Polyamines are required for virulence in Salmonella enterica serovar Typhimurium

Sensing and responding to environmental cues is a fundamental characteristic of bacterial physiology and virulence. Here we identify polyamines as novel environmental signals essential for virulence of Salmonella enterica serovar Typhimurium, a major intracellular pathogen and a model organism for studying typhoid fever. Central to its virulence are two major virulence loci Salmonella Pathogenicity Island 1 and 2 (SPI1 and SPI2). SPI1 promotes invasion of epithelial cells, whereas SPI2 enables S. Typhimurium to survive and proliferate within specialized compartments inside host cells. In this study, we show that an S. Typhimurium polyamine mutant is defective for invasion, intracellular survival, killing of the nematode Caenorhabditis elegans and systemic infection of the mouse model of typhoid fever. Virulence of the mutant could be restored by genetic complementation, and invasion and intracellular survival could, as well, be complemented by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection. Interestingly, intracellular survival of the polyamine mutant was significantly enhanced above the wild type level by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection, indicating that these polyamines function as an environmental signal that primes S. Typhimurium for intracellular survival. Accordingly, experiments addressed at elucidating the roles of these polyamines in infection revealed that expression of genes from both of the major virulence loci SPI1 and SPI2 responded to exogenous polyamines and was reduced in the polyamine mutant. Together our data demonstrate that putrescine and spermidine play a critical role in controlling virulence in S. Typhimurium most likely through stimulation of expression of essential virulence loci. Moreover, our data implicate these polyamines as key signals in S. Typhimurium virulence.     

Mig-14 is an inner membrane-associated protein that promotes Salmonella typhimurium resistance to CRAMP, survival within activated macrophages and persistent infection

Salmonella enterica serovar Typhimurium (S. typhimurium) infects a wide variety of mammalian hosts and in rodents causes a typhoid-like systemic disease involving replication of bacteria inside macrophages within reticuloendothelial tissues. Previous studies demonstrated that the mig-14 and virK genes of Salmonella enterica are important in bacterial resistance to anti-microbial peptides and are necessary for continued replication of S. typhimurium in the liver and spleen of susceptible mice after orogastric inoculation. In this work we report that inflammatory signalling via interferon-gamma (IFN-gamma) is crucial to controlling replication of mig-14 mutant bacteria within the liver and spleen of mice after oral infection. Using a Salmonella persistence model recently developed in our laboratory, we further demonstrate that mig-14 contributes to long-term persistence of Salmonella in the spleen and mesenteric lymph nodes of chronically infected mice. Both mig-14 and virK contribute to the survival of Salmonella in macrophages treated with IFN-gamma and are necessary for resistance to cathelin-related anti-microbial peptide (CRAMP), an anti-microbial peptide expressed at high levels in activated mouse macrophages. We also show that both Mig-14 and VirK inhibit the binding of CRAMP to Salmonella, and demonstrate that Mig-14 is an inner membrane-associated protein. We further demonstrate by transmission electron microscopy that the primary locus of CRAMP activity appears to be intracytoplasmic, rather than at the outer membrane, suggesting that Mig-14 may prevent the penetration of the inner membrane by CRAMP. Together, these data indicate an important role for mig-14 in anti-microbial peptide resistance in vivo, and show that this resistance is important to the survival of Salmonella in systemic sites during both acute and persistent infection.     

Intracellular microbes and haemophagocytosis

Haemophagocytosis (hemophagocytosis) is the phenomenon of activated macrophage consumption of red and white blood cells, including professional phagocytes and lymphocytes. It can occur in patients with severe cases of intracellular microbial infection, including avian influenza, leishmaniasis, tuberculosis and typhoid fever. While well-known to physicians since at least the mid-1800s, haemophagocytosis has been little studied due to a paucity of tractable animal and cell culture models. Recently, haemophagocytosis has been described in a mouse model of typhoid fever, and it was noted that the infectious agent, Salmonella enterica, resides within haemophagocytic macrophages in mice. In addition, a cell culture model for haemophagocytosis revealed that S. enterica preferentially replicate in haemophagocytic macrophages. This review describes how, at the molecular and cellular levels, S. enterica may promote and take advantage of haemophagocytosis to establish long-term systemic infections in mammals. The role, relevance and possible molecular mechanisms of haemophagocytosis are discussed within the context of other microbial infections and of genetic deficiencies in which haemophagocytosis occurs and is associated with morbidity.     

Intracellular activities of Salmonella enterica in murine dendritic cells

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.     

A mouse model for the human pathogen Salmonella typhi

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a life-threatening human disease. The lack of animal models due to S. Typhi's strict human host specificity has hindered its study and vaccine development. We find that immunodeficient Rag2(-/-) γc(-/-) mice engrafted with human fetal liver hematopoietic stem and progenitor cells are able to support?S. Typhi replication and persistent infection. A?S. Typhi mutant in a gene required for virulence in humans was unable to replicate in these mice. Another mutant unable to produce typhoid toxin exhibited increased replication, suggesting a role for this toxin in the establishment of persistent infection. Furthermore, infected animals mounted human innate and adaptive immune responses to S. Typhi, resulting in the production of cytokines and pathogen-specific antibodies. We expect that this mouse model will be a useful resource for understanding S.?Typhi pathogenesis and for evaluating potential vaccine candidates against typhoid fever.     

The cytokine balance in the maintenance of a persistent infection with Salmonella enterica serovar Typhimurium in mice

ClpXP, serine protease-disrupted mutant of Salmonella enterica serovar Typhimurium chi3306 exhibits attenuated but persistent infection in mice. During infection with S. enterica serovar Typhimurium ClpXP-disrupted mutant, gamma interferon (IFN-gamma) produced by CD4+ cells was up-regulated on day 10 and tumor necrosis factor-alpha (TNF-alpha) produced by CD8+ cells was up-regulated on day 30 after infection. Treatment of monoclonal antibodies against cytokines showed that IFN-gamma and interleukin 10 (IL-10) were involved in maintenance of growth of S. Typhimurium mutant on day 10 after infection, and IFN-gamma, TNF-alpha and transforming growth factor-beta (TGF-beta) were involved in maintenance of growth of this bacterium on day 30 after infection. During persistent infection of S. Typhimurium mutant, IFN-gamma, TNF-alpha, IL-10 and TGF-beta may play different roles to maintain the persistent infection. The cytokine balance might be important in persistent infection with ClpXP-disrupted S. enterica serovar Typhimurium.     

Differential contribution of sodC1 and sodC2 to intracellular survival and pathogenicity of Salmonella enterica serovar Choleraesuis

Several of the most virulent Salmonella enterica strains possess two genes encoding periplasmic Cu,Zn superoxide dismutase, sodC1 and sodC2, located on a lambdoid prophage and on the chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide generated by the host's phagocytes. To investigate the respective contributions of sodC1 and sodC2 to the virulence of a clinical isolate of Salmonella enterica serovar Choleraesuis (S. choleraesuis), we have analyzed both the intracellular survival of wild type and sodC mutant strains within J774 macrophages and Caco-2 cells, and their ability to proliferate in intraperitoneally-infected mice in competition assays. In agreement with previous studies, mutant strains lacking one or both sodC genes were equally impaired in their ability to survive within activated macrophages. However, when macrophage killing experiments were carried out with non-opsonized bacteria, sodC2 contributed to intracellular survival more than sodC1, indicating that changes in the pathways of bacterial uptake can modify the relative role of the two sodC genes. More unexpectedly, we have found that the ability of S. choleraesuis to survive within Caco-2 cells was severely affected by inactivation of sodC genes, sodC2 being more important than sodC1. As Caco-2 cells actively produce superoxide, this suggests that oxygen radical production by colonic cells has a role in controlling proliferation of facultative intracellular bacteria. Mouse infection studies confirmed that, in the S. choleraesuis strain under investigation, both sodC genes are required to confer full virulence, sodC2 contributing slightly more than sodC1 to Salmonella pathogenesis. Our findings contrast with the results of other studies carried out in S. enterica serovar Typhimurium and suggest that the relative contributions of sodC1 and sodC2 to host-pathogen interactive biology may vary depending on the Salmonella serovar or strain.     

Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes disease in a variety of hosts. S. Typhimurium actively invade host cells and typically reside within a membrane-bound compartment called the Salmonella-containing vacuole (SCV). The bacteria modify the fate of the SCV using two independent type III secretion systems (TTSS). TTSS are known to damage eukaryotic cell membranes and S. Typhimurium has been suggested to damage the SCV using its Salmonella pathogenicity island (SPI)-1 encoded TTSS. Here we show that this damage gives rise to an intracellular bacterial population targeted by the autophagy system during in vitro infection. Approximately 20% of intracellular S. Typhimurium colocalized with the autophagy marker GFP-LC3 at 1 h postinfection. Autophagy of S. Typhimurium was dependent upon the SPI-1 TTSS and bacterial protein synthesis. Bacteria targeted by the autophagy system were often associated with ubiquitinated proteins, indicating their exposure to the cytosol. Surprisingly, these bacteria also colocalized with SCV markers. Autophagy-deficient (atg5-/-) cells were more permissive for intracellular growth by S. Typhimurium than normal cells, allowing increased bacterial growth in the cytosol. We propose a model in which the host autophagy system targets bacteria in SCVs damaged by the SPI-1 TTSS. This serves to retain intracellular S. Typhimurium within vacuoles early after infection to protect the cytosol from bacterial colonization. Our findings support a role for autophagy in innate immunity and demonstrate that Salmonella infection is a powerful model to study the autophagy process.     

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.     

Nutritional and Metabolic Requirements for the Infection of HeLa Cells by Salmonella enterica Serovar Typhimurium

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food - and water - borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.     

Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles

Autophagy is responsible for the degradation of cytosolic components within eukaryotic cells. Interestingly, autophagy also appears to play a role in recognizing invading intracellular pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that normally resides and replicates within the Salmonella-containing vacuole (SCV). However, during in vitro infection a population of S. Typhimurium damage and escape from the SCV to enter the cytosol. We have observed that some intracellular S. Typhimurium are recognized by autophagy under in vitro infection conditions. Immunofluorescence studies revealed that autophagy recognizes the population of S. Typhimurium within damaged SCVs early after infection. The consequences of autophagic recognition of S. Typhimurium are still being elucidated, though a restrictive effect on intracellular bacterial replication has been demonstrated. Results of our in vitro infection studies are consistent with autophagy playing a role in cellular defense against S. Typhimurium that become exposed to the cytosol.     

Autophagy Recognizes Intracellular Salmonella enterica serovar Typhimurium in Damaged Vacuoles

Autophagy is responsible for the degradation of cytosolic components within eukaryotic cells. Interestingly, autophagy also appears to play a role in recognizing invading intracellular pathogens. Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that normally resides and replicates within the Salmonella-containing vacuole (SCV). However, during in vitro infection a population of S. Typhimurium damage and escape from the SCV to enter the cytosol. We have observed that some intracellular S. Typhimurium are recognized by autophagy under in vitro infection conditions. Immunofluorescence studies revealed that autophagy recognizes the population of S.Typhimurium within damaged SCVs early after infection. The consequences of autophagic recognition of S. Typhimurium are still being elucidated, though a restrictive effect on intracellular bacterial replication has been demonstrated. Results of our in vitro infection studies are consistent with autophagy playing a role in cellular defense against S. Typhimurium that become exposed to the cytosol.     

Selection of Salmonella enterica serovar Typhi genes involved during interaction with human macrophages by screening of a transposon mutant library

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 10(5) transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated.     

Macrophages inhibit Salmonella typhimurium replication through MEK/ERK kinase and phagocyte NADPH oxidase activities

Host responses during the later stages of Salmonella-macrophage interactions are critical to controlling infection but have not been well characterized. After 24 h of infection, nearly half of interferon-gamma-primed murine RAW 264.7 macrophage-like cells infected by Salmonella enterica serovar Typhimurium contained filamentous bacteria. Bacterial filamentation indicates a defect in completing replication and has been previously observed in bacteria responding to a variety of stresses. To understand whether macrophage gene expression was responsible for this effect on Salmonella Typhimurium replication, we used gene arrays to profile interferon-gamma-primed RAW 264.7 cell gene expression following infection. We observed an increase in MEK1 kinase mRNA at 8 h, an increase in MEK protein at 24 h, and measured phosphorylation of MEK's downstream target kinase, ERK1/2, throughout the 24-h infection period. Treatment of cells with MEK kinase inhibitors significantly reduced numbers of filamentous bacteria observed within macrophages after 24 h and increased the number of intracellular colony-forming units. Phagocyte NADPH oxidase inhibitors and antioxidants also significantly reduced bacterial filamentation. Either MEK kinase or phagocyte oxidase inhibitors could be added 4-8 h after infection and still significantly decrease bacterial filamentation. Oxidase activity appears to mediate bacterial filamentation in parallel to MEK kinase signaling, while inducible nitric-oxide synthase inhibitors had no significant effect on bacterial morphology. In summary, Salmonella Typhimurium infection of interferon-gamma-primed macrophages triggers a MEK kinase cascade at later infection times, and both MEK kinase and phagocyte NADPH oxidase activity impair bacterial replication. These two signaling pathways mediate a host bacteriostatic pathway and may play an important role in innate host defense against intracellular pathogens.     

Dynamics of Salmonella small RNA expression in non-growing bacteria located inside eukaryotic cells

Small non-coding regulatory RNAs (sRNAs) have been studied in many bacterial pathogens during infection. However, few studies have focused on how intracellular pathogens modulate sRNA expression inside eukaryotic cells. Here, we monitored expression of all known sRNAs of Salmonella enterica serovar Typhimurium (S. Typhimurium) in bacteria located inside fibroblasts, a host cell type in which this pathogen restrains growth. sRNA sequences known in S. Typhimurium and Escherichia coli were searched in the genome of S. Typhimurium virulent strain SL1344, the subject of this study. Expression of 84 distinct sRNAs was compared in extra- and intracellular bacteria. Non-proliferating intracellular bacteria upregulated six sRNAs, including IsrA, IsrG, IstR-2, RyhB-1, RyhB-2 and RseX while repressed the expression of the sRNAs DsrA, GlmZ, IsrH-1, IsrI, SraL, SroC, SsrS(6S) and RydC. Interestingly, IsrH-1 was previously reported as an sRNA induced by S. Typhimurium inside macrophages. Kinetic analyses unraveled changing expression patterns for some sRNAs along the infection. InvR and T44 expression dropped after an initial induction phase while IstR-2 was induced exclusively at late infection times (> 6 h). Studies focused on the Salmonella-specific sRNA RyhB-2 revealed that intracellular bacteria use this sRNA to regulate negatively YeaQ, a cis-encoded protein of unknown function. RyhB-2, together with RyhB-1, contributes to attenuate intracellular bacterial growth. To our knowledge, these data represent the first comprehensive study of S. Typhimurium sRNA expression in intracellular bacteria and provide the first insights into sRNAs that may direct pathogen adaptation to a non-proliferative state inside the host cell.