scholarly journals Identification of MglA-Regulated Genes Reveals Novel Virulence Factors in Francisella tularensis

2006 ◽  
Vol 74 (12) ◽  
pp. 6642-6655 ◽  
Author(s):  
Anna Brotcke ◽  
David S. Weiss ◽  
Charles C. Kim ◽  
Patrick Chain ◽  
Stephanie Malfatti ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Virulence Factors ◽  
Francisella Tularensis ◽  
Type Strain ◽  
Virulence Genes ◽  
Wild Type Strain ◽  
Transcriptional Profiling ◽  
Intracellular Bacterium ◽  
Wild Type

ABSTRACT The facultative intracellular bacterium Francisella tularensis causes the zoonotic disease tularemia. F. tularensis resides within host macrophages in vivo, and this ability is essential for pathogenesis. The transcription factor MglA is required for the expression of several Francisella genes that are necessary for replication in macrophages and for virulence in mice. We hypothesized that the identification of MglA-regulated genes in the Francisella genome by transcriptional profiling of wild-type and mglA mutant bacteria would lead to the discovery of new virulence factors utilized by F. tularensis. A total of 102 MglA-regulated genes were identified, the majority of which were positively regulated, including all of the Francisella pathogenicity island (FPI) genes. We mutated novel MglA-regulated genes and tested the mutants for their ability to replicate and induce cytotoxicity in macrophages and to grow in mice. Mutations in MglA-regulated genes within the FPI (pdpB and cds2) as well as outside the FPI (FTT0989, oppB, and FTT1209c) were either attenuated or hypervirulent in macrophages compared to the wild-type strain. All of these mutants exhibited decreased fitness in vivo in competition experiments with wild-type bacteria. We have identified five new Francisella virulence genes, and our results suggest that characterizations of additional MglA-regulated genes will yield further insights into the pathogenesis of this bacterium.

scholarly journals Deletion of the Bacillus anthracis capB homologue in Francisella tularensis subspecies tularensis generates an attenuated strain that protects mice against virulent tularaemia

2010 ◽  
Vol 59 (11) ◽  
pp. 1275-1284 ◽  
Author(s):  
Stephen L. Michell ◽  
Rachel E. Dean ◽  
Jim E. Eyles ◽  
Margaret Gill Hartley ◽  
Emma Waters ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Virulence Factors ◽  
Francisella Tularensis ◽  
Causative Agent ◽  
Sequence Homology ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Infectious Dose ◽  
Homologous Challenge

As there is currently no licensed vaccine against Francisella tularensis, the causative agent of tularaemia, the bacterium is an agent of concern as a potential bioweapon. Although F. tularensis has a low infectious dose and high associated mortality, it possesses few classical virulence factors. An analysis of the F. tularensis subspecies tularensis genome sequence has revealed the presence of a region containing genes with low sequence homology to part of the capBCADE operon of Bacillus anthracis. We have generated an isogenic capB mutant of F. tularensis subspecies tularensis SchuS4 and shown it to be attenuated. Furthermore, using BALB/c mice, we have demonstrated that this capB strain affords protection against significant homologous challenge with the wild-type strain. These data have important implications for the development of a defined and efficacious tularaemia vaccine.

scholarly journals Identification and Testing of Porphyromonas gingivalis Virulence Genes with a pPGIVET System

2002 ◽  
Vol 70 (2) ◽  
pp. 928-937 ◽  
Author(s):  
Yi Wu ◽  
Seok-Woo Lee ◽  
Jeffrey D. Hillman ◽  
Ann Progulske-Fox
Keyword(s):  
Mutant Strain ◽  
Porphyromonas Gingivalis ◽  
Type Strain ◽  
Virulence Genes ◽  
Wild Type Strain ◽  
Receptor Protein ◽  
Wild Type ◽  
Kb Cells ◽  
Mutant Strains

ABSTRACT An in vivo expression technology (IVET) system was designed to identify previously unknown virulence genes of Porphyromonas gingivalis. Fourteen ivi (for in vivo induced) genes that are induced during infection in a mouse abscess model were identified in our study. Of these, seven had homology to genes in the NCBI database, and the rest had no homology to reported DNA sequences. In order to determine virulence-related properties of these genes, three mutant strains, deleted of ivi8 (no homology to genes in the database), ivi10 (homologous to a putative TonB-dependent outer membrane receptor protein), and ivi11 (an immunoreactive 33-kDa antigen PG125 in P. gingivalis), were created. The mutants were tested in a mouse abscess model for alterations in virulence relative to the wild type by a competition assay in BALB/c mice. After 5 days we observed the enrichment of the wild-type strain over mutant strains Δivi10 and Δivi11, which indicated that mutant strains Δivi10 and Δivi11 are less able to survive in this model than the wild-type strain, while Δivi8 survives as well as the wild-type strain. We propose that knockout of these ivi genes reduced the ability of the mutated P. gingivalis to survive and cause infection compared to the wild-type strain at the site of injection. Also, in separate experiments, groups of mice were challenged with subcutaneous injections of each individual mutant strain (Δivi8, Δivi10, and Δivi11) or with the wild-type strain alone and were then examined to assess their general health status. The results showed that knockout of these ivi genes conferred a reduction in virulence. The ability of the mutants to invade KB cells compared to the wild type was also determined. Interestingly, the CFU counts of the mutant strain Δivi10 recovered from KB cells were eight times lower than those of the wild type, indicating that this mutant has a lower capacity for invasion. These results demonstrate that IVET is a powerful tool in discovering virulence genes and the significant role that ivi genes play in the pathogenesis of this species.

scholarly journals In VivoEfficacy of Human Simulated Regimens of Carbapenems and Comparator Agents against NDM-1-Producing Enterobacteriaceae

2013 ◽  
Vol 58 (3) ◽  
pp. 1671-1677 ◽  
Author(s):  
Dora E. Wiskirchen ◽  
Patrice Nordmann ◽  
Jared L. Crandon ◽  
David P. Nicolau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Similar Efficacy ◽  
Infection Model ◽  
High Dose ◽  
Wild Type ◽  
Prolonged Infusion ◽  
Content Type

ABSTRACTDoripenem and ertapenem have demonstrated efficacy against several NDM-1-producing isolatesin vivo, despite having high MICs. In this study, we sought to further characterize the efficacy profiles of humanized regimens of standard (500 mg given every 8 h) and high-dose, prolonged infusion of doripenem (2 g given every 8 h, 4-h infusion) and 1 g of ertapenem given intravenously every 24 h and the comparator regimens of ceftazidime at 2 g given every 8 h (2-h infusion), levofloxacin at 500 mg every 24 h, and aztreonam at 2 g every 6 h (1-h infusion) against a wider range of isolates in a murine thigh infection model. An isogenic wild-type strain and NDM-1-producingKlebsiella pneumoniaeand eight clinical NDM-1-producing members of the familyEnterobacteriaceaewere tested in immunocompetent- and neutropenic-mouse models. The wild-type strain was susceptible to all of the agents, while the isogenic NDM-1-producing strain was resistant to ceftazidime, doripenem, and ertapenem. Clinical NDM-1-producing strains were resistant to nearly all five of the agents (two were susceptible to levofloxacin). In immunocompetent mice, all of the agents produced ≥1-log10CFU reductions of the isogenic wild-type and NDM-1-producing strains after 24 h. Minimal efficacy of ceftazidime, aztreonam, and levofloxacin against the clinical NDM-1-producing strains was observed. However, despitein vitroresistance, ≥1-log10CFU reductions of six of eight clinical strains were achieved with high-dose, prolonged infusion of doripenem and ertapenem. Slight enhancements of doripenem activity over the standard doses were obtained with high-dose, prolonged infusion for three of the four isolates tested. Similar efficacy observations were noted in neutropenic mice. These data suggest that carbapenems are a viable treatment option for infections caused by NDM-1-producingEnterobacteriaceae.

scholarly journals Analysis and Manipulation of Aspartate Pathway Genes for l-Lysine Overproduction from Methanol by Bacillus methanolicus

2011 ◽  
Vol 77 (17) ◽  
pp. 6020-6026 ◽  
Author(s):  
Ingemar Nærdal ◽  
Roman Netzer ◽  
Trond E. Ellingsen ◽  
Trygve Brautaset
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Feedback Inhibition ◽  
Wild Type ◽  
Content Type ◽  
Bacillus Methanolicus ◽  
Semialdehyde Dehydrogenase ◽  
Aspartate Pathway ◽  
Lysine Overproduction

ABSTRACTWe investigated the regulation and roles of six aspartate pathway genes inl-lysine overproduction inBacillus methanolicus:dapG, encoding aspartokinase I (AKI);lysC, encoding AKII;yclM, encoding AKIII;asd, encoding aspartate semialdehyde dehydrogenase;dapA, encoding dihydrodipicolinate synthase; andlysA, encodingmeso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed thatin vivo lysCtranscription was repressed 5-fold byl-lysine and induced 2-fold bydl-methionine added to the growth medium. Surprisingly,yclMtranscription was repressed 5-fold bydl-methionine, while thedapG,asd,dapA, andlysAgenes were not significantly repressed by any of the aspartate pathway amino acids. We show that thel-lysine-overproducing classicalB. methanolicusmutant NOA2#13A52-8A66 has—in addition to ahom-1mutation—chromosomal mutations in thedapGcoding region and in thelysApromoter region. No mutations were found in itsdapA,lysC,asd, andyclMgenes. The mutantdapGgene product had abolished feedback inhibition bymeso-diaminopimelatein vitro, and thelysAmutation was accompanied by an elevated (6-fold)lysAtranscription levelin vivo. Moreover,yclMtranscription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important forl-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increasedl-lysine production levels.

scholarly journals Contribution of Urease to Colonization by Shiga Toxin-Producing Escherichia coli

2012 ◽  
Vol 80 (8) ◽  
pp. 2589-2600 ◽  
Author(s):  
Susan R. Steyert ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Type Strain ◽  
Nitrogen Availability ◽  
Wild Type Strain ◽  
Clinical Manifestations ◽  
Maximum Activity ◽  
Wild Type ◽  
Urease Enzyme

ABSTRACTShiga toxin-producingEscherichia coli(STEC) is a food-borne pathogen with a low infectious dose that colonizes the colon in humans and can cause severe clinical manifestations such as hemolytic-uremic syndrome. The urease enzyme, encoded in the STEC chromosome, has been demonstrated to act as a virulence factor in other bacterial pathogens. The NH3produced as urease hydrolyzes urea can aid in buffering bacteria in acidic environments as well as provide an easily assimilated source of nitrogen that bacteria can use to gain a metabolic advantage over intact microflora. Here, we explore the role of urease in STEC pathogenicity. The STEC urease enzyme exhibited maximum activity near neutral pH and during the stationary-growth phase. Experiments altering growth conditions performed with three phylogenetically distinct urease-positive strains demonstrated that the STECuregene cluster is inducible by neither urea nor pH but does respond to nitrogen availability. Quantitative reverse transcription-PCR (qRT-PCR) data indicate that nitrogen inhibits the transcriptional response. The deletion of theuregene locus was constructed in STEC strain 88-0643, and theuremutant was used with the wild-type strain in competition experiments in mouse models to examine the contribution of urease. The wild-type strain was twice as likely to survive passage through the acidic stomach and demonstrated an enhanced ability to colonize the intestinal tract compared to theuremutant strain. Thesein vivoexperiments reveal that, although the benefit STEC gains from urease expression is modest and not absolutely required for colonization, urease can contribute to the pathogenicity of STEC.

scholarly journals Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

2006 ◽  
Vol 188 (17) ◽  
pp. 6269-6276 ◽  
Author(s):  
Sofiane Ghorbel ◽  
Aleksey Smirnov ◽  
Hichem Chouayekh ◽  
Brice Sperandio ◽  
Catherine Esnault ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sufficient Conditions ◽  
Streptomyces Lividans ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild

ABSTRACT The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

scholarly journals Phenotypic Characterization of Auxotrophic Mutant of Nontyphoidal Salmonella and Determination of Its Cytotoxicity, Tumor Inhibiting Cytokine Gene Expression in Cell Line Models

2021 ◽  
Author(s):  
Kadeeja Jazeela ◽  
Anirban Chakraborty ◽  
Akshatha Kotian ◽  
Vankadari Aditya ◽  
Krishna Kumar Ballamoole ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Type Strain ◽  
Virulence Genes ◽  
Wild Type Strain ◽  
Auxotrophic Mutant ◽  
Cytokine Gene Expression ◽  
Wild Type ◽  
Cytokine Genes

Abstract An auxotrophic mutant of non-typhoidal Salmonella (NTS) strain was phenotypically characterized in this study. The characterization was based on phenotype, morphology, motility, biofilm forming ability, growth kinetics, etc. The phenotypic results from the above experiments determined that the mutant showed variation in phenotypic characters from that of wild-type strain. Subsequently, mutant and wild-type NTS were subjected to epithelial cell invasion and intracellular replication assays. The real time PCR analysis was also performed to analyse expression of tumor inhibiting cytokine genes and virulence genes post bacterial infection in cell lines. The mutant showed highest invasion potential than wild-type NTS whereas the replication of mutant was slower in both the cell lines. Similar to the wild-type strain, the mutant also retained the cytotoxic potential when analysed in vitro. Furthermore, the expression of proinflammatory cytokine genes such as TNF-α and IL-1β was upsurged with the downregulation of anti-inflammatory cytokine genes like TGF-β, IL-6 and IL-10 post infection of the mutant strain in cell lines. In addition, virulence genes of Salmonella pathogenicity island one and two of mutant were downregulated in vitro except invA in HeLa cell line. Therefore, the auxotrophic mutant showed positive attributes of a potential antitumor agent in terms of expressing tumor inhibiting cytokine genes when assessed in vitro. Though the study did not check the tumor inhibitory effect of NTS strain directly, findings of the study emphasizes on the development of a novel strain of NTS with less virulence and more immunogenic traits to inhibit tumor cells.

scholarly journals A Single Amino Acid at Residue 188 of Hexon Protein is Responsible for the Pathogenicity of the Emerging Novel Fowl Adenovirus 4

2021 ◽  
Author(s):  
Yu Zhang ◽  
Aijing Liu ◽  
Yanan Wang ◽  
Hongyu Cui ◽  
Yulong Gao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Molecular Basis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Live Attenuated Vaccine ◽  
Hexon Protein

Since 2015, severe hydropericardium-hepatitis syndrome (HHS) associated with a novel fowl adenovirus 4 (FAdV-4) has emerged in China, representing a new challenge for the poultry industry. Although various highly pathogenic FAdV-4 strains have been isolated, the virulence factor and the pathogenesis of novel FAdV-4 are unclear. In our previous studies, we reported that a large genomic deletion (1966 bp) is not related to increased virulence. In this study, two recombinant chimeric viruses, rHN20 strain and rFB2 strain, were generated from a highly pathogenic FAdV-4 strain by replacing hexon or fiber-2 gene of a non-pathogenic FAdV-4, respectively. Both chimeric strains showed similar titers to the wild type strain in vitro . Notably, rFB2 and the wild type strain induced 100% mortality, while no mortality or clinical signs appeared in chickens inoculated with rHN20, indicating that hexon, but not fiber-2, determines the novel FAdV-4 virulence. Furthermore, an R188I mutation in the hexon protein identified residue 188 as the key amino acid for the reduced pathogenicity. The rR188I mutant strain was significantly neutralized by chicken serum in vitro and in vivo , whereas the wild type strain was able to replicate efficiently. Finally, the immunogenicity of the rescued rR188I was investigated. Non-pathogenic rR188I provided full protection against lethal FAdV-4 challenge. Collectively, these findings provide an in-depth understanding of the molecular basis of novel FAdV-4 pathogenicity and present rR188I as a potential live attenuated vaccine candidate or a novel vaccine vector for HHS vaccines. Importance HHS associated with a novel FAdV-4 infection in chickens has caused huge economic losses to the poultry industry in China since 2015. The molecular basis for the increased virulence remains largely unknown. Here, we demonstrate that the hexon gene is vital for FAdV-4 pathogenicity. Furthermore, we show that the amino acid residue at position 188 of the hexon protein is responsible for pathogenicity. Importantly, the rR188I mutant strain was neutralized by chicken serum in vitro and in vivo , whereas the wild type strain was not. Further, the rR188I mutant strain provided complete protection against FAdV-4 challenge. Our results provide a molecular basis of the increased virulence of novel FAdV-4. We propose that the rR188I mutant is a potential live attenuated vaccine against HHS and a new vaccine vector for HHS-combined vaccines.

Porphyromonas gingivalis Induces Insulin Resistance by Increasing BCAA Levels in Mice

2020 ◽  
Vol 99 (7) ◽  
pp. 839-846 ◽  
Author(s):  
J. Tian ◽  
C. Liu ◽  
X. Zheng ◽  
X. Jia ◽  
X. Peng ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Sensitivity ◽  
Plasma Level ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Alveolar Bone ◽  
Alveolar Bone Loss ◽  
Wild Type

Insulin resistance is one of the critical pathogeneses of type 2 diabetes mellitus (T2DM). Elevated levels of plasma branched-chain amino acids (BCAAs) are associated with insulin resistance. Recent studies have demonstrated the role of Porphyromonas gingivalis in the development of insulin resistance. However, the mechanisms by which P. gingivalis induces insulin resistance are still unclear. The purpose of this study was to investigate whether P. gingivalis induces insulin resistance through BCAA biosynthesis. We established a murine model of periodontitis by infecting mice with P. gingivalis. Alveolar bone loss, insulin sensitivity, and the plasma level of BCAAs were measured. A P. gingivalis BCAA aminotransferase-deficient strain ( ∆bcat) was constructed, and its kinetic growth, biofilm formation, and in vivo colonization were compared with its wild-type strain. Alveolar bone loss, insulin sensitivity, and the plasma level of BCAAs of the mice infected with either wild-type strain or ∆bcat strain were further measured. We found that periodontal infection with P. gingivalis significantly upregulated the plasma level of BCAAs and aggravated the high-fat diet (HFD)–induced insulin resistance. Bcat deletion did not alter the growth, biofilm formation, and in vivo colonization of P. gingivalis. More important, the ∆bcat strain was unable to upregulate the plasma level of BCAAs and induce insulin resistance in HFD-fed mice. These findings suggest that the BCAA biosynthesis of P. gingivalis plays a critical role in the development of insulin resistance in the HFD-fed mice. The BCAA biosynthesis pathways may provide a potential target for the disruption of linkage between periodontitis and T2DM.

scholarly journals Genes Involved in Galactooligosaccharide Metabolism in Lactobacillus reuteri and Their Ecological Role in the Gastrointestinal Tract

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Monchaya Rattanaprasert ◽  
Jan-Peter van Pijkeren ◽  
Amanda E. Ramer-Tait ◽  
Maria Quintero ◽  
Car Reen Kok ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Lactobacillus Reuteri ◽  
Selective Advantage ◽  
Wild Type ◽  
Carbohydrate Source ◽  
Content Type ◽  
Gnotobiotic Mice

ABSTRACT Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo. In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two β-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway. IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

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