Priming of Salmonella enterica Serovar Typhi-Specific CD8+ T Cells by Suicide Dendritic Cell Cross-Presentation in Humans

Background

The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8⁺ T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-γ secretion. However, how S. Typhi regulates the development of specific CD8⁺ responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8⁺ T cells.

Methodology/Principal Findings

We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-α, but low levels of IL-12 p70 and IFN-γ. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-γ and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3⁺CD8⁺CD45RA⁻CD62L⁻ effector/memory T cells.

Conclusions/Significance

This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8⁺ cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.     

Mycobacterium marinum MgtC Plays a Role in Phagocytosis but is Dispensable for Intracellular Multiplication

MgtC is a virulence factor involved in intramacrophage growth that has been reported in several intracellular pathogens, including Mycobacterium tuberculosis and Salmonella enterica serovar Typhimurium. MgtC participates also in adaptation to Mg²⁺ deprivation. Herein, we have constructed a mgtC mutant in Mycobacterium marinum to further investigate the role of MgtC in mycobacteria. We show that the M. marinum mgtC gene (Mma mgtC) is strongly induced upon Mg²⁺ deprivation and is required for optimal growth in Mg²⁺-deprived medium. The behaviour of the Mma mgtC mutant has been investigated in the Danio rerio infection model using a transgenic reporter zebrafish line that specifically labels neutrophils. Although the mgtC mutant is not attenuated in the zebrafish embryo model based on survival curves, our results indicate that phagocytosis by neutrophils is enhanced with the mgtC mutant compared to the wild-type strain following subcutaneous injection. Increased phagocytosis of the mutant strain is also observed ex vivo with the murine J774 macrophage cell line. On the other hand, no difference was found between the mgtC mutant and the wild-type strain in bacterial adhesion to macrophages and in the internalization into epithelial cells. Unlike the role reported for MgtC in other intracellular pathogens, Mma MgtC does not contribute significantly to intramacrophage replication. Taken together, these results indicate an unanticipated function of Mma MgtC at early step of infection within phagocytic cells. Hence, our results indicate that although the MgtC function is conserved among pathogens regarding adaptation to Mg²⁺ deprivation, its role towards phagocytic cells can differ, possibly in relation with the specific pathogen''s lifestyles.     

MgtC as a Horizontally-Acquired Virulence Factor of Intracellular Bacterial Pathogens: Evidence from Molecular Phylogeny and Comparative Genomics

MgtC is a virulence factor required for intramacrophage survival and growth in low Mg²⁺ medium in two pathogens that are not phylogenetically related, Salmonella typhimurium and Mycobacterium tuberculosis. In S. typhimurium, mgtC is carried by the SPI-3 pathogenicity island and hybridization studies have suggested that the distribution of mgtC among enterobacteria is limited. In the present study, we searched for the presence of mgtC-like sequences in eubacterial genomes. Analyses of MgtC-like proteins phylogeny and mgtC-like chromosomal context support the hypothesis that mgtC has been acquired by horizontal gene transfer repeatedly throughout bacterial evolution. In addition, the phylogenetic analysis revealed the existence of a subgroup of proteins, that includes the S. typhimurium and M. tuberculosis MgtC proteins, as well as MgtC-related proteins from other pathogens that are able to survive in macrophages, B. melitensis and Y. pestis. We propose that MgtC has a similar function in all these distantly related pathogens, most likely providing the ability to grow in a low Mg²⁺ environment. Present address: (Anne-Béatrice Blanc-Potard) Inserm U431, Faculté de Médecine, 30900 Nîmes, France     

A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens

Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg²⁺ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg²⁺ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.     

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg²⁺ deprivation, but previous work suggested that MgtC is not a Mg²⁺ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg²⁺ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg²⁺ concentration, indicating that it does not bind Mg²⁺. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg²⁺ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.     

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, σ^S (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σ^S protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σ^S, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Comparative Virulence Genotyping and Antimicrobial Susceptibility Profiling of Environmental and Clinical <Emphasis Type="Italic">Salmonella</Emphasis><Emphasis Type="Italic">enterica</Emphasis> from Cochin,India

Salmonella enterica serotype Newport is an important cause of non-typhoidal salmonellosis, a clinically less severe infection than typhoid fever caused by S. enterica serotype Typhi. In this investigation, the virulence genotypes of S. enterica Newport isolated from a backwater environment were compared with Salmonella Typhi from clinical cases in the same region where salmonellosis is endemic. Genotyping was done by PCR screening for virulence markers associated with Salmonella pathogenicity islands (SPIs) and plasmids. The virulence genes associated with SPIs I–VI were detected in 95–100% of all the isolates, while the viaB locus representing SPI-7 was detectable in 66 and 73% of the environmental and clinical isolates, respectively. A significant number of Salmonella Newport lacked virulence genes shdA and sopE compared to S. Typhi. All S. Typhi and S. Newport isolates lacked large plasmid-borne virulence genes spvR and pefA. Further investigations into the antimicrobial resistance of S. Newport revealed multiple drug resistance to ampicillin, amoxicillin/clavulanic acid, trimethorprim-sulfamethoxazole, chloramphenicol, tetracycline, cephalothin, and cephalexin. In comparison, S. Typhi were susceptible to all clinically relevant antimicrobials. The results of this study will help in understanding the spread of virulence genotypes and antibiotic resistance in S. Newport in the region of study.     

Selected Lactic Acid-Producing Bacterial Isolates with the Capacity to Reduce Salmonella Translocation and Virulence Gene Expression in Chickens

Background

Probiotics have been used to control Salmonella colonization/infection in chickens. Yet the mechanisms of probiotic effects are not fully understood. This study has characterized our previously-selected lactic acid-producing bacterial (LAB) isolates for controlling Salmonella infection in chickens, particularly the mechanism underlying the control.

Methodology/Principal Findings

In vitro studies were conducted to characterize 14 LAB isolates for their tolerance to low pH (2.0) and high bile salt (0.3–1.5%) and susceptibility to antibiotics. Three chicken infection trials were subsequently carried out to evaluate four of the isolates for reducing the burden of Salmonella enterica serovar Typhimurium in the broiler cecum. Chicks were gavaged with LAB cultures (10^6–7 CFU/chick) or phosphate-buffered saline (PBS) at 1 day of age followed by Salmonella challenge (10⁴ CFU/chick) next day. Samples of cecal digesta, spleen, and liver were examined for Salmonella counts on days 1, 3, or 4 post-challenge. Salmonella in the cecum from Trial 3 was also assessed for the expression of ten virulence genes located in its pathogenicity island-1 (SPI-1). These genes play a role in Salmonella intestinal invasion. Tested LAB isolates (individuals or mixed cultures) were unable to lower Salmonella burden in the chicken cecum, but able to attenuate Salmonella infection in the spleen and liver. The LAB treatments also reduced almost all SPI-1 virulence gene expression (9 out of 10) in the chicken cecum, particularly at the low dose. In vitro treatment with the extracellular culture fluid from a LAB culture also down-regulated most SPI-1 virulence gene expression.

Conclusions/Significance

The possible correlation between attenuation of Salmonella infection in the chicken spleen and liver and reduction of Salmonella SPI-1 virulence gene expression in the chicken cecum by LAB isolates is a new observation. Suppression of Salmonella virulence gene expression in vivo can be one of the strategies for controlling Salmonella infection in chickens.     

Live Recombinant Salmonella Typhi Vaccines Constructed to Investigate the Role of rpoS in Eliciting Immunity to a Heterologous Antigen

We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (χ9639 and χ9640) were derived from the rpoS mutant strain Ty2 and one (χ9633) from the RpoS⁺ strain ISP1820. In χ9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS⁺ vaccines induced a balanced Th1/Th2 immune response while the RpoS⁻ strain χ9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS⁺ strain χ9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, χ9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human hosts.     

A rule governing the FtsH-mediated proteolysis of the MgtC virulence protein from <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

A tightly controlled turnover of membrane proteins is required for lipid bilayer stability, cell metabolism, and cell viability. Among the energy-dependent AAA⁺ proteases in Salmonella, FtsH is the only membrane-bound protease that contributes to the quality control of membrane proteins. FtsH preferentially degrades the C-terminus or N-terminus of misfolded, misassembled, or damaged proteins to maintain physiological functions. We found that FtsH hydrolyzes the Salmonella MgtC virulence protein when we substitute the MgtC 226^th Trp, which is well conserved in other intracellular pathogens and normally protects MgtC from the FtsH-mediated proteolysis. Here we investigate a rule determining the FtsH-mediated proteolysis of the MgtC protein at Trp226 residue. Substitution of MgtC tryptophan 226^th residue to alanine, glycine, or tyrosine leads to MgtC proteolysis in a manner dependent on the FtsH protease whereas substitution to phenylalanine, methionine, isoleucine, leucine, or valine resists MgtC degradation by FtsH. These data indicate that a large and hydrophobic side chain at 226^th residue is required for protection from the FtsH-mediated MgtC proteolysis.     

Sodium Ion Cycle in Bacterial Pathogens: Evidence from Cross-Genome Comparisons

Analysis of the bacterial genome sequences shows that many human and animal pathogens encode primary membrane Na⁺ pumps, Na⁺-transporting dicarboxylate decarboxylases or Na⁺-translocating NADH:ubiquinone oxidoreductase, and a number of Na⁺-dependent permeases. This indicates that these bacteria can utilize Na⁺ as a coupling ion instead of or in addition to the H⁺ cycle. This capability to use a Na⁺ cycle might be an important virulence factor for such pathogens as Vibrio cholerae, Neisseria meningitidis, Salmonella enterica serovar Typhi, and Yersinia pestis. In Treponema pallidum, Chlamydia trachomatis, and Chlamydia pneumoniae, the Na⁺ gradient may well be the only energy source for secondary transport. A survey of preliminary genome sequences of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Treponema denticola indicates that these oral pathogens also rely on the Na⁺ cycle for at least part of their energy metabolism. The possible roles of the Na⁺ cycling in the energy metabolism and pathogenicity of these organisms are reviewed. The recent discovery of an effective natural antibiotic, korormicin, targeted against the Na⁺-translocating NADH:ubiquinone oxidoreductase, suggests a potential use of Na⁺ pumps as drug targets and/or vaccine candidates. The antimicrobial potential of other inhibitors of the Na⁺ cycle, such as monensin, Li⁺ and Ag⁺ ions, and amiloride derivatives, is discussed.     

Crystal Structure of the Salmonella enterica Serovar Typhimurium Virulence Factor SrfJ,a Glycoside Hydrolase Family Enzyme

To cause infection, Salmonella enterica serovar Typhimurium uses type III secretion systems, which are encoded on two Salmonella pathogenicity islands, SPI-1 and SPI-2, the latter of which is thought to play a crucial role in bacterial proliferation in Salmonella-containing vacuoles (SCVs) after invading cells. S. Typhimurium SrfJ, located outside SPI-2, is also known to be involved in Salmonella pathogenicity and has high amino acid sequence homology with human lysosomal glucosylceramidase (GlcCerase). We present the first crystal structure of SrfJ at a resolution of 1.8 Å. The overall fold of SrfJ shares high structure similarities with that of human GlcCerase, comprising two distinctive domains: a (β/α)₈-barrel catalytic domain and a β-sandwich domain. As in human GlcCerase, the pocket-shaped active site of SrfJ is located on the C-terminal side of the barrel, and two conserved glutamic acid residues are used for the enzyme catalysis. Moreover, a glycerol-bound form of SrfJ reveals that the glucose ring moiety of the substrate might similarly bind to the enzyme as to human GlcCerase, suggesting that SrfJ might function as a glycoside hydrolase. Although some structural differences are observed between SrfJ and human GlcCerase in the substrate entrance of the active site, we speculate that, based on the high structural similarities to human GlcCerase in the overall fold and the active-site environment, SrfJ might have a GlcCerase activity and use the activity to enhance Salmonella virulence by modifying SCV membrane lipids.Gram-negative bacterial pathogens deliver effector proteins into host cells through type III secretion systems (TTSS). The TTSS apparatus is a molecular syringe which spans the inner and outer membranes of pathogens and secretes translocon and effector proteins. Translocon proteins locate at the tip of the needle structure and are involved in the translocation of effector proteins by forming pores in the host cell membrane (3). The translocated effector proteins function to manipulate diverse host cellular processes such as cytoskeleton assembly, vesicle transport, and signal transduction, thereby promoting bacterial virulence (9).Salmonella enteric serovar Typhimurium (S. Typhimurium) causes a systemic infection in mice and is an intensively studied model of typhoid fever. This gram-negative bacterium can invade host cells and then survive by replicating within a membrane-bound compartment known as the Salmonella-containing vacuole (SCV) (16). Both invasion and intracellular survival are mediated by numerous virulence genes, which are clustered within the pathogenicity islands, SPI-1 and SPI-2 (18). The regulation of virulence proteins encoded by each pathogenicity island depends on the different stages of infection. While most of the genes within SPI-1 are required for the invading host cells and the early stages of SCV development (8), the genes in SPI-2 play a crucial role in bacterial proliferation in SCVs after cell invasion (23). The SsrA-SsrB two-component regulatory system is known to regulate the expression of genes within SPI-2 for bacterial virulence (4). Recent works have shown that several genes located outside of SPI-2 are under the control of the SsrA-SsrB regulator as well, and these have been proposed as putative virulence factors (10, 25).S. Typhimurium SrfJ was initially identified as a gene that is strongly activated by SsrB outside SPI-2 (25). Furthermore, a mutation on srfJ leads to mild attenuation of virulence in mice (22). Interestingly, SrfJ shares high amino acid sequence similarity with human lysosomal glucosylceramidase (GlcCerase) (25), which is a peripheral membrane protein catalyzing the hydrolysis of glucosylceramide (GlcCer) to β-glucose and ceramide in the presence of the modulator protein saposin C and lipid (11). Inherited defects in GlcCerase result in lysosomal GlcCer accumulation and, as a consequence, Gaucher disease, the most common lysosomal storage disease (19). Both human GlcCerase and SrfJ have been grouped into glycoside hydrolase (GH) family 30 containing GlcCerase (EC 3.2.1.45), β-1,6-glucanse (EC 3.2.1.75), and β-xylosidase (EC 3.2.1.37) of the GH-A clan in the CAZy database (http://afmb.cnrs-mrs.fr/CAZY). Among the members of GH family 30, structural information is available only on the human enzyme. The biochemical function of SrfJ and its role in Salmonella virulence remain to be elucidated. In order to better understand the function of SrfJ, we have determined the crystal structure of SrfJ from S. Typhimurium at a resolution of 1.8 Å.     

Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonella’s intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still controlled intracellular Salmonella burden. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages.     

Comparative Proteomic Analysis of the PhoP Regulon in Salmonella enterica Serovar Typhi Versus Typhimurium

Background

S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica.

Methodology/Principal Findings

Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of phoP⁻/Q⁻ mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions.

Conclusions/Significance

This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800).     

Host–Pathogen Interaction in Invasive Salmonellosis

Salmonella enterica infections result in diverse clinical manifestations. Typhoid fever, caused by S. enterica serovar Typhi (S. Typhi) and S. Paratyphi A, is a bacteremic illness but whose clinical features differ from other Gram-negative bacteremias. Non-typhoidal Salmonella (NTS) serovars cause self-limiting diarrhea with occasional secondary bacteremia. Primary NTS bacteremia can occur in the immunocompromised host and infants in sub-Saharan Africa. Recent studies on host–pathogen interactions in Salmonellosis using genome sequencing, murine models, and patient studies have provided new insights. The full genome sequences of numerous S. enterica serovars have been determined. The S. Typhi genome, compared to that of S. Typhimurium, harbors many inactivated or disrupted genes. This can partly explain the different immune responses both serovars induce upon entering their host. Similar genome degradation is also observed in the ST313 S. Typhimurium strain implicated in invasive infection in sub-Saharan Africa. Virulence factors, most notably, type III secretion systems, Vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and various factors essential for the intracellular life cycle of S. enterica have been characterized. Genes for these factors are commonly carried on Salmonella Pathogenicity Islands (SPIs). Plasmids also carry putative virulence-associated genes as well as those responsible for antimicrobial resistance. The interaction of Salmonella pathogen-associated molecular patterns (PAMPs) with Toll-like receptors (TLRs) and NOD-like receptors (NLRs) leads to inflammasome formation, activation, and recruitment of neutrophils and macrophages and the production of pro-inflammatory cytokines, most notably interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and interferon-gamma (IFN)-γ. The gut microbiome may be an important modulator of this immune response. S. Typhimurium usually causes a local intestinal immune response, whereas S. Typhi, by preventing neutrophil attraction resulting from activation of TLRs, evades the local response and causes systemic infection. Potential new therapeutic strategies may lead from an increased understanding of infection pathogenesis.     

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.