scholarly journals The Vibrio parahaemolyticus ToxRS Regulator Is Required for Stress Tolerance and Colonization in a Novel Orogastric Streptomycin-Induced Adult Murine Model

2012 ◽  
Vol 80 (5) ◽  
pp. 1834-1845 ◽  
Author(s):  
W. Brian Whitaker ◽  
Michelle A. Parent ◽  
Aoife Boyd ◽  
Gary P. Richards ◽  
E. Fidelma Boyd
Keyword(s):  
Stress Tolerance ◽  
Murine Model ◽  
Vibrio Parahaemolyticus ◽  
Sodium Dodecyl ◽  
Virulence Gene ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Content Type ◽  
Virulence Gene Expression

ABSTRACTVibrio parahaemolyticus, a marine bacterium, is the causative agent of gastroenteritis associated with the consumption of seafood. It contains a homologue of thetoxRSoperon that inV. choleraeis the key regulator of virulence gene expression. We examined a nonpolar mutation intoxRSto determine the role of these genes inV. parahaemolyticusRIMD2210633, an O3:K6 isolate, and showed that compared to the wild type, ΔtoxRSwas significantly more sensitive to acid, bile salts, and sodium dodecyl sulfate stresses. We demonstrated that ToxRS is a positive regulator ofompUexpression, and that the complementation of ΔtoxRSwithompUrestores stress tolerance. Furthermore, we showed that ToxRS also regulates type III secretion system genes in chromosome I via the regulation of theleuOhomologue VP0350. We examined the effect of ΔtoxRS in vivousing a new orogastric adult murine model of colonization. We demonstrated that streptomycin-treated adult C57BL/6 mice experienced prolonged intestinal colonization along the entire intestinal tract by the streptomycin-resistantV. parahaemolyticus. In contrast, no colonization occurred in non-streptomycin-treated mice. A competition assay between the ΔtoxRSand wild-typeV. parahaemolyticusstrains marked with the β-galactosidase genelacZdemonstrated that the ΔtoxRSstrain was defective in colonization compared to the wild-type strain. This defect was rescued by ectopically expressingompU. Thus, the defect in stress tolerance and colonization in ΔtoxRSis solely due to OmpU. To our knowledge, the orogastric adult murine model reported here is the first showing sustained intestinal colonization byV. parahaemolyticus.

scholarly journals MetR-Regulated Vibrio cholerae Metabolism Is Required for Virulence

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ryan W. Bogard ◽  
Bryan W. Davies ◽  
John J. Mekalanos
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Virulence Factor ◽  
Current Knowledge ◽  
Intestinal Colonization ◽  
Wild Type ◽  
Pathogenic Potential ◽  
Content Type ◽  
Mouse Intestine

ABSTRACTLysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel ofV. choleraeEl Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed thatglyA1andmetJwere also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation ofglyA1, indicating that misregulation ofglyA1is likely responsible for the colonization defect observed in themetRmutant. TheglyA1mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor.IMPORTANCEVibrio choleraecontinues to be a severe cause of morbidity and mortality in developing countries. Identification ofV. choleraefactors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons inV. choleraeto be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required byV. choleraeto colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements ofV. choleraewithin the mouse intestine.

scholarly journals Role of BrnQ1 and BrnQ2 in Branched-Chain Amino Acid Transport and Virulence in Staphylococcus aureus

2014 ◽  
Vol 83 (3) ◽  
pp. 1019-1029 ◽  
Author(s):  
Julienne C. Kaiser ◽  
Sameha Omer ◽  
Jessica R. Sheldon ◽  
Ian Welch ◽  
David E. Heinrichs
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Defined Medium ◽  
Infection Model ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Branched Chain Amino Acid ◽  
Branched Chain

The branched-chain amino acids (BCAAs; Ile, Leu, and Val) not only are important nutrients for the growth ofStaphylococcus aureusbut also are corepressors for CodY, which regulates virulence gene expression, implicating BCAAs as an important link between the metabolic state of the cell and virulence. BCAAs are either synthesized intracellularly or acquired from the environment.S. aureusencodes three putative BCAA transporters, designated BrnQ1, BrnQ2, and BrnQ3; their functions have not yet been formally tested. In this study, we mutated all threebrnQparalogs so as to characterize their substrate specificities and their roles in growthin vitroandin vivo. We demonstrated that in the community-associated, methicillin-resistantS. aureus(CA-MRSA) strain USA300, BrnQ1 is involved in uptake of all three BCAAs, BrnQ2 transports Ile, and BrnQ3 does not have a significant role in BCAA transport under the conditions tested. Of the three, only BrnQ1 is essential for USA300 to grow in a chemically defined medium that is limited for Leu or Val. Interestingly, we observed that abrnQ2mutant grew better than USA300 in media limited for Leu and Val, owing to the fact that this mutation leads to overexpression ofbrnQ1. In a murine infection model, thebrnQ1mutant was attenuated, but in contrast,brnQ2mutants had significantly increased virulence compared to that of USA300, a phenotype we suggest is at least partially linked to enhancedin vivoscavenging of Leu and Val through BrnQ1. These data uncover a hitherto-undiscovered connection between nutrient acquisition and virulence in CA-MRSA.

scholarly journals 18β-Glycyrrhetinic Acid Inhibits Methicillin-Resistant Staphylococcus aureus Survival and Attenuates Virulence Gene Expression

2012 ◽  
Vol 57 (1) ◽  
pp. 241-247 ◽  
Author(s):  
Danyelle R. Long ◽  
Julia Mead ◽  
Jay M. Hendricks ◽  
Michele E. Hardy ◽  
Jovanka M. Voyich
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Virulence Gene ◽  
Glycyrrhetinic Acid ◽  
Licorice Root ◽  
Methicillin Resistant ◽  
Content Type ◽  
Virulence Gene Expression

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) has become a major source of infection in hospitals and in the community. Increasing antibiotic resistance inS. aureusstrains has created a need for alternative therapies to treat disease. A component of the licorice rootGlycyrrhizaspp., 18β-glycyrrhetinic acid (GRA), has been shown to have antiviral, antitumor, and antibacterial activity. This investigation explores thein vitroandin vivoeffects of GRA on MRSA pulsed-field gel electrophoresis (PFGE) type USA300. GRA exhibited bactericidal activity at concentrations exceeding 0.223 μM. Upon exposure ofS. aureusto sublytic concentrations of GRA, we observed a reduction in expression of key virulence genes, includingsaeRandhla. In murine models of skin and soft tissue infection, topical GRA treatment significantly reduced skin lesion size and decreased the expression ofsaeRandhlagenes. Our investigation demonstrates that at high concentrations GRA is bactericidal to MRSA and at sublethal doses it reduces virulence gene expression inS. aureusbothin vitroandin vivo.

scholarly journals Lactate Dehydrogenase Is the Key Enzyme for Pneumococcal Pyruvate Metabolism and Pneumococcal Survival in Blood

2014 ◽  
Vol 82 (12) ◽  
pp. 5099-5109 ◽  
Author(s):  
Paula Gaspar ◽  
Firas A. Y. Al-Bayati ◽  
Peter W. Andrew ◽  
Ana Rute Neves ◽  
Hasan Yesilkaya
Keyword(s):  
Lactate Dehydrogenase ◽  
Lactate Production ◽  
Virulence Gene ◽  
Redox Balance ◽  
Central Metabolism ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Key Enzyme

ABSTRACTStreptococcus pneumoniaeis a fermentative microorganism and causes serious diseases in humans, including otitis media, bacteremia, meningitis, and pneumonia. However, the mechanisms enabling pneumococcal survival in the host and causing disease in different tissues are incompletely understood. The available evidence indicates a strong link between the central metabolism and pneumococcal virulence. To further our knowledge on pneumococcal virulence, we investigated the role of lactate dehydrogenase (LDH), which converts pyruvate to lactate and is an essential enzyme for redox balance, in the pneumococcal central metabolism and virulence using an isogenicldhmutant. Loss of LDH led to a dramatic reduction of the growth rate, pinpointing the key role of this enzyme in fermentative metabolism. The pattern of end products was altered, and lactate production was totally blocked. The fermentation profile was confirmed byin vivonuclear magnetic resonance (NMR) measurements of glucose metabolism in nongrowing cell suspensions of theldhmutant. In this strain, a bottleneck in the fermentative steps is evident from the accumulation of pyruvate, revealing LDH as the most efficient enzyme in pyruvate conversion. An increase in ethanol production was also observed, indicating that in the absence of LDH the redox balance is maintained through alcohol dehydrogenase activity. We also found that the absence of LDH renders the pneumococci avirulent after intravenous infection and leads to a significant reduction in virulence in a model of pneumonia that develops after intranasal infection, likely due to a decrease in energy generation and virulence gene expression.

scholarly journals Role of RelA and SpoT in Burkholderia pseudomallei Virulence and Immunity

2012 ◽  
Vol 80 (9) ◽  
pp. 3247-3255 ◽  
Author(s):  
Claudia M. Müller ◽  
Laura Conejero ◽  
Natasha Spink ◽  
Matthew E. Wand ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Galleria Mellonella ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Live Attenuated Vaccine ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Control And Prevention

ABSTRACTBurkholderia pseudomalleiis a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created arelA spoTdouble mutant inB. pseudomalleistrain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotypein vitroandin vivo. TheB. pseudomalleiΔrelAΔspoTmutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in theGalleria mellonellainsect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the ΔrelAΔspoTmutant resulted in partial protection against infection with wild-typeB. pseudomallei. In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation inB. pseudomallei, and the ΔrelAΔspoTmutant may be a promising live-attenuated vaccine candidate.

scholarly journals New Pathogenesis Mechanisms and Translational Leads Identified by Multidimensional Analysis of Necrotizing Myositis in Primates

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Priyanka Kachroo ◽  
Jesus M. Eraso ◽  
Randall J. Olsen ◽  
Luchang Zhu ◽  
Samantha L. Kubiak ◽  
...  
Keyword(s):  
Nonhuman Primates ◽  
Virulence Gene ◽  
Multidimensional Analysis ◽  
Rna Seq ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Genome Wide ◽  
Necrotizing Myositis ◽  
Pathogen Fitness

ABSTRACT A fundamental goal of contemporary biomedical research is to understand the molecular basis of disease pathogenesis and exploit this information to develop targeted and more-effective therapies. Necrotizing myositis caused by the bacterial pathogen Streptococcus pyogenes is a devastating human infection with a high mortality rate and few successful therapeutic options. We used dual transcriptome sequencing (RNA-seq) to analyze the transcriptomes of S. pyogenes and host skeletal muscle recovered contemporaneously from infected nonhuman primates. The in vivo bacterial transcriptome was strikingly remodeled compared to organisms grown in vitro, with significant upregulation of genes contributing to virulence and altered regulation of metabolic genes. The transcriptome of muscle tissue from infected nonhuman primates (NHPs) differed significantly from that of mock-infected animals, due in part to substantial changes in genes contributing to inflammation and host defense processes. We discovered significant positive correlations between group A streptococcus (GAS) virulence factor transcripts and genes involved in the host immune response and inflammation. We also discovered significant correlations between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness, as assessed by previously conducted genome-wide transposon-directed insertion site sequencing (TraDIS). By integrating the bacterial RNA-seq data with the fitness data generated by TraDIS, we discovered five new pathogen genes, namely, S. pyogenes 0281 (Spy0281 [dahA]), ihk-irr, slr, isp, and ciaH, that contribute to necrotizing myositis and confirmed these findings using isogenic deletion-mutant strains. Taken together, our study results provide rich new information about the molecular events occurring in severe invasive infection of primate skeletal muscle that has extensive translational research implications. IMPORTANCE Necrotizing myositis caused by Streptococcus pyogenes has high morbidity and mortality rates and relatively few successful therapeutic options. In addition, there is no licensed human S. pyogenes vaccine. To gain enhanced understanding of the molecular basis of this infection, we employed a multidimensional analysis strategy that included dual RNA-seq and other data derived from experimental infection of nonhuman primates. The data were used to target five streptococcal genes for pathogenesis research, resulting in the unambiguous demonstration that these genes contribute to pathogen-host molecular interactions in necrotizing infections. We exploited fitness data derived from a recently conducted genome-wide transposon mutagenesis study to discover significant correlation between the magnitude of bacterial virulence gene expression in vivo and pathogen fitness. Collectively, our findings have significant implications for translational research, potentially including vaccine efforts.

scholarly journals Loss of Sigma Factor RpoN Increases Intestinal Colonization of Vibrio parahaemolyticus in an Adult Mouse Model

2013 ◽  
Vol 82 (2) ◽  
pp. 544-556 ◽  
Author(s):  
W. Brian Whitaker ◽  
Gary P. Richards ◽  
E. Fidelma Boyd
Keyword(s):  
Mouse Model ◽  
Sigma Factor ◽  
Vibrio Parahaemolyticus ◽  
Wild Type ◽  
Carbon Utilization ◽  
Content Type ◽  
Wide Range ◽  
Competition Assays

ABSTRACTVibrio parahaemolyticusis the leading cause of bacterial seafood-borne gastroenteritis worldwide, yet little is known about how this pathogen colonizes the human intestine. The alternative sigma factor RpoN/sigma-54 is a global regulator that controls flagellar synthesis, as well as a wide range of nonflagellar genes. We constructed an in-frame deletion mutation inrpoN(VP2670) inV. parahaemolyticusRIMD2210633, a clinical serogroup O3:K6 isolate, and examined the effectsin vivousing a streptomycin-treated mouse model of colonization. We confirmed that deletion ofrpoNrenderedV. parahaemolyticusnonmotile, and it caused reduced biofilm formation and an apparent defect in glutamine synthetase production. Inin vivocompetition assays between therpoNmutant and a wild-type RIMD2210633 strain marked with the β-galactosidase genelacZ(WBWlacZ), the mutant colonized significantly more proficiently. Intestinal persistence competition assays also demonstrated that therpoNmutant had enhanced fitness and outcompeted WBWlacZ. Mutants defective in the polar flagellum biosynthesis FliAP sigma factor also outcompeted WBWlacZ but not to the same level as therpoNmutant, which suggested that lack of motility is not the sole cause of the fitness effect. In anin vitrogrowth competition assay in mouse intestinal mucus, therpoNmutant also outcompeted the wild type and exhibited faster doubling times when grown in mucus and on individual components of mucus. Genes in the pathways for the catabolism of mucus sugars also had significantly higher expression levels in a ΔrpoNmutant than in the wild type. These data suggest that inV. parahaemolyticus, RpoN plays an important role in carbon utilization regulation, which may significantly affect host colonization.

scholarly journals Quorum Sensing Regulators Are Required for Metabolic Fitness in Vibrio parahaemolyticus

2017 ◽  
Vol 85 (3) ◽  
Author(s):  
Sai Siddarth Kalburge ◽  
Megan R. Carpenter ◽  
Sharon Rozovsky ◽  
E. Fidelma Boyd
Keyword(s):  
Quorum Sensing ◽  
Cell Density ◽  
Vibrio Parahaemolyticus ◽  
Double Mutant ◽  
Regulatory Region ◽  
Sugar Transport ◽  
Wild Type ◽  
Content Type ◽  
Metabolic Fitness

ABSTRACT Quorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO is active and regulates the two QS master regulators AphA, which is induced, and OpaR, which is repressed. At high cell density the opposite occurs: LuxO is inactive, and therefore OpaR is induced while AphA is repressed. In Vibrio parahaemolyticus, a significant enteric pathogen of humans, the roles of these regulators in pathogenesis are less known. We examined deletion mutants of luxO, opaR, and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to levels in the wild type. The opaR mutant did not show any defect in vivo. Colonization was restored to wild-type levels in a luxO opaR double mutant and was also increased in an opaR aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. Transcriptome sequencing (RNA-Seq) analysis of the wild type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.

scholarly journals Significant Role for ladC in Initiation of Legionella pneumophila Infection

2008 ◽  
Vol 76 (7) ◽  
pp. 3075-3085 ◽  
Author(s):  
Hayley J. Newton ◽  
Fiona M. Sansom ◽  
Jenny Dao ◽  
Christel Cazalet ◽  
Holger Bruggemann ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Acanthamoeba Castellanii ◽  
Virulence Gene ◽  
Early Time ◽  
Dominant Negative ◽  
Wild Type ◽  
Virulence Gene Expression ◽  
Mammalian Cell Lines

ABSTRACT Previously, we identified ladC in a cohort of genes that were present in Legionella pneumophila but absent in other Legionella species. Here we constructed a ladC mutant of L. pneumophila and assessed its ability to replicate in mammalian cell lines and Acanthamoeba castellanii. The ladC mutant was recovered in significantly lower numbers than wild-type L. pneumophila at early time points, which was reversed upon transcomplementation with ladC but not ladC N430A/R434A, encoding a putative catalytically inactive derivative of the protein. In fact, complementation of ladC::Km with ladC N430A/R434A resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the ladC mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the ladC mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type L. pneumophila. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that ladC does not influence the transcriptional profile of L. pneumophila in vitro or during A. castellanii infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in L. pneumophila infection.

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

2020 ◽  
Vol 139 ◽  
pp. 153-160
Author(s):  
S Peeralil ◽  
TC Joseph ◽  
V Murugadas ◽  
PG Akhilnath ◽  
VN Sreejith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Genes ◽  
Vibrio Harveyi ◽  
Penaeus Monodon ◽  
Challenge Test ◽  
Virulence Gene ◽  
Economic Losses ◽  
Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

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