scholarly journals Flavohemoglobin Hmp Protects Salmonella enterica Serovar Typhimurium from Nitric Oxide-Related Killing by Human Macrophages

2002 ◽  
Vol 70 (8) ◽  
pp. 4399-4405 ◽  
Author(s):  
Tânia M. Stevanin ◽  
Robert K. Poole ◽  
Eric A. G. Demoncheaux ◽  
Robert C. Read
Keyword(s):  
Nitric Oxide ◽  
Salmonella Enterica ◽  
Nitrosative Stress ◽  
Invasive Disease ◽  
Wild Type ◽  
Human Macrophages ◽  
Microbicidal Activity ◽  
Enhanced Sensitivity ◽  
Serovar Typhimurium

ABSTRACT Survival of macrophage microbicidal activity is a prerequisite for invasive disease caused by the enteric pathogen Salmonella enterica serovar Typhimurium. Flavohemoglobins, such as those of Escherichia coli, Salmonella, and yeast, play vital roles in protection of these microorganisms in vitro from nitric oxide (NO) and nitrosative stress. A Salmonella hmp mutant defective in flavohemoglobin (Hmp) synthesis exhibits growth that is hypersensitive to nitrosating agents. We found that respiration of this mutant exhibited increased inhibition by NO, whereas wild-type cells pregrown with sodium nitroprusside or S-nitrosoglutathione showed enhanced tolerance of NO. Most significantly, hmp mutants internalized by primary human peripheral monocyte-derived macrophages survived phagocytosis relatively poorly compared with similarly bound and internalized wild-type cells. That the enhanced sensitivity to macrophage microbicidal activity is due primarily to the failure of Salmonella to detoxify NO was suggested by the ability of l-N G-monomethyl arginine—an inhibitor of NO synthase—to eliminate the difference in killing between wild-type and hmp mutant Salmonella cells. These observations suggest that Salmonella Hmp contributes to protection from NO-mediated inhibition by human macrophages.

scholarly journals Caveolin-1-Deficient Mice Show Defects in Innate Immunity and Inflammatory Immune Response during Salmonella enterica Serovar Typhimurium Infection

2006 ◽  
Vol 74 (12) ◽  
pp. 6665-6674 ◽  
Author(s):  
Freddy A. Medina ◽  
Cecilia J. de Almeida ◽  
Elliott Dew ◽  
Jiangwei Li ◽  
Gloria Bonuccelli ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Salmonella Enterica ◽  
Inflammatory Responses ◽  
Systemic Infection ◽  
Bacterial Invasion ◽  
Chemokine Production ◽  
Caveolin 1 ◽  
Serovar Typhimurium ◽  
Deficient Mice

ABSTRACT A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in Salmonella pathogenesis. Cav-1−/− mice displayed a significant decrease in survival when challenged with Salmonella enterica serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1−/− mice. However, infection of Cav-1−/− macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1−/− mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1−/− mice were unable to control the systemic infection of Salmonella. The increased chemokine production in Cav-1−/− mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1−/− macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to Salmonella LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to S. enterica serovar Typhimurium.

scholarly journals Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

2001 ◽  
Vol 69 (12) ◽  
pp. 7413-7418 ◽  
Author(s):  
Tahar van der Straaten ◽  
Angela van Diepen ◽  
Kitty Kwappenberg ◽  
Sjaak van Voorden ◽  
Kees Franken ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Function ◽  
Salmonella Enterica ◽  
Host Cells ◽  
Wild Type ◽  
Intracellular Replication ◽  
Oxygen Intermediates ◽  
Serovar Typhimurium

ABSTRACT Upon contact with host cells, the intracellular pathogenSalmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104 to 107bacteria in C3H/HeN and 101 to 104 bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.

scholarly journals Flagellar Phase Variation of Salmonella enterica Serovar Typhimurium Contributes to Virulence in the Murine Typhoid Infection Model but Does Not InfluenceSalmonella-Induced Enteropathogenesis

2001 ◽  
Vol 69 (5) ◽  
pp. 3021-3030 ◽  
Author(s):  
Jack S. Ikeda ◽  
Clare K. Schmitt ◽  
Stephen C. Darnell ◽  
Patricia R. Watson ◽  
Jennifer Bispham ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Salmonella Enterica ◽  
Phase Variation ◽  
Selective Advantage ◽  
Peritoneal Macrophages ◽  
Infection Model ◽  
Wild Type ◽  
Two Phase ◽  
Serovar Typhimurium

ABSTRACT Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC+phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.

scholarly journals Modulation of Virulence by Two Acidified Nitrite-Responsive Loci of Salmonella enterica Serovar Typhimurium

2003 ◽  
Vol 71 (6) ◽  
pp. 3196-3205 ◽  
Author(s):  
Charles C. Kim ◽  
Denise Monack ◽  
Stanley Falkow
Keyword(s):  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner ◽  
Inducible Promoters ◽  
Serovar Typhimurium ◽  
Bacterial Genes ◽  
Green Fluorescent

ABSTRACT Two acidified nitrite-inducible genes of Salmonella enterica serovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designated nipAB and nipC, which correspond to hcp-hcr (hybrid cluster protein) of Escherichia coli and norA of Alcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. The nipAB promoter was regulated by oxygen in an Fnr-dependent manner. The nipC promoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although the nipAB and nipC mutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.

scholarly journals Thioredoxin 1 Promotes Intracellular Replication and Virulence of Salmonella enterica Serovar Typhimurium

2006 ◽  
Vol 74 (9) ◽  
pp. 5140-5151 ◽  
Author(s):  
Eva Bjur ◽  
Sofia Eriksson-Ygberg ◽  
Fredrik Åslund ◽  
Mikael Rhen
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Salmonella Enterica ◽  
Nitric Oxide Donor ◽  
Intracellular Replication ◽  
Thioredoxin System ◽  
Serovar Typhimurium ◽  
Thioredoxin 1 ◽  
Disulfide Bond Isomerase

ABSTRACT The effect of the cytoplasmic reductase and protein chaperone thioredoxin 1 on the virulence of Salmonella enterica serovar Typhimurium was evaluated by deleting the trxA, trxB, or trxC gene of the cellular thioredoxin system, the grxA or gshA gene of the glutathione/glutaredoxin system, or the dsbC gene coding for a thioredoxin-dependent periplasmic disulfide bond isomerase. Mutants were tested for tolerance to oxidative and nitric oxide donor substances in vitro, for invasion and intracellular replication in cultured epithelial and macrophage-like cells, and for virulence in BALB/c mice. In these experiments only the gshA mutant, which was defective in glutathione synthesis, exhibited sensitization to oxidative stress in vitro and a small decrease in virulence. In contrast, the trxA mutant did not exhibit any growth defects or decreased tolerance to oxidative or nitric oxide stress in vitro, yet there were pronounced decreases in intracellular replication and mouse virulence. Complementation analyses using defined catalytic variants of thioredoxin 1 showed that there is a direct correlation between the redox potential of thioredoxin 1 and restoration of intracellular replication of the trxA mutant. Attenuation of mouse virulence that was caused by a deficiency in thioredoxin 1 was restored by expression of wild-type thioredoxin 1 in trans but not by expression of a catalytically inactive variant. These results clearly imply that in S. enterica serovar Typhimurium, the redox-active protein thioredoxin 1 promotes virulence, whereas in vitro tolerance to oxidative stress depends on production of glutathione.

scholarly journals Single passage in mouse organs enhances the survival and spread of Salmonella enterica

2015 ◽  
Vol 12 (113) ◽  
pp. 20150702 ◽  
Author(s):  
Richard Dybowski ◽  
Olivier Restif ◽  
Alexandre Goupy ◽  
Duncan J. Maskell ◽  
Piero Mastroeni ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Probability Generating Function ◽  
Systemic Infection ◽  
Original Method ◽  
Wild Type ◽  
Immigration Process ◽  
Serovar Typhimurium ◽  
Intravenous Inoculation

Intravenous inoculation of Salmonella enterica serovar Typhimurium into mice is a prime experimental model of invasive salmonellosis. The use of wild-type isogenic tagged strains (WITS) in this system has revealed that bacteria undergo independent bottlenecks in the liver and spleen before establishing a systemic infection. We recently showed that those bacteria that survived the bottleneck exhibited enhanced growth when transferred to naive mice. In this study, we set out to disentangle the components of this in vivo adaptation by inoculating mice with WITS grown either in vitro or in vivo . We developed an original method to estimate the replication and killing rates of bacteria from experimental data, which involved solving the probability-generating function of a non-homogeneous birth–death–immigration process. This revealed a low initial mortality in bacteria obtained from a donor animal. Next, an analysis of WITS distributions in the livers and spleens of recipient animals indicated that in vivo -passaged bacteria started spreading between organs earlier than in vitro -grown bacteria. These results further our understanding of the influence of passage in a host on the fitness and virulence of Salmonella enterica and represent an advance in the power of investigation on the patterns and mechanisms of host–pathogen interactions.

scholarly journals Engineering and Preclinical Evaluation of Attenuated Nontyphoidal Salmonella Strains Serving as Live Oral Vaccines and as Reagent Strains

2011 ◽  
Vol 79 (10) ◽  
pp. 4175-4185 ◽  
Author(s):  
Sharon M. Tennant ◽  
Jin-Yuan Wang ◽  
James E. Galen ◽  
Raphael Simon ◽  
Marcela F. Pasetti ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Lethal Dose ◽  
Invasive Disease ◽  
Oral Immunization ◽  
Oral Vaccines ◽  
Wild Type ◽  
Lethal Challenge ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTWhile nontyphoidalSalmonella(NTS) has long been recognized as a cause of self-limited gastroenteritis, it is becoming increasingly evident that multiple-antibiotic-resistant strains are also emerging as important causes of invasive bacteremia and focal infections, resulting in hospitalizations and deaths. We have constructed attenuatedSalmonella entericaserovar Typhimurium andSalmonella entericaserovar Enteritidis strains that can serve as live oral vaccines and as “reagent strains” for subunit vaccine production in a safe and economical manner. Prototype attenuated vaccine strains CVD 1921 and CVD 1941, derived from the invasive wild-type strainsS. TyphimuriumI77 andS. EnteritidisR11, respectively, were constructed by deletingguaBA, encoding guanine biosynthesis, andclpP, encoding a master protease regulator. TheclpPmutation resulted in a hyperflagellation phenotype. An additional deletion infliDyielded reagent strains CVD 1923 and CVD 1943, respectively, which export flagellin monomers. Oral 50% lethal dose (LD50) analyses showed that the NTS vaccine strains were all highly attenuated in mice. Oral immunization with CVD 1921 or CVD 1923 protected mice against lethal challenge with wild-typeS. TyphimuriumI77. Immunization with CVD 1941 but not CVD 1943 protected mice against lethal infection withS. EnteritidisR11. Immune responses induced by these strains included high levels of serum IgG anti-lipopolysaccharide (LPS) and anti-flagellum antibodies, with titers increasing progressively during the immunization schedule. SinceS. TyphimuriumandS. Enteritidisare the most common NTS serovars associated with invasive disease, these findings can pave the way for development of a highly effective, broad-spectrum vaccine against invasive NTS.

scholarly journals Role of B7 Costimulatory Molecules in Mediating Systemic and Mucosal Antibody Responses to Attenuated Salmonella enterica Serovar Typhimurium and Its Cloned Antigen

2004 ◽  
Vol 72 (10) ◽  
pp. 5824-5831 ◽  
Author(s):  
Carlos A. Garcia ◽  
Michael Martin ◽  
Suzanne M. Michalek
Keyword(s):  
Salmonella Enterica ◽  
Immunoglobulin A ◽  
Costimulatory Molecules ◽  
Antibody Responses ◽  
Wild Type ◽  
Igg Antibody ◽  
Functional Roles ◽  
Serovar Typhimurium

ABSTRACT The purpose of the present study was to evaluate the ability of an attenuated Salmonella enterica serovar Typhimurium vaccine strain to up-regulate B7-1 and B7-2 on antigen-presenting cells and to examine the functional roles these costimulatory molecules play in mediating immune responses to Salmonella and to an expressed cloned antigen, the saliva-binding region (SBR) of antigen I/II. In vitro stimulation of B cells (B220+), macrophages (CD11b+), and dendritic cells (CD11c+) with S. enterica serovar Typhimurium induced an up-regulation of B7-2 and, especially, B7-1 expression. The in vivo functional roles of B7-1, B7-2, and B7-1/2 were evaluated in BALB/c wild-type and B7-1, B7-2, and B7-1/2 knockout (KO) mice following intranasal immunization with the Salmonella expressing the cloned SBR. Differential requirements for B7-1 and B7-2 were observed upon primary and secondary immunizations. Compared to wild-type controls, B7-1 and B7-2 KO mice had reduced mucosal and systemic anti-Salmonella antibody responses after a single immunization, while only B7-1 KO mice exhibited suppressed anti-Salmonella antibody responses following the second immunization. Mucosal and systemic antibody responses to SBR were reduced following the primary immunization, whereas a compensatory role for either B7-1 or B7-2 was observed after the second immunization. B7-1/2 double KO mice failed to induce detectable levels of mucosal or systemic immunoglobulin A (IgA) or IgG antibody responses to either Salmonella or SBR. These findings demonstrate that B7-1 and B7-2 can play distinct as well as redundant roles for mediating mucosal and systemic antibody responses, which are likely dependent upon the nature of the antigen.

scholarly journals Rapid Protection of Gnotobiotic Pigs against Experimental Salmonellosis following Induction of Polymorphonuclear Leukocytes by Avirulent Salmonella enterica

2003 ◽  
Vol 71 (4) ◽  
pp. 2182-2191 ◽  
Author(s):  
N. Foster ◽  
M. A. Lovell ◽  
K. L. Marston ◽  
S. D. Hulme ◽  
A. J. Frost ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Clinical Symptoms ◽  
Invasive Disease ◽  
Polymorphonuclear Neutrophils ◽  
Intestinal Lumen ◽  
Epithelial Cell Line ◽  
Intestinal Pathology ◽  
Serovar Typhimurium ◽  
Gnotobiotic Pigs

ABSTRACT Oral inoculation of 5-day-old gnotobiotic pigs with Salmonella enterica serovar Typhimurium strain F98 resulted in severe enteritis and invasive disease. Preinoculation 24 h earlier with an avirulent mutant of Salmonella enterica serovar Infantis (1326/28) completely prevented disease for up to 14 days (when the experiment was terminated). S. enterica serovar Infantis colonized the alimentary tract well, with high bacterial counts in the intestinal lumen but with almost no invasion into the tissues. Unprotected pigs had high S. enterica serovar Typhimurium counts in the intestines, blood, and major nonintestinal organs. Recovery of this strain from the blood and major organs in S. enterica serovar Infantis-protected pigs was substantially reduced despite the fact that intestinal counts were also very high. Protection against disease thus did not involve a colonization exclusion phenomenon. Significant (P < 0.05) infiltration of monocytes/macrophages was observed in the submucosal regions of the intestines of both S. enterica serovar Infantis-protected S. enterica serovar Typhimurium-challenged pigs and unprotected S. enterica serovar Typhimurium-challenged pigs. However, only polymorphonuclear neutrophils (PMNs) were observed throughout the villus, where significant (P < 0.05) numbers infiltrated the lamina propria and the subnuclear and supranuclear regions of the epithelia, indicating that PMN induction and positioning following S. enterica serovar Infantis inoculation was consistent with rapid protection against the challenge strain. Similarly, in vitro experiments using a human fetal intestinal epithelial cell line (INT 407) demonstrated that, although significantly (P < 0.05) fewer S. enterica serovar Infantis than S. enterica serovar Typhimurium organisms invaded the monolayers, S. enterica serovar Infantis induced an NF-κB response and significantly (P < 0.05) raised interleukin 8 levels and transmigration of porcine PMN. The results of this study suggest that attenuated Salmonella strains can protect the immature intestine against clinical salmonellosis by PMN induction. They also demonstrate that PMN induction is not necessarily associated with clinical symptoms and/or intestinal pathology.

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