scholarly journals A Mutation Upstream of the rplN-rpsD Ribosomal Operon Downregulates Bordetella pertussis Virulence Factor Production without Compromising Bacterial Survival within Human Macrophages

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Jakub Novák ◽  
David Jurnečka ◽  
Irena Linhartová ◽  
Jana Holubová ◽  
Ondřej Staněk ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Infected Mouse ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Content Type ◽  
Human Macrophages ◽  
Mouse Lungs ◽  
Virulence Factor Production

ABSTRACT The BvgS/BvgA two-component system controls expression of ∼550 genes of Bordetella pertussis, of which, ∼245 virulence-related genes are positively regulated by the BvgS-phosphorylated transcriptional regulator protein BvgA (BvgA∼P). We found that a single G-to-T nucleotide transversion in the 5′-untranslated region (5′-UTR) of the rplN gene enhanced transcription of the ribosomal protein operon and of the rpoA gene and provoked global dysregulation of B. pertussis genome expression. This comprised overproduction of the alpha subunit (RpoA) of the DNA-dependent RNA polymerase, downregulated BvgA and BvgS protein production, and impaired production and secretion of virulence factors by the mutant. Nonetheless, the mutant survived like the parental bacteria for >2 weeks inside infected primary human macrophages and persisted within infected mouse lungs for a longer period than wild-type B. pertussis. These observations suggest that downregulation of virulence factor production by bacteria internalized into host cells may enable persistence of the whooping cough agent in the airways. IMPORTANCE We show that a spontaneous mutation that upregulates transcription of an operon encoding ribosomal proteins and causes overproduction of the downstream-encoded α subunit (RpoA) of RNA polymerase causes global effects on gene expression levels and proteome composition of Bordetella pertussis. Nevertheless, the resulting important downregulation of the BvgAS-controlled expression of virulence factors of the whooping cough agent did not compromise its capacity to persist for prolonged periods inside primary human macrophage cells, and it even enhanced its capacity to persist in infected mouse lungs. These observations suggest that the modulation of BvgAS-controlled expression of virulence factors may occur also during natural infections of human airways by Bordetella pertussis and may possibly account for long-term persistence of the pathogen within infected cells of the airways.

scholarly journals BspR/BtrA, an Anti-σ Factor, Regulates the Ability ofBordetella bronchisepticaTo Cause Cough in Rats

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Keiji Nakamura ◽  
Noriko Shinoda ◽  
Yukihiro Hiramatsu ◽  
Shinya Ohnishi ◽  
Shigeki Kamitani ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Bordetella Pertussis ◽  
Rat Model ◽  
Whooping Cough ◽  
Respiratory Infections ◽  
Wild Type ◽  
Content Type ◽  
Infection Models ◽  
Natural Hosts ◽  
Σ Factor

ABSTRACTBordetella pertussis,B. parapertussis, andB. bronchisepticacause respiratory infections, many of which are characterized by coughing of the infected hosts. The pathogenesis of the coughing remains to be analyzed, mainly because there were no convenient infection models of small animals that replicate coughing afterBordetellainfection. Here, we present a coughing model of rats infected withB. bronchiseptica. Rats, which are one of natural hosts ofB. bronchiseptica, were readily infected with the organisms and showed frequent coughing.B. pertussisalso caused coughing in rats, which is consistent with previous reports, but the cough response was less apparent than theB. bronchiseptica-induced cough. By using the rat model, we demonstrated that adenylate cyclase toxin, dermonecrotic toxin, and the type III secretion system are not involved in cough production, but BspR/BtrA (different names for the same protein), an anti-σ factor, regulates the production of unknown factor(s) to cause coughing. Rat coughing was observed by inoculation of not only the living bacteria but also the bacterial lysates. Infection withbspR(btrA)-deficient strains caused significantly less frequent coughing than the wild type; however, intranasal inoculation of the lysates from abspR(btrA)-deficient strain caused coughing similarly to the wild type, suggesting that BspR/BtrA regulates the production of the cough factor(s) only when the bacteria colonize host bodies. Moreover, the cough factor(s) was found to be heat labile and produced byB. bronchisepticain the Bvg+phase. We consider that our rat model provides insight into the pathogenesis of cough induced by theBordetellainfection.IMPORTANCEWhooping cough is a contagious respiratory disease caused byBordetella pertussis. This disease is characterized by severe paroxysmal coughing, which becomes a heavy burden for patients and occasionally results in death; however, its pathogenesis remains largely unknown. The major obstacle to analyzingBordetella-induced coughing is the lack of conventional animal models that replicate coughing. AsBordetella pertussisis highly adapted to humans, infection models in experimental animals are not considered to be well established. In the present study, we examined coughing in rats infected withB. bronchiseptica, which shares many virulence factors withB. pertussis. Using this rat model, we demonstrated that some of the major virulence factors ofBordetellaare not involved in cough production, but an anti-σ factor, BspR/BtrA, ofB. bronchisepticaregulates the production of unknown cough-causing bacterial factor(s). Our results provide important clues to understand the mechanism by whichBordetellainduces cough.

scholarly journals Staphylococcal Virulence Factors on the Skin of Atopic Dermatitis Patients

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Mary C. Moran ◽  
Michael P. Cahill ◽  
Matthew G. Brewer ◽  
Takeshi Yoshida ◽  
Sara Knowlden ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Atopic Dermatitis ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Protein Level ◽  
Content Type ◽  
Factor Production ◽  
First Time ◽  
Virulence Factor Production ◽  
Specific Virulence

ABSTRACT Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, infective endocarditis, osteoarticular, pleuropulmonary, and device-related infections. Virulence factors secreted by S. aureus, including superantigens and cytotoxins, play significant roles in driving disease. The ability to identify virulence factors present at the site of infection will be an important tool in better identifying and understanding how specific virulence factors contribute to disease. Previously, virulence factor production has been determined by culturing S. aureus isolates and detecting the mRNA of specific virulence factors. We demonstrated for the first time that virulence factors can be directly detected at the protein level from human samples, removing the need to first culture isolated bacteria. Superantigens and cytotoxins were detected and quantified with a Western dot blot assay by using reconstituted skin swabs obtained from patients with atopic dermatitis. This methodology will significantly enhance our ability to investigate the complex host-microbe environment and the effects various therapies have on virulence factor production. Overall, the ability to directly quantify virulence factors present at the site of infection or colonization will enhance our understanding of S. aureus-related diseases and help identify optimal treatments. IMPORTANCE For the first time, we show that secreted staphylococcal virulence factors can be quantified at the protein level directly from skin swabs obtained from the skin of atopic dermatitis patients. This technique eliminates the need to culture Staphylococcus aureus and then test the strain’s potential to produce secreted virulence factors. Our methodology shows that secreted virulence factors are present on the skin of atopic patients and provides a more accurate means of evaluating the physiological impact of S. aureus in inflammatory diseases such as atopic dermatitis.

scholarly journals The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP

2016 ◽  
Vol 198 (6) ◽  
pp. 951-963 ◽  
Author(s):  
Manita Guragain ◽  
Michelle M. King ◽  
Kerry S. Williamson ◽  
Ailyn C. Pérez-Osorio ◽  
Tatsuya Akiyama ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Regulatory System ◽  
Dependent Manner ◽  
Content Type ◽  
Factor Production ◽  
Two Component ◽  
Intracellular Ca ◽  
Virulence Factor Production

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections,P. aeruginosaoften encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed thatP. aeruginosaresponds to externally added Ca2+through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+homeostasis are not known. Here, we characterized the genome-wide transcriptional response ofP. aeruginosato elevated [Ca2+] in both planktonic cultures and biofilms. Among the genes induced by CaCl2in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here calledcarSandcarR), while the closely related two-component regulatorsphoPQandpmrABwere repressed by CaCl2addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant ofcarRand performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2are the predicted periplasmic OB-fold protein, PA0320 (here calledcarO), and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here calledcarP). Mutations in bothcarOandcarPaffected Ca2+homeostasis, reducing the ability ofP. aeruginosato export excess Ca2+. In addition, a mutation incarPhad a pleotropic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+and responding through its regulatory targets that modulate Ca2+homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin.IMPORTANCEDuring infectious disease,Pseudomonas aeruginosaencounters environments with high calcium (Ca2+) concentrations, yet the cells maintain intracellular Ca2+at levels that are orders of magnitude less than that of the external environment. In addition, Ca2+signalsP. aeruginosato induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca2+homeostasis or how Ca2+acts as a signal. In this study, we identified a two-component regulatory system inP. aeruginosaPAO1, termed CarRS, that is induced at elevated Ca2+levels. CarRS modulates Ca2+signaling and Ca2+homeostasis through its regulatory targets, CarO and CarP. The results demonstrate thatP. aeruginosauses a two-component regulatory system to sense external Ca2+and relays that information for Ca2+-dependent cellular processes.

scholarly journals The Eukaryotic Host Factor 14-3-3 Inactivates Adenylate Cyclase Toxins ofBordetella bronchisepticaandB. parapertussis, but NotB. pertussis

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Aya Fukui-Miyazaki ◽  
Hirono Toshima ◽  
Yukihiro Hiramatsu ◽  
Keisuke Okada ◽  
Keiji Nakamura ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Bordetella Pertussis ◽  
Host Factor ◽  
Target Cells ◽  
Content Type ◽  
Functional Differences ◽  
Adenylate Cyclase Toxin

ABSTRACTBordetella pertussis,Bordetella bronchiseptica, andBordetella parapertussisshare highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles inBordetellainfections. We herein demonstrate that CyaAs function as virulence factors differently betweenB. bronchiseptica/B. parapertussisandB. pertussis.B.bronchisepticaCyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly toB.pertussisCyaA. The hemolytic activity ofB.bronchisepticaCyaA on sheep erythrocytes was also preserved. However, in nucleated target cells,B.bronchisepticaCyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects ofB.bronchisepticaCyaA based on its enzyme activity were markedly attenuated.B.parapertussisCyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly toB.bronchisepticaCyaA; however,B.pertussisCyaA has Phe375instead of Ser, and thus, was not affected by 14-3-3. In addition,B.pertussisCyaA impaired the barrier function of epithelial cells, whereasB.bronchisepticaCyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity betweenB. bronchiseptica/B.parapertussisandB. pertussis.IMPORTANCEBordetella pertussis,B. bronchiseptica, andB. parapertussisare bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by eachBordetellaspecies. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor ofBordetella. The toxins ofB. bronchisepticaand presumablyB. parapertussiswere inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas theB. pertussistoxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences inBordetellaare attributed to the different activities of adenylate cyclase toxins.

scholarly journals Bordetella pertussis Lipid A Glucosamine Modification Confers Resistance to Cationic Antimicrobial Peptides and Increases Resistance to Outer Membrane Perturbation

2014 ◽  
Vol 58 (8) ◽  
pp. 4931-4934 ◽  
Author(s):  
Nita R. Shah ◽  
Robert E. W. Hancock ◽  
Rachel C. Fernandez
Keyword(s):  
Immune System ◽  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Human Immune System ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Membrane Perturbation

ABSTRACTBordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

scholarly journals Indole Inhibits ToxR Regulon Expression inVibrio cholerae

2019 ◽  
Vol 87 (3) ◽  
Author(s):  
Mondraya F. Howard ◽  
X. Renee Bina ◽  
James E. Bina
Keyword(s):  
Virulence Factor ◽  
Virulence Gene ◽  
Dependent Manner ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Type Vi Secretion ◽  
Biofilm Production ◽  
Factor Production ◽  
Periplasmic Domain ◽  
Virulence Factor Production

ABSTRACTIndole is a degradation product of tryptophan that functions as a signaling molecule in many bacteria. This includesVibrio cholerae, where indole was shown to regulate biofilm and type VI secretion in nontoxigenic environmental isolates. Indole is also produced by toxigenicV. choleraestrains in the human intestine, but its significance in the host is unknown. We investigated the effects of indole on toxigenicV. choleraeO1 El Tor during growth under virulence inducing conditions. The indole transcriptome was defined by RNA sequencing and showed widespread changes in the expression of genes involved in metabolism, biofilm production, and virulence factor production. In contrast, genes involved in type VI secretion were not affected by indole. We subsequently found that indole repressed genes involved inV. choleraepathogenesis, including the ToxR virulence regulon. Consistent with this, indole inhibited cholera toxin and toxin-coregulated pilus production in a dose-dependent manner. The effects of indole on virulence factor production and biofilm were linked to ToxR and the ToxR-dependent regulator LeuO. The expression ofleuOwas increased by exogenous indole and linked to repression of the ToxR virulence regulon. This process was dependent on the ToxR periplasmic domain, suggesting that indole was a ToxR agonist. This conclusion was further supported by results showing that the ToxR periplasmic domain contributed to indole-mediated increased biofilm production. Collectively, our results suggest that indole may be a niche-specific cue that can function as a ToxR agonist to modulate virulence gene expression and biofilm production inV. cholerae.

scholarly journals The RNA Chaperone Hfq Is Required for Virulence of Bordetella pertussis

2013 ◽  
Vol 81 (11) ◽  
pp. 4081-4090 ◽  
Author(s):  
Ilona Bibova ◽  
Karolina Skopova ◽  
Jiri Masin ◽  
Ondrej Cerny ◽  
David Hot ◽  
...  
Keyword(s):  
Bordetella Pertussis ◽  
Posttranscriptional Regulation ◽  
Noncoding Rna ◽  
Whooping Cough ◽  
Lethal Dose ◽  
Rna Chaperone ◽  
Content Type ◽  
Small Noncoding Rna ◽  
Mrna Targets

ABSTRACTBordetella pertussisis a Gram-negative pathogen causing the human respiratory disease called pertussis or whooping cough. Here we examined the role of the RNA chaperone Hfq inB. pertussisvirulence. Hfq mediates interactions between small regulatory RNAs and their mRNA targets and thus plays an important role in posttranscriptional regulation of many cellular processes in bacteria, including production of virulence factors. We characterized anhfqdeletion mutant (Δhfq) ofB. pertussis18323 and show that the Δhfqstrain produces decreased amounts of the adenylate cyclase toxin that plays a central role inB. pertussisvirulence. Production of pertussis toxin and filamentous hemagglutinin was affected to a lesser extent.In vitro, the ability of the Δhfqstrain to survive within macrophages was significantly reduced compared to that of the wild-type (wt) strain. The virulence of the Δhfqstrain in the mouse respiratory model of infection was attenuated, with its capacity to colonize mouse lungs being strongly reduced and its 50% lethal dose value being increased by one order of magnitude over that of the wt strain. In mixed-infection experiments, the Δhfqstrain was then clearly outcompeted by the wt strain. This requirement for Hfq suggests involvement of small noncoding RNA regulation inB. pertussisvirulence.

scholarly journals Overexpression of the RNA Polymerase Alpha Subunit Reduces Transcription of Bvg-Activated Virulence Genes inBordetella pertussis

2000 ◽  
Vol 182 (2) ◽  
pp. 529-531 ◽  
Author(s):  
Nicholas H. Carbonetti ◽  
Alla Romashko ◽  
Teresa J. Irish
Keyword(s):  
Rna Polymerase ◽  
Virulence Factor ◽  
Pertussis Toxin ◽  
Bordetella Pertussis ◽  
Virulence Genes ◽  
Transcriptional Activator ◽  
Alpha Subunit ◽  
Terminal Domain

ABSTRACT Overexpression of the RNA polymerase alpha subunit inBordetella pertussis reduces expression of the virulence factor pertussis toxin. Here we show that this reduction is at the level of transcription, is reversed by overexpression of the transcriptional activator BvgA, and is dependent on the C-terminal domain of alpha.

scholarly journals Staphylococcus aureus Formyl-Methionyl Transferase Mutants Demonstrate Reduced Virulence Factor Production and Pathogenicity

2013 ◽  
Vol 57 (7) ◽  
pp. 2929-2936 ◽  
Author(s):  
Thomas Lewandowski ◽  
Jianzhong Huang ◽  
Frank Fan ◽  
Shannon Rogers ◽  
Daniel Gentry ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factor ◽  
Survival Rates ◽  
Extracellular Proteins ◽  
High Survival ◽  
Invasive Infection ◽  
Loss Of Function ◽  
Content Type ◽  
Factor Production ◽  
Virulence Factor Production

ABSTRACTInhibitors of peptide deformylase (PDF) represent a new class of antibacterial agents with a novel mechanism of action. Mutations that inactivate formyl methionyl transferase (FMT), the enzyme that formylates initiator methionyl-tRNA, lead to an alternative initiation of protein synthesis that does not require deformylation and are the predominant cause of resistance to PDF inhibitors inStaphylococcus aureus. Here, we report that loss-of-function mutations in FMT impart pleiotropic effects that include a reduced growth rate, a nonhemolytic phenotype, and a drastic reduction in production of multiple extracellular proteins, including key virulence factors, such as α-hemolysin and Panton-Valentine leukocidin (PVL), that have been associated withS. aureuspathogenicity. Consequently,S. aureusFMT mutants are greatly attenuated in neutropenic and nonneutropenic murine pyelonephritis infection models and show very high survival rates compared with wild-typeS. aureus. These newly discovered effects on extracellular virulence factor production demonstrate that FMT-null mutants have a more severe fitness cost than previously anticipated, leading to a substantial loss of pathogenicity and a restricted ability to produce an invasive infection.

scholarly journals Analysis of BvgA Activation of the Pertactin Gene Promoter in Bordetella pertussis

1999 ◽  
Vol 181 (17) ◽  
pp. 5234-5241 ◽  
Author(s):  
Susan M. Kinnear ◽  
Philip E. Boucher ◽  
Scott Stibitz ◽  
Nicholas H. Carbonetti
Keyword(s):  
Virulence Factors ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Start Site ◽  
Reading Frame ◽  
Dnase I Footprinting ◽  
Rapid Amplification

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the bvgregulatory locus. It has been shown by activation kinetics that several of the virulence factors are differentially regulated. fhais transcribed at 10 min following an inducing signal, whileptx is not transcribed until 2 to 4 h after the inducing signal. We present data indicating that prn is transcribed at 1 h, an intermediate time compared to those offha and ptx. We have identifiedcis-acting sequences necessary for expression ofprn in B. pertussis by usingprn-lac fusions containing alterations in the sequence upstream of the prn open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream ofprn to activate transcription from the promoter. Our genetic data indicate that the region critical for prnactivation extends upstream to position −84. However, these data do not support the location of the prn transcription start site as previously published. We used a number of methods, includingprn-lac fusions, reverse transcriptase PCR, and 5′ rapid amplification of cDNA ends, to localize and identify thebvg-dependent 5′ end of the prn transcript to the cytosine at −125 with respect to the published start site.

Sign in / Sign up

Export Citation Format

Share Document