scholarly journals Impact of the Listeria monocytogenes Protein InlC on Infection in Mice

2013 ◽  
Vol 81 (4) ◽  
pp. 1334-1340 ◽  
Author(s):  
Nelly Leung ◽  
Antonella Gianfelice ◽  
Scott D. Gray-Owen ◽  
Keith Ireton
Keyword(s):  
Listeria Monocytogenes ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Bacterial Pathogen ◽  
Adaptor Protein ◽  
Single Amino Acid ◽  
Wild Type ◽  
Content Type

ABSTRACTThe bacterial pathogenListeria monocytogenescauses serious food-borne illnesses in pregnant women and the immunocompromised.L. monocytogenespromotes its internalization into host epithelial cells and then uses an F-actin-dependent motility process to spread from infected cells to surrounding healthy cells. In cultured enterocytes, efficient spread ofL. monocytogenesrequires the secreted bacterial protein InlC. InlC promotes dissemination by physically interacting with and antagonizing the function of the human adaptor protein Tuba. Here we examine the role of InlC and its interaction with host Tuba during infection in mice. The study took advantage of a single-amino-acid substitution (K173A) in InlC that impairs binding to human Tuba but does not affect InlC-mediated inhibition of the NF-κB pathway. Mice were inoculated intravenously with the wild-typeL. monocytogenesstrain EGD, an isogenic strain deleted for theinlCgene (ΔinlC), or a strain expressing K173A mutant InlC (inlC.K173A). The 50% lethal doses (LD50) for the ΔinlCorinlC.K173Amutant strain were approximately 4- or 6-fold greater than that for the wild-type strain, indicating a role forinlCin virulence. Compared to the wild-type strain, theinlC.K173Amutant strain exhibited lower bacterial loads in the liver. Histological analysis of livers indicated that the twoinlCmutant strains produced smaller foci of infection than did the wild-type strain. These smaller foci are consistent with a role for InlC in cell-to-cell spreadin vivo. Taken together, these results provide evidence that interaction of InlC with host Tuba is important for full virulence.

scholarly journals Titan Cell Production Enhances the Virulence of Cryptococcus neoformans

2012 ◽  
Vol 80 (11) ◽  
pp. 3776-3785 ◽  
Author(s):  
Juliet N. Crabtree ◽  
Laura H. Okagaki ◽  
Darin L. Wiesner ◽  
Anna K. Strain ◽  
Judith N. Nielsen ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Formation ◽  
Yeast Cells ◽  
Wild Type ◽  
Content Type ◽  
Cell Production

ABSTRACTInfection withCryptococcus neoformansbegins when desiccated yeast cells or spores are inhaled and lodge in the alveoli of the lungs. A subset of cryptococcal cells in the lungs differentiate into enlarged cells, referred to as titan cells. Titan cells can be as large as 50 to 100 μm in diameter and exhibit a number of features that may affect interactions with host immune defenses. To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously describedC. neoformansmutant, thegpr4Δgpr5Δ mutant, which has minimal titan cell productionin vivo. Thegpr4Δgpr5Δ mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with thegpr4Δgpr5Δ mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor inC. neoformansthat promotes establishment of the initial pulmonary infection and plays a key role in disease progression.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

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 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 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.

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.

scholarly journals Characterization and Evaluation of a Salmonella enterica Serotype Senftenberg Mutant Created by Deletion of Virulence-Related Genes for Use as a Live Attenuated Vaccine

2016 ◽  
Vol 23 (10) ◽  
pp. 802-812 ◽  
Author(s):  
Nitin M. Kamble ◽  
John Hwa Lee
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Protective Efficacy ◽  
Control Group ◽  
Intestinal Loop ◽  
Wild Type ◽  
Exchange Method ◽  
Content Type

ABSTRACTNatural infections of chickens withSalmonella entericasubsp.entericaserovar Senftenberg (S.Senftenberg) are characterized by low-level intestinal invasiveness and insignificant production of antibodies. In this study, we investigated the potential effects oflonandcpxRgene deletions on the invasiveness ofS. Senftenberg into the intestinal epithelium of chickens and its ability to induce an immune response, conferring protection againstS. Senftenberg infection. With the allelic exchange method, we developed JOL1596 (Δlon), JOL1571 (ΔcpxR), and JOL1587 (ΔlonΔcpxR) deletion mutants from wild-typeS. Senftenberg. Deletion of thelongene fromS. Senftenberg produced increased frequency of elongated cells, with significantly greater amounts of exopolysaccharide (EPS) than in thecpxR-deleted strain and the wild-type strain. Thein vivointestinal loop invasion assay showed a significant increase in epithelial invasiveness for JOL1596 (Δlon) and JOL1587 (ΔlonΔcpxR), compared to JOL1571 (ΔcpxR) and the wild-type strain. Furthermore, theS. Senftenberg wild-type and mutant strains were internalized at high levels inside activated abdominal macrophages from chicken. Thein vivoinoculation of JOL1587 (ΔlonΔcpxR) into chickens led to colonization of the liver, spleen, and cecum for a short time. Chickens inoculated with JOL1587 (ΔlonΔcpxR) showed significant increases in humoral, mucosal, and cellular immune responses specific toS. Senftenberg antigens. Postchallenge, compared to the control group, the JOL1587 (ΔlonΔcpxR)-inoculated chickens showed not only lower persistence but also faster clearance of wild-typeS. Senftenberg from the cecum. We conclude that the increased intestinal invasiveness and colonization of internal organs exhibited by JOL1587 (ΔlonΔcpxR) led to the establishment of immunogenicity and conferred protective efficacy againstS. Senftenberg infections in chickens.

scholarly journals Role of a New Intimin/Invasin-Like Protein in Yersinia pestis Virulence

2012 ◽  
Vol 80 (10) ◽  
pp. 3559-3569 ◽  
Author(s):  
Keun Seok Seo ◽  
Jong Wan Kim ◽  
Joo Youn Park ◽  
Austin K. Viall ◽  
Scott S. Minnich ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Imaging System ◽  
Lethal Dose ◽  
Insertion Site ◽  
Surface Proteins ◽  
Wild Type ◽  
Content Type

ABSTRACTA comprehensive TnphoAmutant library was constructed inYersinia pestisKIM6 to identify surface proteins involved inY. pestishost cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants ofilpwere generated inY. pestisstrains KIM5(pCD1+) Pgm−(pigmentation negative)/, KIM6(pCD1−) Pgm+, and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cellsin vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion ofilphad no effect on bacterial blockage of flea blood feeding or colonization. TheY. pestisKIM5 Δilpstrain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P< 0.05). Following intravenous challenge withY. pestisKIM5 Δilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored byin vivoimaging using aluxreporter system (in vivoimaging system [IVIS]) and bacterial counts. Intranasal challenge in mice withY. pestisCO92 Δilphad a 55-fold increase in the 50% lethal dose ([LD50] 1.64 × 104CFU) compared to the parental wild-type strain LD50(2.98 × 102CFU). These findings identified Ilp as a novel virulence factor ofY. pestis.

scholarly journals The RNA Chaperone Hfq Promotes Fitness of Actinobacillus pleuropneumoniae during Porcine Pleuropneumonia

2013 ◽  
Vol 81 (8) ◽  
pp. 2952-2961 ◽  
Author(s):  
Sargurunathan Subashchandrabose ◽  
Rhiannon M. Leveque ◽  
Roy N. Kirkwood ◽  
Matti Kiupel ◽  
Martha H. Mulks
Keyword(s):  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Actinobacillus Pleuropneumoniae ◽  
Wild Type ◽  
Content Type ◽  
Porcine Pleuropneumonia ◽  
Porcine Lungs

ABSTRACTActinobacillus pleuropneumoniaeis the etiological agent of porcine pleuropneumonia, an economically important disease of pigs. Thehfqgene inA. pleuropneumoniae, encoding the RNA chaperone and posttranscriptional regulator Hfq, is upregulated during infection of porcine lungs. To investigate the role of thisin vivo-induced gene inA. pleuropneumoniae, anhfqmutant strain was constructed. Thehfqmutant was defective in biofilm formation on abiotic surfaces. The level ofpgaCtranscript, encoding the biosynthesis of poly-β-1,6-N-acetylglucosamine (PNAG), a major biofilm matrix component, was lower and PNAG content was 10-fold lower in thehfqmutant than in the wild-type strain. When outer membrane proteins were examined, cysteine synthase, implicated in resistance to oxidative stress and tellurite, was not found at detectable levels in the absence of Hfq. Thehfqmutant displayed enhanced sensitivity to superoxide generated by methyl viologen and tellurite. These phenotypes were readily reversed by complementation with thehfqgene expressed from its native promoter. The role of Hfq in the fitness ofA. pleuropneumoniaewas assessed in a natural host infection model. Thehfqmutant failed to colonize porcine lungs and was outcompeted by the wild-type strain (median competitive index of 2 × 10−5). Our data demonstrate that thein vivo-induced genehfqis involved in the regulation of PNAG-dependent biofilm formation, resistance to superoxide stress, and the fitness and virulence ofA. pleuropneumoniaein pigs and begin to elucidate the role of anin vivo-induced gene in the pathogenesis of pleuropneumonia.

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