Modifying bacterial flagellin to evade Nod-like Receptor CARD 4 recognition enhances protective immunity against Salmonella.

Pattern recognition receptors (PRRs) expressed in antigen-presenting cells are thought to shape pathogen-specific immunity by inducing secretion of costimulatory cytokines during T-cell activation, yet data to support this notion in vivo are scarce. Here, we show that the cytosolic PRR Nod-like Receptor CARD 4 (NLRC4) suppresses, rather than facilitates, effector and memory CD4+ T-cell responses against Salmonella in mice. NLRC4 negatively regulates immunological memory by preventing delayed activation of the cytosolic PRR NLR pyrin domain 3 (NLRP3) that would otherwise amplify the production of cytokines important for the generation of Th1 immunity such as intereukin-18. Consistent with a role for NLRC4 in memory immunity, primary challenge with Salmonella expressing flagellin modified to largely evade NLRC4 recognition notably increases protection against lethal rechallenge. This finding suggests flagellin modification to reduce NLRC4 activation enhances protective immunity, which could have important implications for vaccine development against flagellated microbial pathogens.

Fish tank granuloma: An emerging skin disease in Iran mimicking Cutaneous Leishmaniasis.

OBJECTIVE: Mycobacterium marinum causes a rare cutaneous disease known as fish tank granuloma (FTG). The disease manifestations resemble those associated with Cutaneous Leishmaniasis (CL). The aim of this study was to determine whether FTG was the cause of cutaneous lesions in patients who were referred to the Parasitology laboratory of Imam Reza Hospital in Mashhad to be investigated for CL. MATERIALS/METHODS: One hundered patients, clinically diagnosed with CL between April 2014 and March 2015, were included in this study. Ziehl-Neelsen staining was performed to identify acid-fast Mycobacterium in addition to bacterial cultures using Löwenstein-Jensen medium. Skin lesion samples were also collected and kept on DNA banking cards for PCR testing. RESULTS: Twenty-nine of the 100 individuals with skin lesions, and therefore suspected of suffering from CL, tested positive for Mycobacterium marinum by PCR. Of these, 21 (72.4%) were male and 8(27.6%) were female. In 97% of these cases the lesions were located on hands and fingers. These patients had a history of manipulating fish and had been in contact with aquarium water. A sporotrichoid appearance was observed in 58.6% of the patients with mycobacterial lesions; 67% of patients had multiple head appearance. CONCLUSION: Patients suspected to have CL and who test negative for CL could be affected by FTG. Therefore, after obtaining an accurate case history, molecular diagnosis is recommended for cases that give a negative result by conventional methods.

Cytotoxicity against tumor cell lines and anti-inflammatory properties of chitinases from Calotropis procera latex.

The role of chitinases from the latex of medicinal shrub Calotropis procera on viability of tumor cell lines and inflammation was investigated. Soluble latex proteins were fractionated in a CM Sepharose Fast-Flow Column and the major peak (LPp1) subjected to ion exchange chromatography using a Mono-Q column coupled to an FPLC system. In a first series of experiments, immortalized macrophages were cultured with LPp1 for 24 h. Then, cytotoxicity of chitinase isoforms (LPp1-P1 to P6) was evaluated against HCT-116 (colon carcinoma), OVCAR-8 (ovarian carcinoma), and SF-295 (glioblastoma) tumor cell lines in 96-well plates. Cytotoxic chitinases had its anti-inflammatory potential assessed through the mouse peritonitis model. We have shown that LPp1 was not toxic to macrophages at dosages lower than 125 µg/mL but induced high messenger RNA expression of IL-6, IL1-ß, TNF-α, and iNOs. On the other hand, chitinase isoform LPp1-P4 retained all LPp1 cytotoxic activities against the tumor cell lines with IC50 ranging from 1.2 to 2.9 µg/mL. The intravenous administration of LPp1-P4 to mouse impaired neutrophil infiltration into the peritoneal cavity induced by carrageenan. Although the contents of pro-inflammatory cytokines IL-6, TNF-α, and IL1-ß were high in the bloodstreams, such effect was reverted by administration of iNOs inhibitors NG-nitro-L-arginine methyl ester and aminoguanidine. We conclude that chitinase isoform LPp1-P4 was highly cytotoxic to tumor cell lines and capable to reduce inflammation by an iNOs-derived NO mechanism.

Effect of in vivo neutralization of tumor necrosis alpha on the efficacy of antibiotic treatment in systemic Salmonella enterica infections.

Immunity can co-operate with antibiotics, but can also antagonize drug efficacy by segregating the bacteria to areas of the body that are less accessible to antimicrobials, and by selecting for subpopulations with low division rates that are often difficult to eradicate. We studied the effect of an anti-inflammatory/immunosuppressive anti-TNFα treatment, which accelerates bacterial growth in the tissues and inhibits or reverses the formation of granulomas, on the efficacy of ampicillin and ciprofloxacin during a systemic Salmonella enterica infection of the mouse. The anti-TNFα treatment neither precluded nor enhanced the efficacy of antibiotic treatment. However, the anti-TNFα treatment rendered the animals susceptible to the rapid relapse of the infection seen after cessation of the antibiotic treatment. Reactivation of an established infection, due to late administration of anti-TNFα antibodies, could be successfully controlled by antibiotics, but full clearance of the bacterial load from the tissues was not achieved. We conclude that the lack of TNFα does not preclude the efficacy of antibiotic treatment and must be monitored with care due to post-treatment relapses. Combinations of anti-cytokine compounds and antibiotic molecules may not be the best way to treat persistent infections with intracellular bacteria like Salmonella.

Genes Required for the Fitness of Salmonella enterica Serovar Typhimurium during Infection of Immunodeficient gp91-/- phox Mice.

Salmonella enterica causes systemic diseases (typhoid and paratyphoid fever), nontyphoidal septicemia (NTS), and gastroenteritis in humans and other animals worldwide. An important but underrecognized emerging infectious disease problem in sub-Saharan Africa is NTS in children and immunocompromised adults. A current goal is to identify Salmonella mutants that are not pathogenic in the absence of key components of the immune system such as might be found in immunocompromised hosts. Such attenuated strains have the potential to be used as live vaccines. We have used transposon-directed insertion site sequencing (TraDIS) to screen mutants of Salmonella enterica serovar Typhimurium for their ability to infect and grow in the tissues of wild-type and immunodeficient mice. This was to identify bacterial genes that might be deleted for the development of live attenuated vaccines that would be safer to use in situations and/or geographical areas where immunodeficiencies are prevalent. The relative fitness of each of 9,356 transposon mutants, representing mutations in 3,139 different genes, was determined in gp91(-/-) phox mice. Mutations in certain genes led to reduced fitness in both wild-type and mutant mice. To validate these results, these genes were mutated by allelic replacement, and resultant mutants were retested for fitness in the mice. A defined deletion mutant of cysE was attenuated in C57BL/6 wild-type mice and immunodeficient gp91(-/-) phox mice and was effective as a live vaccine in wild-type mice.

Dynamics of Salmonella infection of macrophages at the single cell level.

Salmonella enterica causes a range of diseases. Salmonellae are intracellular parasites of macrophages, and the control of bacteria within these cells is critical to surviving an infection. The dynamics of the bacteria invading, surviving, proliferating in and killing macrophages are central to disease pathogenesis. Fundamentally important parameters, however, such as the cellular infection rate, have not previously been calculated. We used two independent approaches to calculate the macrophage infection rate: mathematical modelling of Salmonella infection experiments, and analysis of real-time video microscopy of infection events. Cells repeatedly encounter salmonellae, with the bacteria often remain associated with the macrophage for more than ten seconds. Once Salmonella encounters a macrophage, the probability of that bacterium infecting the cell is remarkably low: less than 5%. The macrophage population is heterogeneous in terms of its susceptibility to the first infection event. Once infected, a macrophage can undergo further infection events, but these reinfection events occur at a lower rate than that of the primary infection.

Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

BACKGROUND: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients. PRINCIPAL FINDINGS: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, ß2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin. SIGNIFICANCE: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.

TLR and B cell receptor signals to B cells differentially program primary and memory Th1 responses to Salmonella enterica.

Protective Th1 responses to Salmonella enterica do not develop in the absence of B cells. Using chimeric mice, we dissect the early (innate) and late (cognate) contributions of B cells to Th programming. B cell-intrinsic MyD88 signaling is required for primary effector Th1 development, whereas Ag-specific BCR-mediated Ag presentation is necessary for the development of memory Th1 populations. Programming of the primary T cell response is BCR/B cell MHC II independent, but requires MyD88-dependent secretion of cytokines by B cells. Chimeras in which B cells lack IFN-gamma or IL-6 genes make impaired Th1 or Th17 responses to Salmonella.

Copper homeostasis in Salmonella is atypical and copper-CueP is a major periplasmic metal complex.

Salmonella enterica sv. typhimurium (S. enterica sv. Typhimurium) has two metal-transporting P(1)-type ATPases whose actions largely overlap with respect to growth in elevated copper. Mutants lacking both ATPases over-accumulate copper relative to wild-type or either single mutant. Such duplication of ATPases is unusual in bacterial copper tolerance. Both ATPases are under the control of MerR family metal-responsive transcriptional activators. Analyses of periplasmic copper complexes identified copper-CueP as one of the predominant metal pools. Expression of cueP was recently shown to be controlled by the same metal-responsive activator as one of the P(1)-type ATPase genes (copA), and copper-CueP is a further atypical feature of copper homeostasis in S. enterica sv. Typhimurium. Elevated copper is detected by a reporter construct driven by the promoter of copA in wild-type S. enterica sv. Typhimurium during infection of macrophages. Double mutants missing both ATPases also show reduced survival inside cultured macrophages. It is hypothesized that elevated copper within macrophages may have selected for specialized copper-resistance systems in pathogenic microorganism such as S. enterica sv. Typhimurium.

Toll-like receptor 4 signalling through MyD88 is essential to control Salmonella enterica serovar typhimurium infection, but not for the initiation of bacterial clearance.

Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF(-/-) mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice in vitro. Pro-inflammatory responses from Mal(-/-) macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF(-/-) macrophages were partially restored by the addition of interferon-gamma (IFN-gamma), and TRIF(-/-) mice produced markedly enhanced IFN-gamma levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4(-/-), MyD88(-/-), TRIF(-/-) and Mal(-/-) mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.

LuxS-based quorum sensing does not affect the ability of Salmonella enterica serovar Typhimurium to express the SPI-1 type 3 secretion system, induce membrane ruffles, or invade epithelial cells.

Bacterial species can communicate by producing and sensing small autoinducer molecules by a process known as quorum sensing. Salmonella enterica produces autoinducer 2 (AI-2) via the luxS synthase gene, which is used by some bacterial pathogens to coordinate virulence gene expression with population density. We investigated whether the luxS gene might affect the ability of Salmonella enterica serovar Typhimurium to invade epithelial cells. No differences were found between the wild-type strain of S. Typhimurium, SL1344, and its isogenic luxS mutant with respect to the number and morphology of the membrane ruffles induced or their ability to invade epithelial cells. The dynamics of the ruffling process were also similar in the wild-type strain (SL1344) and the luxS mutant. Furthermore, comparing the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion profiles of wild-type SL1344 and the luxS mutant by Western blotting and measuring the expression of a single-copy green fluorescent protein fusion to the prgH (an essential SPI-1 gene) promoter indicated that SPI-1 expression and activity are similar in the wild-type SL1344 and luxS mutant. Genetic deletion of luxS did not alter the virulence of S. Typhimurium in the mouse model, and therefore, it appears that luxS does not play a significant role in regulating invasion of Salmonella in vitro or in vivo.

B cell intrinsic MyD88 signals drive IFN-gamma production from T cells and control switching to IgG2c.

The question of whether Ab responses to T-dependent Ags require B cell intrinsic signaling via the main TLR adaptor (MyD88) has become embroiled in confusion. In part this may be related to the methods used to analyze B cell intrinsic signaling. We have used a mixed bone marrow chimera model to generate mice in which the B cell compartment is completely deficient in MyD88 expression, while the other hematopoietic lineages are largely normal. These mice were immunized with T-dependent Ags or infected with Salmonella. We found that the Ag-specific IgG2c primary response was absolutely dependent on MyD88 signaling to B cells, while other Ig classes were not (IgG1 and IgG3) or much less so (IgG2b, IgA). The MyD88(B-/-) chimeric mice exhibited an impairment of development of IFN-gamma effector T cells, a likely contributory factor in the lack of IgG2c. We also found that B cell intrinsic MyD88 signals are required for the production of natural Abs. The data emphasize the nonredundant role of B cells as programmers of T cell differentiation in vivo.

Virulent Salmonella enterica infections can be exacerbated by concomitant infection of the host with a live attenuated S. enterica vaccine via Toll-like receptor 4-dependent interleukin-10 production with the involvement of both TRIF and MyD88.

During systemic disease in mice, Salmonella enterica grows intracellularly within discrete foci of infection in the spleen and liver. In concomitant infections, foci containing different S. enterica strains are spatially separated. We have investigated whether functional interactions between bacterial populations within the same host can occur despite the known spatial separation of the foci and independence of growth of salmonellae residing in different foci. In this study we have demonstrated that bacterial numbers of virulent S. enterica serovar Typhimurium C5 strain in mouse tissues can be increased by the presence of the attenuated aroA S. Typhimurium SL3261 vaccine strain in the same tissue. Disease exacerbation does not require simultaneous coinjection of the attenuated bacteria. SL3261 can be administered up to 48 hr after or 24 hr before the administration of C5 and still determine higher tissue numbers of the virulent bacteria. This indicates that intravenous administration of a S. enterica vaccine strain could potentially exacerbate an established infection with wild-type bacteria. These data also suggest that the severity of an infection with a virulent S. enterica strain can be increased by the prior administration of a live attenuated vaccine strain if infection occurs within 48 hr of vaccination. Exacerbation of the growth of C5 requires Toll-like receptor 4-dependent interleukin-10 production with the involvement of both Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-beta and myeloid differentiation factor 88.

Salmonella infection of afferent lymph dendritic cells.

The interactions of Salmonella enterica subspecies I serotype Abortusovis (S. Abortusovis) with ovine afferent lymph dendritic cells (ALDCs) were investigated for their ability to deliver Maedi visna virus (MVV) GAG p25 antigens to ALDCs purified from afferent lymph. Salmonellae were found to enter ALDC populations by a process of cell invasion, as confirmed by electron and confocal microscopy. This led to phenotypical changes in ALDC populations, as defined by CD1b and CD14 expression. No differences in the clearance kinetics of intracellular aroA-negative Salmonella from CD1b+ CD14lo and CD1b+ CD14(-) ALDC populations were noted over 72 h. ALDCs were also shown to present MVV GAG p25 expressed by aroA-negative S. Abortusovis to CD4+ T lymphocytes. Thus, the poor immune responses that Salmonella vaccines elicited in large animal models compared with mice are neither a result of an inability of Salmonella to infect large animal DCs nor an inability of these DCs to present delivered antigens. However, the low efficiency of infection of ALDC compared with macrophages or monocyte-derived DCs may account for the poor immune responses induced in large animal models.

The MHP36 line of murine neural stem cells expresses functional CXCR1 chemokine receptors that initiate chemotaxis in vitro.

Chemokines help to establish cerebral inflammation after ischemia, which comprises a major component of secondary brain injury. The CXCR4 chemokine receptor system induces neural stem cell migration, and hence has been implicated in brain repair. We show that CXCR1 and interleukin-8 also stimulate chemotaxis in murine neural stem cells from the MHP36 cell line. The presence of CXCR1 was confirmed by reverse transcriptase PCR and immunohistochemistry. Interleukin-8 evoked intracellular calcium currents, upregulated doublecortin (a protein expressed by migrating neuroblasts), and elicited positive chemotaxis in vitro. Therefore, effectors of the early innate immune response may also influence brain repair mechanisms.

Effect of immune serum and role of individual Fcgamma receptors on the intracellular distribution and survival of Salmonella enterica serovar Typhimurium in murine macrophages.

Immune serum has a protective role against Salmonella infections in mice, domestic animals and humans. In this study, the effect of antibody on the interaction between murine macrophages and S. enterica serovar Typhimurium was examined. Detailed analysis at the single-cell level demonstrated that opsonization of the bacteria with immune serum enhanced bacterial uptake and altered bacterial distribution within individual phagocytic cells. Using gene-targeted mice deficient in individual Fc gamma receptors it was shown that immune serum enhanced bacterial internalization by macrophages via the high-affinity immunoglobulin G (IgG) receptor, Fc gamma receptor I. Exposure of murine macrophages to S. enterica serovar Typhimurium opsonized with immune serum resulted in increased production of superoxide, leading to enhanced antibacterial functions of the infected cells. However, opsonization of bacteria with immune serum did not increase either nitric oxide production in response to S. enterica serovar Typhimurium or fusion of phagosomes with lysosomes.

Cytokine and chemokine responses associated with clearance of a primary Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to rechallenge.

Infection of poultry with Salmonella enterica serovar Typhimurium poses a significant risk to public health through contamination of meat from infected animals. Vaccination has been proposed to control infections in chickens. However, the vaccines are currently largely empirical, and our understanding of the mechanisms that underpin immune clearance and protection in avian salmonellosis is not complete. In this study we describe the cytokine, chemokine, and antibody responses and cellular changes in primary and secondary infections of chickens with Salmonella serovar Typhimurium. Infection of 1-week-old chickens induced early expression of a macrophage inflammatory protein (MIP) family chemokine in the spleen and liver, followed by increased expression of gamma interferon accompanied by increased numbers of both CD4(+) and CD8(+) T cells and the formation of granuloma-like follicular lesions. This response correlated with a Th1-mediated clearance of the systemic infection. Primary infection also induced specific immunoglobulin M (IgM), IgG, and IgA antibody responses. In contrast to previously published studies performed with newly hatched chicks, the expression levels of proinflammatory cytokines in the gastrointestinal tract were not greatly increased following infection. However, significant expression of the anti-inflammatory cytokine transforming growth factor beta4 was detected in the gut early in infection. Following secondary challenge, the birds were fully protected against systemic infection and showed a high level of protection against gastrointestinal colonization. Rapid expression of the MIP family chemokine and interleukin-6 was detected in the guts of these birds and was accompanied by an influx of lymphocytes. Increased levels of serum IgA-specific antibodies were also found following rechallenge. These findings suggest that cellular responses, particularly Th1 responses, play a crucial role in immune clearance in avian salmonellosis and that protection against rechallenge involves the rapid recruitment of cells to the gastrointestinal tract. Additionally, the high levels of inflammatory response found following Salmonella serovar Typhimurium infection of newly hatched chicks were not observed following infection of older birds (1 week old), in which the expression of regulatory cytokines appeared to limit inflammation.

Activation of murine dendritic cells and macrophages induced by Salmonella enterica serovar Typhimurium.

Macrophages and dendritic cells (DCs) are antigen-presenting cells (APCs), and the direct involvement of both cell types in the immune response to Salmonella has been identified. In this study we analysed the phenotypic and functional changes that take place in murine macrophages and DCs in response to live and heat-killed Salmonella enterica serovar Typhimurium. Both types of cell secreted proinflammatory cytokines and nitric oxide (NO) in response to live and heat-killed salmonellae. Bacterial stimulation also resulted in up-regulation of costimulatory molecules on macrophages and DCs. The expression of major histocompatibility complex (MHC) class II molecules by macrophages and DCs was differentially regulated by interferon (IFN)-gamma and salmonellae. Live and heat-killed salmonellae as well as lipopolysaccharide (LPS) inhibited the up-regulation of MHC class II expression induced by IFN-gamma on macrophages but not on DCs. Macrophages as well as DCs presented Salmonella-derived antigen to CD4 T cells, although DCs were much more efficient than macrophages at stimulating CD4 T-cell cytokine release. Macrophages are effective in the uptake and killing of bacteria whilst DCs specialize in antigen presentation. This study showed that the viability of salmonellae was not essential for activation of APCs but, unlike live bacteria, prolonged contact with heat-killed bacteria was necessary to obtain maximal expression of the activation markers studied.

Interactions of proteoliposomes from serogroup B Neisseria meningitidis with bone marrow-derived dendritic cells and macrophages: adjuvant effects and antigen delivery.

Exposure to proteoliposomes from serogroup B Neisseria meningitidis (PL) induced up-regulation of MHC-II, MHC-I, CD40, CD80 and CD86 expression on the surface of murine bone marrow-derived dendritic cells (DC). CD40, CD80 and CD86 were up-regulated on bone marrow-derived macrophages (MPhi) upon stimulation with PL. Both DC and MPhi released TNFalpha, but only DC produced IL12(p70) in response to PL. A small increase in the expression of MHC-II, CD40 and CD86, as well as production of IL12(p70), was observed on the cell surface of DC, but not MPhi from LPS-non-responder C3H/HeJ after exposure to PL. DC, but not MPhi, incubated with PL containing ovalbumin (PL-OVA) presented OVA-specific peptides to CD4+ and CD8+ OVA-specific T-cell hybridomas. These data clearly indicate that PL exert an immunomodulatory effect on DC and MPhi, with some contribution of non-LPS components besides the main role of LPS. The work also shows the potential of PL as a general system to deliver antigens to DC for presentation to CD4+ and CD8+ T-cells.

Attenuated Salmonella typhimurium htrA mutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers.

Salmonella live vaccine strains harbouring mutations in htrA, a stress protein gene, display increased susceptibility to oxidative stress in vitro. This is believed to be connected to their reduced virulence, perhaps due to impaired survival inside phagocytes, although this has never been formally proven. We report that the in vitro phenotype of increased susceptibility to oxidative stress of Salmonella typhimurium htrA mutants newly prepared by transduction is rapidly lost on subculture, with the mutants becoming as resistant as the parent for reasons that remain unclear. However, despite this change, htrA mutants are still attenuated in normal mice. In contrast, they were found to be lethal for gene targeted gp91phox-/- mice deficient in NADPH oxidase, as was a S. typhimurium SPI-2 mutant known to be virulent in gp9lphox-/- mice. Infection with htrA mutants caused little damage to primary bone marrow macrophage cultures from normal mice; conversely, they caused extensive damage to macrophages from gp9lphox-/- mice, with more than 60% reduction in cell numbers 2.5h after being infected. The parental wild type strain similarly caused extensive damage to macrophages from both normal and gp9lphox-/- mice, whereas an aroA live vaccine strain had no effect on either normal or gp9lphox-/- macrophages. Taken collectively, the present results suggest that htrA is somehow involved in resistance to oxidative stress in vivo, with the avirulence of htrA mutants in mice being due to mechanisms which involve NADPH oxidase and suppression of bacterial growth within macrophages.