Type IV Secretion-Dependent Activation of Host MAP Kinases Induces an Increased Proinflammatory Cytokine Response to Legionella pneumophila

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-κB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.     

Temporal and Anatomical Host Resistance to Chronic Salmonella Infection Is Quantitatively Dictated by Nramp1 and Influenced by Host Genetic Background

The lysosomal membrane transporter, Nramp1, plays a key role in innate immunity and resistance to infection with intracellular pathogens such as non-typhoidal Salmonella (NTS). NTS-susceptible C57BL/6 (B6) mice, which express the mutant Nramp1^D169 allele, are unable to control acute infection with Salmonella enterica serovar Typhimurium following intraperitoneal or oral inoculation. Introducing functional Nramp1^G169 into the B6 host background, either by constructing a congenic strain carrying Nramp1^G169 from resistant A/J mice (Nramp-Cg) or overexpressing Nramp1^G169 from a transgene (Nramp-Tg), conferred equivalent protection against acute Salmonella infection. In contrast, the contributions of Nramp1 for controlling chronic infection are more complex, involving temporal and anatomical differences in Nramp1-dependent host responses. Nramp-Cg, Nramp-Tg and NTS-resistant 129×1/SvJ mice survived oral Salmonella infection equally well for the first 2–3 weeks, providing evidence that Nramp1 contributes to the initial control of NTS bacteremia preceding establishment of chronic Salmonella infection. By day 30, increased host Nramp1 expression (Tg>Cg) provided greater protection as indicated by decreased splenic bacterial colonization (Tg<Cg). However, despite controlling bacterial growth within MLN as effectively as 129×1/SvJ mice, Nramp-Cg and Nramp-Tg mice eventually succumbed to infection. These data indicate: 1) discrete, anatomically localized host resistance is conferred by Nramp1 expression in NTS-susceptible mice, 2) restriction of systemic bacterial growth in the spleens of NTS-susceptible mice is enhanced by Nramp1 expression and dose-dependent, and 3) host genes other than Nramp1 also contribute to the ability of NTS-resistant 129×1/SvJ mice to control bacterial replication during chronic infection.     

More than Motility: Salmonella Flagella Contribute to Overriding Friction and Facilitating Colony Hydration during Swarming

We show in this study that Salmonella cells, which do not upregulate flagellar gene expression during swarming, also do not increase flagellar numbers per μm of cell length as determined by systematic counting of both flagellar filaments and hooks. Instead, doubling of the average length of a swarmer cell by suppression of cell division effectively doubles the number of flagella per cell. The highest agar concentration at which Salmonella cells swarmed increased from the normal 0.5% to 1%, either when flagella were overproduced or when expression of the FliL protein was enhanced in conjunction with stator proteins MotAB. We surmise that bacteria use the resulting increase in motor power to overcome the higher friction associated with harder agar. Higher flagellar numbers also suppress the swarming defect of mutants with changes in the chemotaxis pathway that were previously shown to be defective in hydrating their colonies. Here we show that the swarming defect of these mutants can also be suppressed by application of osmolytes to the surface of swarm agar. The “dry” colony morphology displayed by che mutants was also observed with other mutants that do not actively rotate their flagella. The flagellum/motor thus participates in two functions critical for swarming, enabling hydration and overriding surface friction. We consider some ideas for how the flagellum might help attract water to the agar surface, where there is no free water.     

Diacylglycerol Kinases Terminate Diacylglycerol Signaling during the Respiratory Burst Leading to Heterogeneous Phagosomal NADPH Oxidase Activation

It is commonly assumed that all phagosomes have identical molecular composition. This assumption has remained largely unchallenged due to a paucity of methods to distinguish individual phagosomes. We devised an assay that extends the utility of nitro blue tetrazolium for detection and quantification of NAPDH oxidase (NOX) activity in individual phagosomes. Implementation of this assay revealed that in murine macrophages there is heterogeneity in the ability of individual phagosomes to generate superoxide, both between and within cells. To elucidate the molecular basis of the variability in NOX activation, we employed genetically encoded fluorescent biosensors to evaluate the uniformity in the distribution of phospholipid mediators of the oxidative response. Despite variability in superoxide generation, the distribution of phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-phosphate, and phosphatidic acid was nearly identical in all phagosomes. In contrast, diacylglycerol (DAG) was not generated uniformly across the phagosomal population, varying in a manner that directly mirrored superoxide production. Modulation of DAG levels suggested that NOX activation is precluded when phagosomes fail to reach a critical DAG concentration. In particular, forced expression of diacylglycerol kinase β abrogated DAG accumulation at the phagosome, leading to impaired respiratory burst. Conversely, pharmacological inhibition of DAG kinases or expression of an inactive diacylglycerol kinase β mutant increased the proportion of DAG-positive phagosomes, concomitantly potentiating phagosomal NOX activity. Our data suggest that diacylglycerol kinases limit the extent of NADPH oxidase activation, curtailing the production of potentially harmful reactive oxygen species. The resulting heterogeneity in phagosome responsiveness could enable the survival of a fraction of invading microorganisms.     

Enterovirus71 (EV71) Utilise Host microRNAs to Mediate Host Immune System Enhancing Survival during Infection

Hand, Foot and Mouth Disease (HFMD) is a self-limiting viral disease that mainly affects infants and children. In contrast with other HFMD causing enteroviruses, Enterovirus71 (EV71) has commonly been associated with severe clinical manifestation leading to death. Currently, due to a lack in understanding of EV71 pathogenesis, there is no antiviral therapeutics for the treatment of HFMD patients. Therefore the need to better understand the mechanism of EV71 pathogenesis is warranted. We have previously reported a human colorectal adenocarcinoma cell line (HT29) based model to study the pathogenesis of EV71. Using this system, we showed that knockdown of DGCR8, an essential cofactor for microRNAs biogenesis resulted in a reduction of EV71 replication. We also demonstrated that there are miRNAs changes during EV71 pathogenesis and EV71 utilise host miRNAs to attenuate antiviral pathways during infection. Together, data from this study provide critical information on the role of miRNAs during EV71 infection.     

The Major Surface-Associated Saccharides of Klebsiella pneumoniae Contribute to Host Cell Association

Analysing the pathogenic mechanisms of a bacterium requires an understanding of the composition of the bacterial cell surface. The bacterial surface provides the first barrier against innate immune mechanisms as well as mediating attachment to cells/surfaces to resist clearance. We utilised a series of Klebsiella pneumoniae mutants in which the two major polysaccharide layers, capsule and lipopolysaccharide (LPS), were absent or truncated, to investigate the ability of these layers to protect against innate immune mechanisms and to associate with eukaryotic cells. The capsule alone was found to be essential for resistance to complement mediated killing while both capsule and LPS were involved in cell-association, albeit through different mechanisms. The capsule impeded cell-association while the LPS saccharides increased cell-association in a non-specific manner. The electrohydrodynamic characteristics of the strains suggested the differing interaction of each bacterial strain with eukaryotic cells could be partly explained by the charge density displayed by the outermost polysaccharide layer. This highlights the importance of considering not only specific adhesin:ligand interactions commonly studied in adherence assays but also the initial non-specific interactions governed largely by the electrostatic interaction forces.     

Circulating Lipoproteins Are a Crucial Component of Host Defense against Invasive Salmonella typhimurium Infection

Background

Circulating lipoproteins improve the outcome of severe Gram-negative infections through neutralizing lipopolysaccharides (LPS), thus inhibiting the release of proinflammatory cytokines.

Methods/Principal Findings

Low density lipoprotein receptor deficient (LDLR−/−) mice, with a 7-fold increase in LDL, are resistant against infection with Salmonella typhimurium (survival 100% vs 5%, p<0.001), and 100 to 1000-fold lower bacterial burden in the organs, compared with LDLR+/+ mice. Protection was not due to differences in cytokine production, phagocytosis, and killing of Salmonella organisms. The differences were caused by the excess of lipoproteins, as hyperlipoproteinemic ApoE−/− mice were also highly resistant to Salmonella infection. Lipoproteins protect against infection by interfering with the binding of Salmonella to host cells, and preventing organ invasion. This leads to an altered biodistribution of the microorganisms during the first hours of infection: after intravenous injection of Salmonella into LDLR+/+ mice, the bacteria invaded the liver and spleen within 30 minutes of infection. In contrast, in LDLR−/− mice, Salmonella remained constrained to the circulation from where they were efficiently cleared, with decreased organ invasion.

Conclusions

plasma lipoproteins are a potent host defense mechanism against invasive Salmonella infection, by blocking adhesion of Salmonella to the host cells and subsequent tissue invasion.     

Host cytokine response and resistance to Salmonella infection

Knowledge of the host response, of the resistance process, and of the mediators committed against Salmonella infection is essential to progress towards better means of prophylaxis and eradication. In this context, the present contribution attempts to interconnect, with the pivotal role of the macrophage, the early resistance process under the control of the Nramp1 gene and the cytokine response for resolving infection. IL-12 produced by macrophages is an inducer of IFN-gamma production, which in turn activates the macrophage antibacterial activity and synergizes its effects with TNF-alpha. All three of these cytokines are powerful actors in the first line of anti-Salmonella defence. It can be pointed out that susceptible and resistant individuals do not seem to see the cytokine environment the same way, the former being unresponsive to IL-1 or GM-CSF treatment and deficient in IFN-gamma production. These discrepancies may rely on cell signalling events that could be defective in macrophages of the susceptible phenotype.     

Endoglin Haploinsufficiency Promotes Fibroblast Accumulation during Wound Healing through Akt Activation

Accurate regulation of dermal fibroblast function plays a crucial role in wound healing. Many fibrotic diseases are characterized by a failure to conclude normal tissue repair and the persistence of fibroblasts inside lesions. In the present study we demonstrate that endoglin haploinsufficiency promotes fibroblast accumulation during wound healing. Moreover, scars from endoglin-heterozygous (Eng^+/−) mice show persisting fibroblasts 12 days after wounding, which could lead to a fibrotic scar. Endoglin haploinsufficiency results in increased proliferation and migration of primary cultured murine dermal fibroblasts (MDFs). Moreover, Eng^+/− MDF have diminished responses to apoptotic signals compared with control cells. Altogether, these modifications could explain the augmented presence of fibroblasts in Eng^+/− mice wounds. We demonstrate that endoglin expression regulates Akt phosphorylation and that PI3K inhibition abolishes the differences in proliferation between endoglin haploinsufficient and control cells. Finally, persistent fibroblasts in Eng^+/− mice wound co-localize with a greater degree of Akt phosphorylation. Thus, endoglin haploinsufficiency seems to promote fibroblast accumulation during wound healing through the activation of the PI3K/Akt pathway. These studies open new non-Smad signaling pathway for endoglin regulating fibroblast cell function during wound healing, as new therapeutic opportunities for the treatment of fibrotic wounds.     

Host and Viral Translational Mechanisms during Cricket Paralysis Virus Infection

The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2α phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2α by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2α phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2α phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5′ untranslated region (5′UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5′UTR and IGR IRES.For productive viral protein expression, viruses have to compete for and hijack the host translational machinery (45). Some viruses such as poliovirus, vesicular stomatitis virus (VSV), and influenza virus selectively antagonize the translation apparatus to shut off host translation, resulting in the release of ribosomes from host mRNAs and the inhibition of antiviral responses. On the other hand, the host cell can counteract through antiviral mechanisms to shutdown viral translation. For instance, viral RNA replication intermediates can trigger PKR, leading to an inhibition of overall translation. To bypass the block in translation, viruses have evolved unique mechanisms to preferentially recruit the ribosome for viral protein synthesis. Thus, the control of the translational machinery during infection is a major focal point in the battle between the host and the virus and often, elucidation of these viral translational shutoff strategies reveals key targets of translational regulation.The majority of cellular mRNAs initiate translation through the recruitment of the cap-binding complex, eukaryotic translation initiation factor 4F (eIF4F), to the 5′ cap of the mRNA (56). eIF4F consists of the cap-binding protein eIF4E, the RNA helicase, eIF4A, and the adaptor protein eIF4G. eIF4G acts as a bridge to join eIF4E and the 40S subunit via eIF3. With the ternary eIF2-Met-tRNA_i-GTP complex bound, the 40S subunit scans in a 5′-to-3′ direction until an AUG start codon is encountered. Here, eIF5 mediates GTP hydrolysis on the ternary complex, releasing the eIFs and subsequently leading to 60S subunit joining to assemble an elongation-competent 80S ribosome. The ternary eIF2-Met-tRNA_i-GTP complex is reactivated for another round of translation by exchange of GDP for GTP, which is mediated by the guanine nucleotide exchange factor, eIF2B. The 3′ poly(A) tail of the mRNA also stimulates translational initiation by binding to the poly(A) binding protein (PABP), which in turn interacts with eIF4G at the 5′end, resulting in a circularized mRNA. PABP has been proposed to enhance eIF4E affinity for the 5′cap and promote 60S joining, indicating that PABP functions at multiple steps of translational initiation (33).A common tactic viruses use to inhibit host translation is to selectively target eIFs. One of the best studied is the cleavage of eIF4G by viral proteases during picornavirus infection. In humans, two isoforms, eIF4GI and eIF4GII, are cleaved early in poliovirus infection by the viral protease 2A, where cleavage of eIFGII correlates more precisely with host translation shutoff (20). Cleavage of eIF4G produces an amino-terminal fragment that binds to eIF4E and a C-terminal fragment that binds to eIF4A and eIF3 (26, 39, 42). PABP is also cleaved by the viral protease 3C during poliovirus infection, thus contributing to shutoff of both host and viral translation and thereby enabling the switch from viral translation to replication (3, 31, 38). Another major target is the availability of the cap-binding protein eIF4E, which is regulated by binding to the repressor protein 4E-BP (21, 41). 4E-BP and eIF4G compete for an overlapping site on eIF4E (42). In its hypophosphorylated state, 4E-BP binds to and sequesters eIF4E, preventing eIF4G recruitment. Dephosphorylation and activation of 4E-BP has been observed during poliovirus, encephalomyocarditis (EMCV), and VSV infections (7, 18).During virus infection, host antiviral responses are triggered that also inhibit translation to counteract viral protein synthesis. An integral antiviral response is phosphorylation at Ser⁵¹ of eIF2α, which reduces the pool of the ternary complex by blocking the eIF2B-dependent exchange of GDP to GTP. In mammals, four known eIF2α kinases exist including the endoplasmic reticulum (ER)-stress-inducible PERK, GCN2, which senses the accumulation of deacylated tRNAs during amino acid starvation conditions; the heme-regulated kinase HRI; and the interferon-inducible double-stranded RNA-binding PKR (64). In mammalian cells, PKR is activated by binding to double-stranded viral RNA replication intermediates, leading to eIF2α phosphorylation and inhibition of overall host and viral translation. PERK and GCN2 have also been shown to be activated during virus infections by VSV and members of the alphavirus family (2, 6, 43, 65, 79). Often, viruses rely on the ER for synthesis and proper folding of viral proteins. The large burden on the ER activates PERK to phosphorylate eIF2α, thereby inhibiting global protein synthesis to reduce the load on the ER (23). Some viruses such as HCV and herpes simplex viruses have adapted to responses that induce eIF2α phosphorylation by producing viral proteins that counteract PKR or modulate the ER stress response (27, 76). Thus, virus infection can trigger several eIF2α kinases that lead to translational shutoff to counteract viral protein synthesis.To circumvent these translation blocks, viruses such as poliovirus and hepatitis C virus utilize internal ribosome entry sites (IRES), which are RNA elements that directly recruit ribosomes in a cap-independent manner and require only a subset of canonical eIFs (15, 25). It is generally thought that IRES-containing viral mRNAs can be translated under conditions when specific eIFs are compromised during infection. Except for a few cases, the specific mechanisms and factors that lead to IRES stimulation is poorly understood. For example, poliovirus and the related EMCV possess an IRES that allows viral translation despite cleavage of eIF4G during infection or inhibiting eIF4E by 4E-BP binding. This type of IRES can still bind to the central domain of eIF4G and mediate 40S subunit recruitment (11, 37, 57).One of the most unique and simplest IRES is found within the intergenic region (IGR) of the Dicistroviridae family (for extensive reviews, see references 28, 36, and 49). Members of this family include the cricket paralysis virus (CrPV), drosophila C virus (DCV), taura syndrome virus, the Plautia stali intestine virus (PSIV), the Rhopalosiphum padi virus (RhPV), and several bee viruses such as the black queen cell virus and the Israeli acute paralysis virus, which has been recently linked to colony collapse disorder (10). The dicistroviruses encode a positive-strand 8- to 10-kb single-stranded RNA genome, which contains two main open reading frames, ORF1 and ORF2, encoding the nonstructural and structural proteins, respectively, separated by an IGR (see Fig. ​Fig.1A).1A). The 5′ end of the CrPV RNA is linked to the viral protein VpG and the 3′ end contains a poly(A) tail (16). Radiolabeling of intracellular RNA in infected cells reveals no subgenomic RNA species smaller than the full-length genomic RNA, and this has been supported by Northern blot analysis (16, 81). Translation of ORF2 is directed by the IGR IRES, whereas ORF1 expression is mediated by an IRES within the 5′ untranslated region (5′UTR) (35, 67, 81, 82). Remarkably, the IGR IRES element can directly recruit the ribosome independently of eIFs or the initiator Met-tRNA_i (29, 30, 54, 80). Furthermore, the IRES occupies the P-site of the ribosome to initiate translation from the ribosomal A-site encoding non-AUG codon (35, 81). Extensive biochemical and structural analyses from several groups have revealed that the IGR IRES mimics a tRNA that occupies the mRNA cleft of the ribosome and sets the ribosome into an elongation state (9, 29, 30, 34, 51, 55, 58, 68, 72, 83). Using reporter constructs, it has also been demonstrated that CrPV IGR IRES-mediated translation is active under a number of cellular conditions when the activity of the ternary complex eIF2-Met-tRNA_i-GTP is compromised (17, 63, 78, 80). Because IGR IRES-mediated translation does not require initiation factors, the IRES can direct translation under a number of cellular conditions when the activity of multiple eIFs is compromised (12). Although the majority of studies have focused on the IGR IRES of CrPV, PSIV, and TSV, it is predicted that the IGRs within this viral family all function similarly based on the predicted conserved RNA structures (28, 36, 49). In contrast, only the 5′UTR IRES mechanism of RhPV has been studied in detail (77). Despite the wealth of studies on the mechanics of these IRES, the mechanisms that lead to translational shutoff during dicistrovirus infection and the interaction of dicistrovirus with the host machinery to allow virus production have been relatively unexplored.Open in a separate windowFIG. 1.Kinetics of host protein synthesis and viral protein expression in CrPV-infected Drosophila S2 cells. (A) Genomic arrangement of the CrPV RNA. The viral open reading frames, ORF1 and ORF2, that encode nonstructural (NS) and structural (S) proteins, respectively, are shown, which are separated by the intergenic internal ribosome entry site (IGR IRES). Translation of ORF1 and ORF2 is directed by the 5′UTR IRES and the IGR IRES, respectively. The first amino acid of ORF2 directed by the IGR IRES is encoded by a GCU alanine codon. (B) Autoradiography of protein lysates resolved on a SDS-12% PAGE gel. The protein lysates were collected from S2 cells that were untreated (U), mock infected (M), CrPV infected (5 FFU/cell), or thapsigargin treated (Tg; 0.4 μM) for the indicated times (h p.i.) and metabolically labeled with [³⁵S]methionine for 30 min at each time point. The migration of proteins with known molecular masses is shown on the left. The expression of detectable nonstructural (NS) and structural (S) proteins is denoted. (C) Quantitation of host protein synthesis during CrPV infection. To calculate the host translation at each time point, the amount of radioactivity of the bands between 55 and 70 kDa in panel A was quantitated by using ImageQuant, and the percent translation was calculated at each time point of virus infection or thapsigargin treatment compared to the mock infection. Shown are averages (± the standard deviation) from at least three independent experiments. (D) Immunoblots of viral ORF1 and ORF2 during CrPV infection at various times postinfection (h p.i.). Antibodies were raised against peptides within ORF1 and ORF2. The expression of ORF1 and ORF2 was quantitated by a LI-COR Odyssey system, plotted against time of infection, and normalized to the amount of ORF1 or ORF2 expression at 6 h p.i. (100%). As a comparison, viral RNA synthesis as detected by Northern blot analysis (see Fig. ​Fig.2B)2B) is plotted on the same graph.Previous studies have shown that the CrPV and the related DCV can infect a wide range of insect hosts, including the Drosophila melanogaster S2 cell line (60, 69). In the present study, we have explored how CrPV infection leads to host translational shutoff in S2 cells. Two steps of translational initiation are targeted during CrPV infection. First, the interaction of deIF4G with deIF4E is disrupted early in infection and remains dissociated during the course of infection. Second, deIF2α is phosphorylated at a time that lags after the initial host translational shutoff during infection. Premature phosphorylation of deIF2α early in infection inhibited translation directed by the 5′UTR IRES, but IGR IRES-mediated translation remained relatively resistant. These results support the model that multiple mechanisms, including impairment of deIF4F complex formation and induction of deIF2α phosphorylation, contribute to the host translational shutoff during CrPV infection. The inhibition of host translation and the release of ribosomes from host mRNAs ensures that translation mediated by the 5′UTR and IGR IRES is optimal to produce sufficient viral nonstructural and structural proteins for proper CrPV maturation and assembly.     

NLRC4 and TLR5 Each Contribute to Host Defense in Respiratory Melioidosis

Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice) and type III secretion system components (in mice and humans). In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1β production in Nlrc4^−/− mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis.     

Activation of Akt/protein kinase B in epithelial cells by the Salmonella typhimurium effector sigD

The serine-threonine kinase Akt is a protooncogene involved in the regulation of cell proliferation and survival. Activation of Akt is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser(473) and Thr(308). We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells. A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. In HeLa cells, wild type S. typhimurium induced translocation of Akt to membrane ruffles and phosphorylation at residues Thr(308) and Ser(473) and increased kinase activity. In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles. Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (C462S), did not restore the Akt activation phenotype. This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB. Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane-associated Akt into its active form. SigD is also the first bacterial effector to be identified as an activator of Akt.     

Temperature Alters Host Genotype-Specific Susceptibility to Chytrid Infection

The cost of parasitism often depends on environmental conditions and host identity. Therefore, variation in the biotic and abiotic environment can have repercussions on both, species-level host-parasite interaction patterns but also on host genotype-specific susceptibility to disease. We exposed seven genetically different but concurrent strains of the diatom Asterionella formosa to one genotype of its naturally co-occurring chytrid parasite Zygorhizidium planktonicum across five environmentally relevant temperatures. We found that the thermal tolerance range of the tested parasite genotype was narrower than that of its host, providing the host with a “cold” and “hot” thermal refuge of very low or no infection. Susceptibility to disease was host genotype-specific and varied with temperature level so that no genotype was most or least resistant across all temperatures. This suggests a role of thermal variation in the maintenance of diversity in disease related traits in this phytoplankton host. The duration and intensity of chytrid parasite pressure on host populations is likely to be affected by the projected changes in temperature patterns due to climate warming both through altering temperature dependent disease susceptibility of the host and, potentially, through en- or disabling thermal host refugia. This, in turn may affect the selective strength of the parasite on the genetic architecture of the host population.