Salmonella effector AvrA regulation of colonic epithelial cell inflammation by deubiquitination

AvrA is a newly described bacterial effector existing in Salmonella. Here, we test the hypothesis that AvrA is a deubiquitinase that removes ubiquitin from two inhibitors of the nuclear factor-kappaB (NF-kappaB) pathway, IkappaBalpha and beta-catenin, thereby inhibiting the inflammatory responses of the host. The role of AvrA was assessed in intestinal epithelial cell models and in mouse models infected with AvrA-deficient and -sufficient Salmonella strains. We also purified AvrA and AvrA mutant proteins and characterized their deubiquitinase activity in a cell-free system. We investigated target gene and inflammatory cytokine expression, as well as effects on epithelial cell proliferation and apoptosis induced by AvrA-deficient and -sufficient bacterial strains in vivo. Our results show that AvrA blocks degradation of IkappaBalpha and beta-catenin in epithelial cells. AvrA deubiquitinates IkappaBalpha, which blocks its degradation and leads to the inhibition of NF-kappaB activation. Target genes of the NF-kappaB pathway, such as interleukin-6, were correspondingly down-regulated during bacterial infection with Salmonella expressing AvrA. AvrA also deubiquitinates and thus blocks degradation of beta-catenin. Target genes of the beta-catenin pathway, such as c-myc and cyclinD1, were correspondingly up-regulated with AvrA expression. Increased beta-catenin further negatively regulates the NF-kappaB pathway. Our findings suggest an important role for AvrA in regulating host inflammatory responses through NF-kappaB and beta-catenin pathways.     

The deubiquitinase activity of the Salmonella pathogenicity island 2 effector, SseL, prevents accumulation of cellular lipid droplets

To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets.     

Characterization of Salmonella-induced cell death in human macrophage-like THP-1 cells

Salmonella strains are facultative intracellular pathogens that produce marked cytopathology during infection of host cells. Different forms of cytopathic effects have been associated with the virulence systems encoded by the two Salmonella pathogenicity islands (SPI-1 and SPI-2) and the spv locus. We used Salmonella enterica serovar Dublin to investigate the induction of cytopathology during infection of the human macrophage-like cell line THP-1. Analysis of host cells by flow cytometry using a fluorescent terminal deoxynucleotidyltransferase dUTP-biotin nick end labeling (TUNEL) assay revealed that 70% of THP-1 cells showed DNA fragmentation after 4 h of infection, increasing to greater than 90% by 5.5 h. Moreover, the results showed that gentamicin-killed or chloramphenicol-treated bacteria did not induce DNA fragmentation. Serovar Dublin strains with mutations in SPI-1, SPI-2, or spvB induced these cytopathic effects similar to wild-type bacteria. In contrast, a mutation in the phoP regulatory gene abolished DNA fragmentation in the TUNEL assay. Caspase-3 activation was detected during Salmonella infection of THP-1 cells, but caspase-8 and caspase-9 activities were not found. However, inhibition of caspase-3 did not block Salmonella-induced DNA fragmentation. These results identify a previously undetected apoptotic effect in Salmonella-infected cells that is dependent on phoP gene function.     

Genetic regulation of host responses to Salmonella infection in mice

Salmonella spp are Gram-negative bacteria capable of infecting a wide range of host species, including humans, domesticated and wild mammals, reptiles, birds and insects. The outcome of an encounter between Salmonella and its host is dependent upon multiple factors including the host genetic background. To facilitate the study of the genetic factors involved in resistance to this pathogen, mouse models of Salmonella infection have been developed and studied for years, allowing identification of several genes and pathways that may influence the disease outcome. In this review, we will cover some of the genes involved in mouse resistance to Salmonella that were identified through the study of congenic mouse strains, cloning of spontaneous mouse mutations, use of site-directed mutagenesis or quantitative trait loci analysis. In parallel, the relevant information pertaining to genes involved in resistance to Salmonella in humans will be discussed.     

Microgravity as a novel environmental signal affecting Salmonella enterica serovar Typhimurium virulence

The effects of spaceflight on the infectious disease process have only been studied at the level of the host immune response and indicate a blunting of the immune mechanism in humans and animals. Accordingly, it is necessary to assess potential changes in microbial virulence associated with spaceflight which may impact the probability of in-flight infectious disease. In this study, we investigated the effect of altered gravitational vectors on Salmonella virulence in mice. Salmonella enterica serovar Typhimurium grown under modeled microgravity (MMG) were more virulent and were recovered in higher numbers from the murine spleen and liver following oral infection compared to organisms grown under normal gravity. Furthermore, MMG-grown salmonellae were more resistant to acid stress and macrophage killing and exhibited significant differences in protein synthesis than did normal-gravity-grown cells. Our results indicate that the environment created by simulated microgravity represents a novel environmental regulatory factor of Salmonella virulence.     

Acid pre-adaptation enhances virulence of Salmonella enterica serovar Typhimurium dam mutant

It is well established that success or failure of bacterial pathogens during infection relies upon its ability to overcome many lethal environments in the host such as acidity, osmolarity and bile salts. In the present study, we have studied the effects of acid adaptation on the virulence of Salmonella enterica serovar Typhimurium dam mutant. Our results indicated that LD(50) of adapted strains were lower than those of control strains. Also, the in vivo assays have shown that the development of a systemic infection is slower for control strains than for adapted strains. In addition, the number of acid-adapted mutants colonizing spleen and liver is higher than control strains. Adhesion and invasion experiments were performed in order to compare the pathogenicity of Salmonella. No significant differences were shown between pre-treated and non-adapted strains. According to these results, we report that acid adaptation of Salmonella enterica serovar Typhimurium dam mutants can increase their in vivo virulence in mice.     

Increased susceptibility to bacterial infection as a sequela of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The effects of subclinical levels of 2,3,7,8-tetrachloridibenzo-p-dioxin (TCDD) on the response of mice to infection with either Salmonella bern or Herpesvirus suis, also known as pseudorabies virus, are reported. TCDD is a contaminant of certain commercially useful chemicals, such as chlorinated phenols or herbicides. It has been shown to cause thymic atrophy and to suppress cell-mediated immunity in laboratory animals. Sublethal levels of TCDD were used: 0.5, 1,5, 10, or 20 mug/kg, given through a gastric tube once weekly for 4 weeks. A significant decrease in weight gain compared with control mice occurred at the 20-mug dosage. Dose schedules of 1 mug or more, followed by salmonella infection, resulted in significant increases in mortality and decreases in the time from infection to dealth. However, TCDD had no significant effect on mortality in the pseudorabies-infected mice. The most important finding in this study is that extremely low levels of TCDD, which do not produce clinical or pathological change, still have the capacity to affect host defense.     

Single gene effects in mouse models of host: pathogen interactions

Inbred mouse strains have been known for many years to vary in their degree of susceptibility to different types of infectious diseases. The genetic basis of these interstrain differences is sometimes simple but often complex. In a few cases, positional cloning has been used successfully to identify single gene effects. The natural resistance-associated macrophage protein 1 (Nramp1) gene (Slc11a1) codes for a metal transporter active at the phagosomal membrane of macrophages, and Nramp1 mutations cause susceptibility to Mycobacterium, Salmonella, and Leishmania. Furthermore, recent advances in gene transfer technologies in transgenic mice have enabled the functional dissection of gene effects mapping to complex, repeated parts of the genome, such as the Lgn1 locus, causing susceptibility to Legionella pneumophila in macrophages. Finally, complex traits such as the genetically determined susceptibility to malaria can sometimes be broken down into multiple single gene effects. One such example is the case of pyruvate kinase, where a loss-of-function mutation was recently shown by our group to be protective against blood-stage infection with Plasmodium chabaudi. In all three cases reviewed, the characterization of the noted gene effect(s) has shed considerable light on the pathophysiology of the infection, including host response mechanisms.     

CD4+ T cells and toll-like receptors recognize Salmonella antigens expressed in bacterial surface organelles

A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysaccharide (LPS). PhoP/PhoQ, a regulon controlling Salmonella virulence and remodeling of LPS to resist innate immunity, coordinately represses production of surface-exposed antigens recognized by CD4+ T cells and TLRs. These data suggest that genetically coordinated surface modifications may provide a growth advantage for Salmonella in host tissues by limiting both innate and adaptive immune recognition.     

Modulation of host immunity by HIV may be partly achieved through usurping host autonomic functions

Modulation of host immunity has been observed in human immunodeficiency virus (HIV) infections. HIV is believed to influence host immunity through a variety of mechanisms including direct effects on host T cell survival, indirect effects on cytokine profile through modulation of immune cells, and modulation of endocrine functions that affect immunity such as steroids. We hypothesize that HIV infection may also alter host immunity through modulation of host sympatho-vagal balance. Specifically, we propose that HIV drives autonomic balance towards sympathetic bias, which can contribute to a T helper (Th)2 type immunity. A variety of paraviral syndromes associated with HIV infection such as QT prolongation, cachexia, cardiomyopathy, and lipodystrophy are consistent with evidence of autonomic dysfunction. Immunomodulatory effects of autonomic dysfunction toward Th2 bias are presented. A plausible mechanism by which HIV can influence autonomic balance through hypothalamic manipulation is offered. Shift to Th2 dominance is associated with HIV disease progression and can be viewed as a viral adaptation to promote its own survival. Autonomic remodeling by HIV may exemplify this phenomenon. Our hypothesis has implications for treatment of HIV and its associated syndromes.     

Analysis of host-pathogen modulators of autophagy during Mycobacterium Tuberculosis infection and therapeutic repercussions

Mycobacterium tuberculosis is one of the most deadly human pathogens known today in modern world, responsible for about 1.5 million deaths annually. Development of TB disease occurs only in 1 out of 10 individuals exposed to the pathogen which indicates that the competent host defense mechanisms exist in majority of the hosts to control the infection. In the last decade, autophagy has emerged as a key host immune defense mechanism against intracellular M. tuberculosis infection. Autophagy has been demonstrated not only as an effective antimicrobial mechanism for the clearance of M. tuberculosis, but the process has also been suggested to prevent excessive inflammation to avoid the adverse effects of infection on host. Nevertheless, increasing evidences also show that in order to enhance its intracellular survival, M. tuberculosis has also evolved multiple strategies to compromise the optimal functioning of host autophagic machinery. This review describes an overview of the various host signaling pathways such as pattern recognition receptors, cytokines, nutrient starvation and other cellular stress that have been implicated in induction of autophagy during M. tuberculosis infection. The review also chalk out the complex interplay of several bacterial factors of M. tuberculosis that are known to be involved in compromising autophagy mediated defense of the host. A comprehensive understanding of the interaction of bacterial and host factors at the intersections of autophagic pathways could provide integrative insights for the development of autophagy-based prophylactics and novel therapeutic interventions for TB.     

The ferritin-like Dps protein is required for Salmonella enterica serovar Typhimurium oxidative stress resistance and virulence

Resistance to phagocyte-derived reactive oxygen species is essential for Salmonella enterica serovar Typhimurium pathogenesis. Salmonella can enhance its resistance to oxidants through the induction of specific genetic pathways controlled by SoxRS, OxyR, sigma(S), sigma(E), SlyA, and RecA. These regulons can be found in a wide variety of pathogenic and environmental bacteria, suggesting that evolutionarily conserved mechanisms defend against oxidative stress both endogenously generated by aerobic respiration and exogenously produced by host phagocytic cells. Dps, a ferritin-like protein found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress by sequestering iron and limiting Fenton-catalyzed oxyradical formation. In Escherichia coli and some other bacterial species, Dps has been shown to accumulate during stationary phase in a sigma(S)-dependent fashion, bind nonspecifically to DNA, and form a crystalline structure that compacts and protects chromatin from oxidative damage. In the present study, we provide evidence that Dps protects Salmonella from iron-dependent killing by hydrogen peroxide, promotes Salmonella survival in murine macrophages, and enhances Salmonella virulence. Reduced numbers of dps mutant bacteria in the livers and spleens of infected mice are consistent with a role of Dps in protecting Salmonella from oxidative stress encountered during infection.     

Signaling through the P38 and ERK pathways: a common link between HIV replication and the immune response

One of the defining characteristics of HIV is its ability to manipulate the human immune response to promote its own replication. Since the beginning of the epidemic, there has been controversy whether a robust immune response to the virus is beneficial or detrimental for the host. Therefore, the effects of HIV on signaling pathways and cytokine production need to be characterized in order to distinguish between protective immune responses and inappropriate immune activation. Cytokine and biomarker expression during HIV infection results from the combined effects of intracellular signaling pathways orchestrated by kinases like P38 and ERK. The P38 and ERK Mitogen-Activated Protein Kinase (MAPK) pathways govern the regulation of cytokines (IL-2, IL-10, and TNF-α) as well biomarkers (PD-1, Fas/FasL, among others) that are skewed in chronic HIV infection. HIV utilizes the P38 and ERK pathways to produce new virions and to deplete CD4+ T cells from the host’s immune system. Understanding the interplay between HIV and the cytokines induced by activation of the P38 and ERK pathways may provide insights into HIV immunopathogenesis and the development of a protective vaccine.     

The role of iron in infections with intracellular bacteria

The requirement for iron as a critical component for cellular processes has long been appreciated. During infection with intracellular bacteria, iron is required by both the host cell and the pathogen that inhabits the host cell. Macrophages require iron as a cofactor for the execution of important antimicrobial effector mechanisms, including the NADPH dependent oxidative burst and the production of nitrogen radicals catalysed by the inducible nitric oxide synthase. On the other side of the equation, intracellular bacteria such as Salmonella typhimurium and Mycobacterium tuberculosis have an obligate requirement for iron to support their growth and survival inside cells. This brief report summarises the background to our work on iron modulation in infections with these two organisms and highlights key observations on how modulation of host iron status disturbs the equilibrium between host and pathogen and can determine the outcome of infection.     

Global regulators and environmental adaptation in Gram-negative pathogens

A powerful combination of single-gene studies and whole genome approaches has provided a wealth of information about the regulatory circuits used by bacteria to adapt to the environmental changes that are encountered during infection. The facultative intracellular pathogen Salmonella enterica will be used to illustrate how global regulators such as the nucleoid-associated proteins Fis and H-NS collaborate with fluctuations in the superhelicity of the DNA template to modify the gene expression profile of the bacterial cell outside and inside the host.     

Yersinia pestis YopJ suppresses tumor necrosis factor alpha induction and contributes to apoptosis of immune cells in the lymph node but is not required for virulence in a rat model of bubonic plague

The virulence of the pathogenic Yersinia species depends on a plasmid-encoded type III secretion system that transfers six Yop effector proteins into host cells. One of these proteins, YopJ, has been shown to disrupt host cell signaling pathways involved in proinflammatory cytokine production and to induce macrophage apoptosis in vitro. YopJ-dependent apoptosis in mesenteric lymph nodes has also been demonstrated in a mouse model of Yersinia pseudotuberculosis infection. These results suggest that YopJ attenuates the host innate and adaptive immune response during infection, but the role of YopJ during bubonic plague has not been completely established. We evaluated the role of Yersinia pestis YopJ in a rat model of bubonic plague following intradermal infection with a fully virulent Y. pestis strain and an isogenic yopJ mutant. Deletion of yopJ resulted in a twofold decrease in the number of apoptotic immune cells in the bubo and a threefold increase in serum tumor necrosis factor alpha levels but did not result in decreased virulence, systemic spread, or colonization levels in the spleen and blood. Our results indicate that YopJ is not essential for bubonic plague pathogenesis, even after peripheral inoculation of low doses of Y. pestis. Instead, the effects of YopJ appear to overlap and augment the immunomodulatory effects of other Y. pestis virulence factors.     

Live attenuated Salmonella: a paradigm of mucosal vaccines

Summary: Two key steps control immune responses in mucosal tissues: the sampling and transepkhelial transport of antigens, and their targeting into professional antigen-presenting cells in mucosa-associated lymphoid tissue. Live Salmonella bacteria use strategies that allow them to cross the epithelial barrier of the gut, to survive in antigen-presenting cells where bacterial antigens are processed and presented to the immune cells, and to express adjuvant activity that prevents induction of oral tolerance. Two Salmonella serovars have been used as vaccines or vectors, S. typhimurium in mice and S. lyph'i in humans. S. lyphimurium causes gasiroenieritis in a broad host range, including humans, while S. typhi infection is restricted to humans. Attenuated S. typhitnurium has been used successfully in mice to induce systemic and mucosal responses against more than 60 heterolo-gous antigens. Tbis review aims to revisit S. typhimurium-based vaccination, as an alternative to S. typhi, with special emphasis on the molecttlar pathogenesis of S. typhimurium and the host response. We then discuss how such knowledge constitutes the basis for the rational design of novel live mucosal vaccines.     

Autoimmunity, anergy, lentiviral immunity and disease

Autoimmune antibodies and autoimmune responses have been characterized in both human HIV infection and the rhesus macaque (RM) non-human primate model of SIV infection and reasoned to contribute to the pathogenesis of AIDS. Many theories for the induction and maintenance of such responses have been entertained including molecular mimicry between HIV proteins and self molecules, CD4+ T cell loss accompanied by loss of normal immune regulation that dictate self-non-self-reactivity, defective negative/positive selection of T cells to name a few. The precise mechanisms that lead to such immune dysfunction is difficult to study in humans. Our lab has been studying such autoimmune responses in both SIV-infected RM and sooty mangabeys (SM), a species from Africa that are naturally infected with SIV but do not display any detectable signs of immune deficiency or autoimmunity. We submit that this model is an important model since it allows for narrowing down those mechanisms and pathways that are a result of lentiviral infection per se from those that specifically cause disease including autoimmunity. During the course of these studies, we have ruled out a role for plasma and cellular viral loads, anti-viral humoral responses and a variety of cell signaling pathways. We have identified select pathways that appear to play roles in the pathogenesis of lentiviral infection and disease. These include pathways involved in innocent bystander killing by apoptosis of CD4+ T cells, role for differential regulation of the cell cycle, and a role for distinct host proteins that get incorporated by the virions as they are assembled and either bud out of CD4+ T cells or exit the cells in the form of multi-vesicular endosomal particles from monocytes/macrophages from SIV-infected disease susceptible RM and disease-resistant SM. We present our current working model and hypotheses that are designed to elucidate differences that are responsible for such distinct outcomes of lentiviral infection, autoimmunity and disease. We believe that such findings have important implications for the design of vaccines against human HIV infection.     

Involvement of Salmonella enterica serovar Typhi RpoS in resistance to NO-mediated host defense against serovar Typhi infection

The involvement of nitric oxide (NO) in host defense and cytoprotective functions in murine salmonellosis has been reported. Salmonella mutants with the altered sigma factor RpoS (sigmaS) are less virulent and are susceptible to various stresses. This study investigated the role of the rpoS gene of Salmonella enterica serovar Typhi in NO-dependent host defense in vitro and in vivo. Wild-type mice and mice deficient in inducible NO synthase (iNOS) were infected intraperitoneally or orally with serovar Typhi strains. iNOS-deficient mice were more susceptible to infection by both wild-type and rpoS mutant strains of serovar Typhi and showed extensive apoptotic liver damage compared with wild-type mice. Intracellular killing of Salmonella was analyzed with RAW 264 macrophage-like cells and primary peritoneal macrophages from wild-type and iNOS-deficient mice after cells were infected with the serovar Typhi parent or rpoS mutant strain. The rpoS mutant was more susceptible to killing by macrophages than was the wild-type strain. Also, the wild-type strain produced more extensive apoptotic changes in macrophages than did rpoS mutant. These effects were nullified in RAW 264 cells treated with an NOS inhibitor and in iNOS-deficient primary macrophages. Peroxynitrite susceptibility assays of these strains were also performed. The rpoS mutant Typhi strain was more sensitive to in vitro peroxynitrite treatment than was the parent strain. Together these data show that NO has a significant host defense function during serovar Typhi infection, and that Salmonella RpoS, because it reacts to the presence of NO or its reactive derivatives, is thought to have a role in the pathogenicity of serovar Typhi.