Nicastro ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Strain ◽

Wild Type Strain ◽

Environmental Changes ◽

Response Regulator ◽

Classical Pathway ◽

Wild Type ◽

Macrophage Infection ◽

Content Type ◽

Successful Infection

ABSTRACT Two-component systems are widespread in bacteria, allowing adaptation to environmental changes. The classical pathway is composed of a histidine kinase that phosphorylates an aspartate residue in the cognate response regulator (RR). RRs lacking the phosphorylatable aspartate also occur, but their function and contribution during host-pathogen interactions are poorly characterized. AtvR (PA14_26570) is the only atypical response regulator with a DNA-binding domain in the opportunistic pathogen Pseudomonas aeruginosa. Macrophage infection with the atvR mutant strain resulted in higher levels of tumor necrosis factor alpha secretion as well as increased bacterial clearance compared to those for macrophages infected with the wild-type strain. In an acute pneumonia model, mice infected with the atvR mutant presented increased amounts of proinflammatory cytokines, increased neutrophil recruitment to the lungs, reductions in bacterial burdens, and higher survival rates in comparison with the findings for mice infected with the wild-type strain. Further, several genes involved in hypoxia/anoxia adaptation were upregulated upon atvR overexpression, as seen by high-throughput transcriptome sequencing (RNA-Seq) analysis. In addition, atvR was more expressed in hypoxia in the presence of nitrate and required for full expression of nitrate reductase genes, promoting bacterial growth under this condition. Thus, AtvR would be crucial for successful infection, aiding P. aeruginosa survival under conditions of low oxygen tension in the host. Taken together, our data demonstrate that the atypical response regulator AtvR is part of the repertoire of transcriptional regulators involved in the lifestyle switch from aerobic to anaerobic conditions. This finding increases the complexity of regulation of one of the central metabolic pathways that contributes to Pseudomonas ubiquity and versatility.

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NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

Journal of Bacteriology ◽

10.1128/jb.00349-15 ◽

2015 ◽

Vol 197 (20) ◽

pp. 3339-3353 ◽

Cited By ~ 14

Author(s):

Jihong Li ◽

John C. Freedman ◽

Bruce A. McClane

Keyword(s):

Sialic Acid ◽

Clostridium Perfringens ◽

Host Cells ◽

Start Codon ◽

Null Mutant ◽

Wild Type ◽

Mobility Shift ◽

Content Type ◽

Type D ◽

Epsilon Toxin

ABSTRACTClostridium perfringenstype D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011,http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, ananInull mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due tonanIgene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions incodYandccpAgene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A doublecodYccpAnull mutant produced even less ETX than acodYorccpAsingle null mutant. CcpA bound directly to sequences upstream of theetxorcodYstart codon, and bioinformatics identified putative CcpA-bindingcresites immediately upstream of both thecodYandetxstart codons, suggesting possible direct CcpA regulatory effects. AccpAmutation also decreasedcodYtranscription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects oncodYtranscription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease.IMPORTANCEClostridium perfringensNanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using singleccpAorcodYnull mutants and accpAcodYdouble null mutant showed thatcodYandccpAregulate ETX production independently of one another but thatccpAalso affectscodYtranscription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulateetxtranscription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producingC. perfringensstrains, via CcpA and CodY, to upregulate ETX production and cause disease.

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Gene Conservation among Endospore-Forming Bacteria Reveals Additional Sporulation Genes in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.01778-12 ◽

2012 ◽

Vol 195 (2) ◽

pp. 253-260 ◽

Cited By ~ 33

Author(s):

Bjorn A. Traag ◽

Antonia Pugliese ◽

Jonathan A. Eisen ◽

Richard Losick

Keyword(s):

Reverse Genetics ◽

Wild Type Strain ◽

Developmental Process ◽

Gene Conservation ◽

Wild Type ◽

Content Type ◽

Gram Positive Bacteria ◽

Gene Regulatory ◽

Phylogenetic Profiling ◽

Sporulation Genes

ABSTRACTThe capacity to form endospores is unique to certain members of the low-G+C group of Gram-positive bacteria (Firmicutes) and requires signature sporulation genes that are highly conserved across members of distantly related genera, such asClostridiumandBacillus. Using gene conservation among endospore-forming bacteria, we identified eight previously uncharacterized genes that are enriched among endospore-forming species. The expression of five of these genes was dependent on sporulation-specific transcription factors. Mutants of none of the genes exhibited a conspicuous defect in sporulation, but mutants of two,ylxYandylyA, were outcompeted by a wild-type strain under sporulation-inducing conditions, but not during growth. In contrast, aylmCmutant displayed a slight competitive advantage over the wild type specific to sporulation-inducing conditions. The phenotype of aylyAmutant was ascribed to a defect in spore germination efficiency. This work demonstrates the power of combining phylogenetic profiling with reverse genetics and gene-regulatory studies to identify unrecognized genes that contribute to a conserved developmental process.

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Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

Infection and Immunity ◽

10.1128/iai.01487-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1600-1605 ◽

Cited By ~ 32

Author(s):

Melissa A. Dean ◽

Randall J. Olsen ◽

S. Wesley Long ◽

Adriana E. Rosato ◽

James M. Musser

Keyword(s):

Staphylococcus Aureus ◽

Pathway Analysis ◽

Molecular Mechanisms ◽

Point Mutations ◽

Biosynthesis Pathway ◽

Wild Type ◽

Small Colony Variants ◽

Content Type ◽

Genes Encoding ◽

Small Colony

ABSTRACTStaphylococcus aureussmall-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of fiveS. aureusSCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

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