Transposon Mutagenesis of Salmonella enterica Serovar Enteritidis Identifies Genes That Contribute to Invasiveness in Human and Chicken Cells and Survival in Egg Albumen

Salmonella enterica serovar Enteritidis is an important food-borne pathogen, and chickens are a primary reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on Salmonella enterica serovar Typhimurium, S. Enteritidis is known to have pathobiology specific to chickens that impacts epidemiology in humans. Therefore, more information is needed about S. Enteritidis pathobiology in comparison to that of S. Typhimurium. We used transposon mutagenesis to identify S. Enteritidis virulence genes by assay of invasiveness in human intestinal epithelial (Caco-2) cells and chicken liver (LMH) cells and survival within chicken (HD-11) macrophages as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of an invasive G1 Nal^r strain were screened using Caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which include genes previously known to contribute to enteric infection (e.g., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR, and rfbMN) in S. Enteritidis and other Salmonella serovars. Several genes or genomic islands that have not been reported previously (e.g., SPI-14, ksgA, SEN0034, SEN2278, and SEN3503) or that are absent in S. Typhimurium or in most other Salmonella serovars (e.g., pegD, SEN1152, SEN1393, and SEN1966) were also identified. Most mutants with reduced Caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells and impaired survival in chicken macrophages and in egg albumen. Consequently, these genes may play an important role during infection of the chicken host and also contribute to successful egg contamination by S. Enteritidis.     

Cathelicidin Antimicrobial Peptide Expression Is Not Induced or Required for Bacterial Clearance during Salmonella enterica Infection of Human Monocyte-Derived Macrophages

Salmonella enterica serovar Typhimurium is able to resist antimicrobial peptide killing by induction of the PhoP-PhoQ and PmrA-PmrB two-component systems and the lipopolysaccharide (LPS) modifications they mediate. Murine cathelin-related antimicrobial peptide (CRAMP) has been reported to inhibit S. Typhimurium growth in vitro and in vivo. We hypothesize that infection of human monocyte-derived macrophages (MDMs) with Salmonella enterica serovar Typhi and S. Typhimurium will induce human cathelicidin antimicrobial peptide (CAMP) production, and exposure to LL-37 (processed, active form of CAMP/hCAP18) will lead to upregulation of PmrAB-mediated LPS modifications and increased survival in vivo. Unlike in mouse macrophages, in which CRAMP is upregulated during infection, camp gene expression was not induced in human MDMs infected with S. Typhi or S. Typhimurium. Upon infection, intracellular levels of ΔphoPQ, ΔpmrAB, and PhoP^c S. Typhi decreased over time but were not further inhibited by the vitamin D₃-induced increase in camp expression. MDMs infected with wild-type (WT) S. Typhi or S. Typhimurium released similar levels of proinflammatory cytokines; however, the LPS modification mutant strains dramatically differed in MDM-elicited cytokine levels. Overall, these findings indicate that camp is not induced during Salmonella infection of MDMs nor is key to Salmonella intracellular clearance. However, the cytokine responses from MDMs infected with WT or LPS modification mutant strains differ significantly, indicating a role for LPS modifications in altering the host inflammatory response. Our findings also suggest that S. Typhi and S. Typhimurium elicit different proinflammatory responses from MDMs, despite being capable of adding similar modifications to their LPS structures.     

Interplay between the QseC and QseE Bacterial Adrenergic Sensor Kinases in Salmonella enterica Serovar Typhimurium Pathogenesis

The bacterial adrenergic sensor kinases QseC and QseE respond to epinephrine and/or norepinephrine to initiate a complex phosphorelay regulatory cascade that modulates virulence gene expression in several pathogens. We have previously shown that QseC activates virulence gene expression in Salmonella enterica serovar Typhimurium. Here we report the role of QseE in S. Typhimurium pathogenesis as well as the interplay between these two histidine sensor kinases in gene regulation. An S. Typhimurium qseE mutant is hampered in the invasion of epithelial cells and intramacrophage replication. The ΔqseC strain is highly attenuated for intramacrophage survival but has only a minor defect in invasion. However, the ΔqseEC strain has only a slight attenuation in invasion, mirroring the ΔqseC strain, and has an intermediary intramacrophage replication defect in comparison to the ΔqseE and ΔqseC strains. The expressions of the sipA and sopB genes, involved in the invasion of epithelial cells, are activated by epinephrine via QseE. The expression levels of these genes are still decreased in the ΔqseEC double mutant, albeit to a lesser extent, congruent with the invasion phenotype of this mutant. The expression level of the sifA gene, important for intramacrophage replication, is decreased in the qseE mutant and the ΔqseEC double mutant grown in vitro. However, as previously reported by us, the epinephrine-dependent activation of this gene occurs via QseC. In the systemic model of S. Typhimurium infection of BALB/c mice, the qseC and qseE mutants are highly attenuated, while the double mutant has an intermediary phenotype. Altogether, these data suggest that both adrenergic sensors play an important role in modulating several aspects of S. Typhimurium pathogenesis.     

Role of different receptors and actin filaments on Salmonella Typhimurium invasion in chicken macrophages

Bacterial attachment to host cell is the first event for pathogen entry. The attachment is mediated through membrane expressed adhesins present on the organism and receptors on the cell surface of host. The objective of this study was to investigate the significance of Fc receptors (FcRs), actin filament polymerization, mannose receptors (MRs), carbohydrate moieties like N-linked glycans and sialic acid on chicken macrophages for invasion of S. Typhimurium. Opsonisation of S. Typhimurium resulted in three folds more invasion in chicken monocyte derived macrophages. Cytochalasin D, an inhibitor of actin filament polymerization prevented uptake of S. Typhimurium. Pre-incubation of macrophages with cytochalasin D, showed severe decrease (28 folds) in S. Typhimurium invasion. Next we attempted to analyse the role of carbohydrate receptors of macrophages in S. Typhimurium invasion. Treatment of macrophages with methyl α-d-mannopyranoside, PNGase F and neuraminidase, showed 2.5, 5 and 2.5 folds decrease in invasion respectively. Our data suggest that deglycosylation of N-linked glycans including sialic acid by PNGase F is more effective in inhibition of S. Typhimurium invasion than neuraminidase which removes only sialic acid. These findings suggested FcRs, actin filament polymerization, MRs, N-linked glycans and sialic acid may act as gateway for entry of S. Typhimurium.     

Characterisation of multidrug-resistant Salmonella Typhimurium 4,[5],12:i:- DT193 strains carrying a novel genomic island adjacent to the thrW tRNA locus

In 2006, monophasic, multidrug-resistant Salmonella enterica spp. enterica serovar 4,[5],12:i:- strains appeared as a novel serotype in Germany, associated with large diffuse outbreaks and increased need for hospitalisation. The emerging 4,[5],12:i:- strains isolated from patients in Germany belong mainly to phage type DT193 according to the Anderson phage typing scheme for S. Typhimurium (STM) and exhibit at least a tetra-drug resistance. The strains have been shown to harbour STM-specific Gifsy-1, Gifsy-2, and ST64B prophages. Furthermore, the extensive sequence similarity of the tRNA regions between one characterised 4,[5],12:i:- phage type DT193 and the S. Typhimurium LT2 strain as well as the STM-specific position of an IS200 element within the fliA-fliB intergenic region ( Echeita et al., 2001) prompted us to classify them as a monophasic variant of S. Typhimurium. In 2008, the monophasic variant represented 42.2% of all S. Typhimurium isolates from human analysed at the National Reference Centre. Searching for insertions in tRNA sites resulted in the detection of an 18.4-kb fragment adjacent to the thrW tRNA locus, exhibiting a lower G+C content compared to the LT2 genome. Sequence analysis identified 17 potential ORFs. Some of them showed high similarity to enterobacterial phage sequences and sequences from Shigella boydii, Sh. dysenteriae, avian pathogenic Escherichia coli and other Escherichia spp. The biological function of this novel island with respect to virulence properties and metabolic functions is under investigation.     

Identification of Two Shigella flexneri Chromosomal Loci Involved in Intercellular Spreading

The ability of Shigella flexneri to multiply within colonic epithelial cells and spread to adjacent cells is essential for production of dysentery. Two S. flexneri chromosomal loci that are required for these processes were identified by screening a pool of TnphoA insertion mutants. These mutants were able to invade cultured epithelial cells but could not form wild-type plaques. Analysis of the nucleotide sequence indicated that the sites of TnphoA insertion were within two different regions that are almost identical to Escherichia coli K-12 chromosomal sequences of unknown functions. One region is located at 70 min on the E. coli chromosome, upstream of murZ, while the other is at 28 min, downstream of tonB. The mutant with the insertion at 70 min was named vpsC because it showed an altered pattern of virulence protein secretion. The vpsC mutant formed pinpoint-sized plaques, was defective in recovery from infected tissue culture cells, and was sensitive to lysis by the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the S. flexneri vpsA, -B, and -C genes complemented all of the phenotypes of the vpsC mutant. A mutation in vpsA resulted in the same phenotype as the vpsC mutation, suggesting that these two genes are part of a virulence operon in S. flexneri. The mutant with the insertion at 28 min was interrupted in the same open reading frame as S. flexneri ispA. This ispA mutant could not form plaques and was defective in bacterial septation inside tissue culture cells.     

Absence of Platelet Endothelial Cell Adhesion Molecule 1, PECAM-1/CD31, In Vivo Increases Resistance to Salmonella enterica Serovar Typhimurium in Mice

PECAM-1/CD31 is known to regulate inflammatory responses and exhibit pro- and anti-inflammatory functions. This study was designed to determine the functional role of PECAM-1 in susceptibility to murine primary in vivo infection with Salmonella enterica serovar Typhimurium and in in vitro inflammatory responses of peritoneal macrophages. Lectin profiling showed that cellular PECAM-1 and recombinant human PECAM-1-Ig chimera contain high levels of mannose sugars and N-acetylglucosamine. Consistent with this carbohydrate pattern, both recombinant human and murine PECAM-1-Ig chimeras were shown to bind S. Typhimurium in a dose-dependent manner in vitro. Using oral and fecal-oral transmission models of S. Typhimurium SL1344 infection, PECAM-1^−/− mice were found to be more resistant to S. Typhimurium infection than wild-type (WT) C57BL/6 mice. While fecal shedding of S. Typhimurium was comparable in wild-type and PECAM-1^−/− mice, the PECAM-1-deficient mice had lower bacterial loads in systemic organs such as liver, spleen, and mesenteric lymph nodes than WT mice, suggesting that extraintestinal dissemination was reduced in the absence of PECAM-1. This reduced bacterial load correlated with reduced tumor necrosis factor (TNF), interleukin-6 (IL-6), and monocyte chemoattractant protein (MCP) levels in sera of PECAM-1^−/− mice. Following in vitro stimulation of macrophages with either whole S. Typhimurium, lipopolysaccharide (LPS) (Toll-like receptor 4 [TLR4] ligand), or poly(I·C) (TLR3 ligand), production of TNF and IL-6 by PECAM-1^−/− macrophages was reduced. Together, these results suggest that PECAM-1 may have multiple functions in resistance to infection with S. Typhimurium, including binding to host cells, extraintestinal spread to deeper tissues, and regulation of inflammatory cytokine production by infected macrophages.     

Polynucleotide phosphorylase (PNPase) is required for Salmonella enterica serovar Typhimurium colonization in swine

The pnp gene encodes polynucleotide phosphorylase, an exoribonuclease involved in RNA processing and degradation. A mutation in the pnp gene was previously identified by our group in a signature-tagged mutagenesis screen designed to search for Salmonella enterica serovar Typhimurium genes required for survival in an ex vivo swine stomach content assay. In the current study, attenuation and colonization potential of a S. Typhimurium pnp mutant in the porcine host was evaluated. Following intranasal inoculation with 10⁹ cfu of either the wild-type S. Typhimurium χ4232 strain or an isogenic derivative lacking the pnp gene (n = 5/group), a significant increase (p < 0.05) in rectal temperature (fever) was observed in the pigs inoculated with wild-type S. Typhimurium compared to the pigs inoculated with the pnp mutant. Fecal shedding of the pnp mutant was significantly reduced during the 7-day study compared to the wild-type strain (p < 0.001). Tissue colonization was also significantly reduced in the pigs inoculated with the pnp mutant compared to the parental strain, including the tonsils, ileocecal lymph nodes, Peyer's Patch region of the ileum, cecum and contents of the cecum (p < 0.05). The data indicate that the pnp gene is required for S. Typhimurium virulence and gastrointestinal colonization of the natural swine host.     

Defining the Core Genome of Salmonella enterica Serovar Typhimurium for Genomic Surveillance and Epidemiological Typing

Salmonella enterica serovar Typhimurium is the most common Salmonella serovar causing foodborne infections in Australia and many other countries. Twenty-one S. Typhimurium strains from Salmonella reference collection A (SARA) were analyzed using Illumina high-throughput genome sequencing. Single nucleotide polymorphisms (SNPs) in 21 SARA strains ranged from 46 to 11,916 SNPs, with an average of 1,577 SNPs per strain. Together with 47 strains selected from publicly available S. Typhimurium genomes, the S. Typhimurium core genes (STCG) were determined. The STCG consist of 3,846 genes, a set that is much larger than that of the 2,882 Salmonella core genes (SCG) found previously. The STCG together with 1,576 core intergenic regions (IGRs) were defined as the S. Typhimurium core genome. Using 93 S. Typhimurium genomes from 13 epidemiologically confirmed community outbreaks, we demonstrated that typing based on the S. Typhimurium core genome (STCG plus core IGRs) provides superior resolution and higher discriminatory power than that based on SCG for outbreak investigation and molecular epidemiology of S. Typhimurium. STCG and STCG plus core IGR typing achieved 100% separation of all outbreaks compared to that of SCG typing, which failed to separate isolates from two outbreaks from background isolates. Defining the S. Typhimurium core genome allows standardization of genes/regions to be used for high-resolution epidemiological typing and genomic surveillance of S. Typhimurium.     

Immunological responses against Salmonella enterica serovar Typhimurium Braun lipoprotein and lipid A mutant strains in Swiss-Webster mice: potential use as live-attenuated vaccines

We generated and characterized Salmonella enterica serovar Typhimurium mutants that were deleted for the genes encoding Braun lipoprotein (lpp) alone or in conjunction with the msbB gene, which codes for an enzyme required for the acylation of the lipid A moiety of lipopolysaccharide. Two copies of the lpp gene, designated as lppA and lppB, exist on the chromosome of S. Typhimurium. These mutants were highly attenuated in a mouse infection model and induced minimal histopathological changes in mouse organs compared to those seen in infection with wild-type (WT) S. Typhimurium. The lppB/msbB and the lppAB/msbB mutants were maximally attenuated, and hence further examined in this study for their ability to induce humoral and cellular immune responses. Importantly, infection of out-bred Swiss-Webster mice with the mutant S. Typhimurium generated superior T helper cell type 2 (Th2) responses compared to WT S. Typhimurium, as determined by measuring IgG subclasses and cytokines. WT S. Typhimurium induced higher levels of IgG2a in sera of infected mice, while the lppB/msbB and lppAB/msbB mutants mounted higher levels of IgG1 as determined by an enzyme-linked immunosorbent assay. Mice immunized with lppB/msbB and lppAB/msbB mutants rapidly cleared WT S. Typhimurium upon subsequent rechallenge, and naïve mice passively immunized with sera from animals infected with S. Typhimurium mutants were protected against subsequent challenge with WT S. Typhimurium. Splenic T cells produced higher levels of interferon-gamma following ex vivo exposure to WT S. Typhimurium, while splenic T cells infected with the above-mentioned two mutants evoked higher levels of interleukin-6. Further, mice infected with lppB/msbB and lppAB/msbB mutants showed much higher levels of splenic T cell activation as measured by CD44⁺ expression on CD4⁺ T cells by flow cytometry and by incorporation of ³H-thymidine compared to mice that were infected with WT S. Typhimurium. We expect the lppB/msbB and lppAB/msbB mutants to be excellent live-attenuated vaccine candidates, because they induced minimal inflammatory responses and evoked stronger and specific antibody and cellular immune responses.     

Toll‐like receptor 4 signalling through MyD88 is essential to control Salmonella enterica serovar Typhimurium infection,but not for the initiation of bacterial clearance

Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF^−/− mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4^−/−, MyD88^−/− and TRIF^−/− mice in vitro. Pro-inflammatory responses from Mal^−/− macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF^−/− macrophages were partially restored by the addition of interferon-γ (IFN-γ), and TRIF^−/− mice produced markedly enhanced IFN-γ levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4^−/−, MyD88^−/−, TRIF^−/− and Mal^−/− mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.