The phage shock protein PspA facilitates divalent metal transport and is required for virulence of Salmonella enterica sv. Typhimurium

The phage shock protein (Psp) system is induced by extracytoplasmic stress and thought to be important for the maintenance of proton motive force. We investigated the contribution of PspA to Salmonella virulence. A pspA deletion mutation significantly attenuates the virulence of Salmonella enterica serovar Typhimurium following intraperitoneal inoculation of C3H/HeN (Ity^r) mice. PspA was found to be specifically required for virulence in mice expressing the natural resistance‐associated macrophage protein 1 (Nramp1) (Slc11a1) divalent metal transporter, which restricts microbial growth by limiting the availability of essential divalent metals within the phagosome. Salmonella competes with Nramp1 by expressing multiple metal uptake systems including the Nramp‐homologue MntH, the ABC transporter SitABCD and the ZIP family transporter ZupT. PspA was found to facilitate Mn²⁺ transport by MntH and SitABCD, as well as Zn²⁺ and Mn²⁺ transport by ZupT. In vitro uptake of ⁵⁴Mn²⁺ by MntH and ZupT was reduced in the absence of PspA. Transport‐deficient mutants exhibit reduced viability in the absence of PspA when grown under metal‐limited conditions. Moreover, the ZupT transporter is required for Salmonella enterica serovar Typhimurium virulence in Nramp1‐expressing mice. We propose that PspA promotes Salmonella virulence by maintaining proton motive force, which is required for the function of multiple transporters mediating bacterial divalent metal acquisition during infection.     

Evaluation of the immunogenicity of Campylobacter jejuni CjaA protein delivered by Salmonella enterica sv. Typhimurium strain with regulated delayed attenuation in chickens

Campylobacter spp. are regarded as the most common bacterial cause of gastroenteritis worldwide, and consumption of chicken meat contaminated by Campylobacter is considered to be one of the most frequent sources of human infection in developed countries. Here we evaluated the immunogenicity and protective efficacy of Salmonella Typhimurium χ9718 producing the Campylobacter jejuni CjaA protein as a chicken anti-Campylobacter vaccine. In this study chickens were orally immunized with a new generation S. Typhimurium strain χ9718 with regulated delayed attenuation in vivo and displaying delayed antigen expression. The immunization with the S. Typhimurium χ9718 strain producing C. jejuni CjaA antigen induced strong immune responses against CjaA in both serum IgY and intestinal IgA, however, it did not result in the significant reduction of intestinal colonization by Campylobacter strain. The low level of protection might arise due to a lack of T cell response. Our results demonstrated that a Salmonella strain with regulated delayed attenuation and displaying regulated delayed antigen expression might be an efficient vector to induce immune response against Campylobacter. It seems that an efficient anti-Campylobacter subunit vaccine should be multicomponent. Since S. Typhimurium χ9718 contains two compatible balanced-lethal plasmids, it can provide the opportunity of cloning several Campylobacter genes encoding immunodominant proteins. It may also be used as a delivery vector of eukaryotic genes encoding immunostimulatory molecules to enhance or modulate functioning of chicken immune system.     

The natural resistance-associated macrophage protein 1 Slc11a1 (formerly Nramp1) and iron metabolism in macrophages

Slc11a1 (solute carrier family 11 member 1) (formerly Nramp1) modulation of iron metabolism in macrophages plays an important role in early phase macrophage activation, and therefore host innate immunity. This review focuses on the role of Nramp1 in intramacrophage iron metabolism, with emphasis on the two prevailing mechanisms of Nramp1 modulation of iron metabolism in macrophages.     

The role of ferritins in the physiology of Salmonella enterica sv. Typhimurium: a unique role for ferritin B in iron-sulphur cluster repair and virulence

Ferritins are ubiquitous iron (Fe) storage proteins that play a fundamental role in cellular Fe homeostasis. The enteric pathogen Salmonella enterica serovar Typhimurium possesses four ferritins: bacterioferritin, ferritin A, ferritin B and Dps. The haem-containing bacterioferritin (Bfr) accounts for the majority of stored Fe, followed by ferritin A (FtnA). Inactivation of bfr elevates the intracellular free Fe concentration and enhances susceptibility to H2O2 stress. The DNA-binding Dps protein provides protection from oxidative damage without affecting the steady-state intracellular free Fe concentration. FtnB appears to be particularly important for the repair of oxidatively damaged Fe-sulphur clusters of aconitase and, in contrast to Bfr and FtnA, is required for Salmonella virulence in mice. Moreover, ftnB and dps are repressed by the Fe-responsive regulator Fur and induced under conditions of Fe limitation, whereas bfr and ftnA are maximally expressed when Fe is abundant. The absence of a conserved ferroxidase domain and the potentiation of oxidative stress by FtnB in some strains lacking Dps suggest that FtnB serves as a facile cellular reservoir of Fe2+.     

The Salmonella enterica sv. Typhimurium smvA,yddG and ompD (porin) genes are required for the efficient efflux of methyl viologen

In Gram-negative bacteria, a subset of inner membrane proteins in the major facilitator superfamily (MFS) acts as efflux pumps to decrease the intracellular concentrations of multiple toxic substrates and confers multidrug resistance. The Salmonella enterica sv. Typhimurium smvA gene encodes a product predicted to be an MFS protein most similar to QacA of Staphylococcus aureus. Like mutations in qacA, mutations in smvA confer increased sensitivity to methyl viologen (MV). Mutations in the adjacent ompD (porin) and yddG (drug/metabolite transporter) genes also confer increased sensitivity to MV, and mutations in smvA are epistatic to mutations in ompD or yddG for this phenotype. YddG and OmpD probably comprise a second efflux pump in which the OmpD porin acts as an outer membrane channel (OMC) protein for the efflux of MV and functions independently of the SmvA pump. In support of this idea, the pump dependent on YddG and OmpD has a different substrate specificity from the pump dependent on SmvA. Mutations in tolC, which encodes an OMC protein, confer increased resistance to MV. TolC apparently facilitates the import of MV, and a subset of OMC proteins including the OmpD porin and TolC may facilitate both import and export of distinct subsets of toxic substrates.     

Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation

Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-alpha signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1.     

Molecular characterization of InJR06, a class 1 integron located in a conjugative plasmid of Salmonella enterica ser. Typhimurium.

The presence of class 1, 2, and 3 integrons was investigated in four pediatric isolates of Salmonella enterica ser. Typhimurium (S. Typhimurium). A class 1 integron was detected in one S. Typhimurium strain, the only one that also showed resistance to various aminoglycoside antibiotics. This integron, called InJR06, and the aminoglycoside resistance determinants were located in pS06, a large (> or = 55 kb) conjugative plasmid. A single mobile cassette (encoding the aminoglycoside adenylyltransferase ANT(3')-Ia) was detected in the variable region of InJR06, while the architecture of the attI1 and attC sites was conserved.     

Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection

Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H(+)ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.     

Oral and intranasal administration of somatostatin DNA vaccine mediated by attenuated Salmonella enterica serovar Typhimurium to promote growth of piglets

An attenuated strain of Salmonella typhimurium has been used as a carrier for oral and intranasal genetic immunization. Here, we evaluate the efficacy of a vaccine strain of S. typhimurium. CSO22 (pGM-CSF/SS, plasmid granulocyte-macrophage colony-stimulating factor/somatostatin) expressing two copies of SS genes. A total of 115 piglets, aged 2 months old, were either orally or intranasally immunized against the vaccine strain CSO22 (pGM-CSF/SS) with three dosages (5 × 10(10) colony forming units (CFU), 5 × 10(9) CFU and 5 × 10(8) CFU). For oral immunization, the specific anti-SS antibodies were detected in the immunized piglets. The levels of SS antibodies in the high-dose immunized group (5 × 10(10) CFU) were significantly higher than that in the phosphate buffered saline immunized group (P < 0.01) and 40% of animals were positive in SS antibodies in the high-dose immunized group. Moreover, the weight gain of the high-dose group was increased by 20.86%, 10.26% and 15.30% during 4, 8 and 12 weeks, respectively, after immunization in comparison to the control. For intranasal immunization, the growth of the low-dose group was increased by 10.23% in the whole test period (12 weeks). In conclusion, our results suggest that the recombinant strain could elicit anti-SS antibodies and improve the growth performance of immunized piglets, and that the oral immunization program is better than the intranasal program.     

Tuberculosis triggers a tissue-dependent program of differentiation and acquisition of effector functions by circulating monocytes

The origin and function of the different myeloid cell subsets that appear in the lung during pulmonary tuberculosis are unknown. Herein we show that adoptively transferred monocytes give rise to many of the macrophage and dendritic cell (DC) subsets that appear following aerosol infection with virulent Mycobacterium tuberculosis. Monocyte differentiation in infected peripheral tissue is surprisingly heterogeneous and results in the formation of five distinct myeloid subsets, including both classically activated macrophages, that produce inducible NO synthase via an IFN-gamma-dependent mechanism, and DC. In contrast, monocytes recruited to draining pulmonary lymph nodes are functionally different and acquire a mature DC phenotype. Thus, while monocytes are recruited to the lungs of uninfected mice, their differentiation and acquisition of myeloid effector functions are dramatically altered in the presence of inflammation and bacteria and are dependent on tissue localization. Therefore, our results support a model in which recruited monocytes are well poised to influence multiple aspects of host immunity to infections in the lungs. This report provides the first direct evidence for monocyte differentiation into both the macrophage and DC lineages in vivo following infection with a live human pathogen.     

Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage.

Salmonella typhimurium is a facultative intracellular pathogen, able both to invade and to survive within eukaryotic cells and to grow in various extracellular environments. To compare the bacterial responses to these disparate environments and to shed light on the nature of the intracellular environment, we have examined the pattern of protein synthesis by two-dimensional polyacrylamide gel electrophoresis. The levels of approximately 40 proteins were observed to increase during growth within macrophage-like U937 cells, while approximately 100 proteins exhibited levels that were repressed relative to those of an extracellular control culture. To aid in the interpretation of these results, the patterns of proteins made by S. typhimurium exposed to various environmental conditions in the laboratory were determined. The intracellular protein pattern was then compared with each of these benchmark protein patterns. This analysis revealed that, as expected, the intracellular environment appears to impose numerous stresses on the bacteria, but unexpectedly, the macrophage-induced response was not a simple sum of the individual stress responses displayed during extracellular growth.     

Genetic detoxification of an aroA Salmonella enterica serovar Typhimurium vaccine strain does not compromise protection against virulent Salmonella and enhances the immune responses towards a protective malarial antigen

Live Salmonella vaccines are limited in use by the inherent toxicity of the lipopolysaccharide. The waaN gene encodes a myristyl transferase required for the secondary acylation of lipid A in lipopolysaccharide. A waaN mutant exhibits reduced induction of the inflammatory cytokines associated with lipopolysaccharide toxicity. Here the characteristics of a Salmonella enterica serovar Typhimurium aroA waaN mutant (SK100) in vitro and in vivo compared with its parent aroA strain (SL3261) were described. Phenotypic analysis of purified lipopolysaccharide obtained from SK100 confirmed that the physical and biological activities of the lipopolysaccharide had been altered. Nevertheless both strains had similar patterns of colonization and persistence in mice and significantly the aroA waaN mutant was equally as effective as the parent at protecting against challenge with wild-type S. Typhimurium. Furthermore, a SK100 strain was constructed expressing both tetanus toxin fragment C and the circumsporozoite protein of a malaria parasite. In marked contrast to its isogenic parent, the new attenuated strain induces significantly enhanced immune responses against the circumsporozoite protein. The waaN mutation enhances the ability of this strain to elicit immune responses towards guest antigens. This study provides important insights into the development of safe and effective multivalent Salmonella vaccines.     

Modulation of host signaling by a bacterial mimic: structure of the Salmonella effector SptP bound to Rac1

Salmonella spp. utilize a specialized protein secretion system to deliver a battery of effector proteins into host cells. Several of these effectors stimulate Cdc42- and Rac1-dependent cytoskeletal changes that promote bacterial internalization. These potentially cytotoxic alterations are rapidly reversed by the effector SptP, a tyrosine phosphatase and GTPase activating protein (GAP) that targets Cdc42 and Rac1. The 2.3 A resolution crystal structure of an SptP-Rac1 transition state complex reveals an unusual GAP architecture that mimics host functional homologs. The phosphatase domain possesses a conserved active site but distinct surface properties. Binding to Rac1 induces a dramatic stabilization in SptP of a four-helix bundle that makes extensive contacts with the Switch I and Switch II regions of the GTPase.     

Uptake and handling of iron from transferrin, lactoferrin and immune complexes by a macrophage cell line.

The murine macrophage-like cell line P388D1 has been used as a model to investigate whether iron acquired simultaneously from different sources (transferrin, lactoferrin, and ovotransferrin-anti-ovotransferrin immune complexes) is handled in the same way. P388D1 cells bound both lactoferrin and transferrin, but over a 6 h incubation period only the latter actually donated iron to the cells. When the cells were incubated with [55Fe]transferrin and [59Fe]ovotransferrin-anti-ovotransferrin immune complexes iron was acquired from both sources. However, there was a difference in the intracellular distribution of the two isotopes, proportionally more 55Fe entering haem compounds and less entering ferritin. When the cells were precultured in a low-iron serum-free medium almost no transferrin-iron was incorporated into ferritin, whereas the proportion of immune complex-derived iron incorporated into ferritin was unchanged. Lactoferrin enhanced the rate of cellular proliferation, as measured by [3H]thymidine incorporation, despite its inability to donate iron to the cells, suggesting a stimulatory effect independent of iron donation. In contrast immune complexes inhibited cell proliferation. These findings indicate that iron acquired from transferrin and iron acquired by scavenging mechanisms are handled differently, and suggest that more than one intracellular iron transit pool may exist.