In Vivo Genetic Analysis Indicates That PhoP-PhoQ and the Salmonella Pathogenicity Island 2 Type III Secretion System Contribute Independently to Salmonella enterica Serovar Typhimurium Virulence

Many virulence factors are required for Salmonella enterica serovar Typhimurium to replicate intracellularly and proliferate systemically within mice. In this work, we have carried out genetic analyses in vivo to determine the functional relationship between two major virulence factors necessary for systemic infection by S. enterica serovar Typhimurium: the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) and the PhoP-PhoQ two-component regulatory system. Although previous work suggested that PhoP-PhoQ regulates SPI-2 TTSS gene expression in vitro, in vivo competitive analysis of mutant strains indicates that these systems contribute independently to S. typhimurium virulence. Our results also suggest that mutation of phoP may compensate partially for defects in the SPI-2 TTSS by deregulating SPI-1 TTSS expression. These results provide an explanation for previous reports showing an apparent functional overlap between these two systems in vitro.     

Expression of STM4467-Encoded Arginine Deiminase Controlled by the STM4463 Regulator Contributes to Salmonella enterica Serovar Typhimurium Virulence

Arginine deiminase (ADI), carbamate kinase (CK), and ornithine transcarbamoylase (OTC) constitute the ADI system. In addition to metabolic functions, the ADI system has been implicated in the virulence of certain pathogens. The pathogenic intracellular bacterium Salmonella enterica serovar Typhimurium possesses the STM4467, STM4466, and STM4465 genes, which are predicted to encode ADI, CK, and OTC, respectively. Here we report that the STM4467 gene encodes an ADI and that ADI activity plays a role in the successful infection of a mammalian host by S. Typhimurium. An STM4467 deletion mutant was defective for replication inside murine macrophages and was attenuated for virulence in mice. We determined that a regulatory protein encoded by the STM4463 gene functions as an activator for STM4467 expression. The expression of the ADI pathway genes was enhanced inside macrophages in a process that required STM4463. Lack of STM4463 impaired the ability of S. Typhimurium to replicate within macrophages. A mutant defective in STM4467-encoded ADI displayed normal production of nitric oxide by macrophages.     

Infection of mice by Salmonella enterica serovar Enteritidis involves additional genes that are absent in the genome of serovar Typhimurium

Salmonella enterica serovar Enteritidis causes a systemic, typhoid-like infection in newly hatched poultry and mice. In the present study, a library of 54,000 transposon mutants of S. Enteritidis phage type 4 (PT4) strain P125109 was screened for mutants deficient in the in vivo colonization of the BALB/c mouse model using a microarray-based negative-selection screening. Mutants in genes known to contribute to systemic infection (e.g., Salmonella pathogenicity island 2 [SPI-2], aro, rfa, rfb, phoP, and phoQ) and enteric infection (e.g., SPI-1 and SPI-5) in this and other Salmonella serovars displayed colonization defects in our assay. In addition, a strong attenuation was observed for mutants in genes and genomic islands that are not present in S. Typhimurium or in most other Salmonella serovars. These genes include a type I restriction/modification system (SEN4290 to SEN4292), the peg fimbrial operon (SEN2144A to SEN2145B), a putative pathogenicity island (SEN1970 to SEN1999), and a type VI secretion system remnant SEN1001, encoding a hypothetical protein containing a lysin motif (LysM) domain associated with peptidoglycan binding. Proliferation defects for mutants in these individual genes and in exemplar genes for each of these clusters were confirmed in competitive infections with wild-type S. Enteritidis. A ΔSEN1001 mutant was defective for survival within RAW264.7 murine macrophages in vitro. Complementation assays directly linked the SEN1001 gene to phenotypes observed in vivo and in vitro. The genes identified here may perform novel virulence functions not characterized in previous Salmonella models.     

Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants

Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA' reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence.     

Salmonella enterica Serovar Enteritidis Pathogenicity Island 1 Is Not Essential for but Facilitates Rapid Systemic Spread in Chickens

Salmonella enterica subsp. enterica serovar Enteritidis is a leading cause of human food-borne illness that is mainly associated with the consumption of contaminated poultry meat and eggs. To cause infection, S. Enteritidis is known to use two type III secretion systems, which are encoded on two salmonella pathogenicity islands, SPI-1 and SPI-2, the first of which is thought to play a major role in invasion and bacterial uptake. In order to study the role of SPI-1 in the colonization of chicken, we constructed deletion mutants affecting the complete SPI-1 region (40 kb) and the invG gene. Both ΔSPI-1 and ΔinvG mutant strains were impaired in the secretion of SipD, a SPI-1 effector protein. In vitro analysis using polarized human intestinal epithelial cells (Caco-2) revealed that both mutant strains were less invasive than the wild-type strain. A similar observation was made when chicken cecal and small intestinal explants were coinfected with the wild-type and ΔSPI-1 mutant strains. Oral challenge of 1-week-old chicken with the wild-type or ΔSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection of the liver and spleen was delayed in birds that were challenged with the ΔSPI-1 strain. These data demonstrate that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of the organism in chickens.Salmonella enterica is a gram-negative enteropathogenic bacterium. Within the S. enterica species, more than 2,300 serovars have been identified, of which the serovars Enteritidis and Typhimurium have been the most frequently associated with human infections (49). S. Enteritidis is a well-known zoonotic pathogen (30), and infected poultry are among the most common reservoir of salmonellae that can be transmitted through the food chain to humans (18). In young chicks, S. Enteritidis infection can lead to increased incidence of illness, while older birds are less susceptible to the effects of this pathogen, often experiencing intestinal colonization and even systemic dissemination without significant morbidity or mortality. Hence, a better understanding of the pathogenesis of S. Enteritidis in chickens may subsequently lower the rates of human disease caused by this pathogen.S. Enteritidis requires a substantial number of genes for virulence, which are clustered in large chromosomal regions known as Salmonella pathogenicity islands (SPI) (40). Two of these pathogenicity islands encode two functionally distinct type III secretion systems (T3SS) that are utilized as “molecular syringes” to translocate virulence determinants, called effector proteins, from the bacterial cytoplasm into (29, 30) or in the vicinity of the target cell (52). Effector proteins delivered by the SPI-1 T3SS are mainly involved in host cell invasion by inducing membrane ruffling and disrupting actin polymerization to facilitate bacterial uptake (17). The SPI-2 T3SS plays a major role in systemic virulence and in facilitating intracellular survival, especially within macrophages. However, recent evidence suggests that the SPI-2 T3SS also plays a role in intestinal colonization (10, 11) and is expressed prior to bacterial uptake (6).Invasion of epithelial cells by Salmonella enterica subspecies enterica serovar Typhimurium has been shown to disrupt tight junctions (15, 31, 54) which, along with other components, form important intercellular junctions found in polarized epithelial cells. Tight junctions regulate the paracellular flow of ions and solutes across the intestinal epithelium. They also maintain distinct apical and basolateral domains with well-defined plasma membrane components (42). SPI-1 effector proteins have been associated with the disruption of tight junctions (5, 31, 54) by activating Cdc42 and Rac-1 (Rho family GTPases), which subsequently activate signal transduction pathways that lead to the reorganization of actin, resulting in the uptake of Salmonella (45). Further, the S. Typhimurium SPI-1 effectors SopB, SopE, SopE2, and SipA have been identified as major contributors in the disruption of tight junctions (5).The contribution of S. Typhimurium SPI-1 to intestinal cell invasion has been studied in cell culture using different SPI-1 deletion mutants. SPI-1 regulatory gene hilA and sipB (SPI-1 effector gene) mutants have been shown to be attenuated in invasion relative to the wild-type strain in porcine intestinal epithelioid (IPI-21) cells. However, the use of polarized porcine intestinal epithelial cells (IPEC-J2) has revealed that, in addition to hilA and sipB mutant strains, a sipA (SPI-1 effector gene) mutant strain was also recovered at a significantly lower rate than the wild-type strain (4), suggesting that SPI-1 is important for efficient invasion. In another study, it was demonstrated that the S. Typhimurium SPI-1 effectors SipA, SopA, SopB, SopD, and SopE2 all contributed to invasion of polarized human colon carcinoma cells (T84). Mutants lacking the genes for the aforementioned effector proteins were less invasive than the wild-type strain (51). Moreover, an S. Typhimurium strain lacking the invG gene (encoding an outer membrane component of the SPI-1 T3SS apparatus) was also impaired in invasion of COS-7 cells (24). Taken together, the results from these invasion studies suggest that SPI-1 plays a role in invasion in cell culture. However, most of the data available is from research that has been conducted using S. Typhimurium.Several in vivo experiments have been performed to investigate the role of SPI-1 during the course of a Salmonella infection in different animal species using deletion mutants in SPI-1 genes. In the murine model of infection, it was observed that S. Typhimurium strains containing mutations in hilA and invG were recovered from intestinal contents and systemic sites at a lower frequency than the wild-type strain (43). Other groups have reported that a functional SPI-1 T3SS is required to induce intestinal inflammation and cause significant histopathological changes in the streptomycin-treated mouse model of infectious enterocolitis (1, 10, 24). Similarly, in the bovine model of enteritis, SPI-1 has been shown to be important for intestinal colonization (20). In addition, it has been demonstrated that S. Typhimurium SPI-1 gene (sipA, sipB, and hilA) mutants were impaired in their ability to colonize the porcine gut in a ligated intestinal loop model (4). However, a recent study has shown that a SPI-1 functional mutant induces intestinal pathology that is very similar to the wild-type strain when studied 5 days postchallenge in a novel bovine ileal loop model (11).Studies investigating the contribution of SPI-1 in chicken during the course of an S. Enteritidis infection are limited in number and have mostly observed colonization and systemic infection over a short time frame. Infection of 1-day-old birds with S. Enteritidis strains containing mutations in the invA, invB, and invC genes (SPI-1 genes) has shown that these organisms are attenuated in the colonization of the gastrointestinal tract, as well as in the systemic spread of the organism over a period of 6 days postinfection (48). In another study, it was observed that S. Enteritidis sipD mutants were unable to colonize spleens compared to the wild-type strain 3 days postinfection in 1-day-old chicks (44). However, the colonization of the spleen was only measured at one time point, making it difficult to predict the effect of the mutant strain prior to and after, the third day after infection. A similar trend was seen in the ceca of 1-day-old chicks challenged with a S. Enteritidis hilA mutant strain over a period of 28 days postinfection (3). However, the hilA mutant strain did not have a significant impact on the infection of the livers and spleens. Recently, the impact of SPI-1 was examined using a S. Typhimurium spaS mutant strain in 1-day-old and 1-week-old birds (32). Inactivation of spaS (SPI-1 structural protein) did not affect colonization of the livers or ceca of 1-day-old birds over a period of 72 h postinfection. In 1-week-old birds, the same strain was recovered at lower levels from the ceca over a period of 14 days postinfection, while the recovery from the liver was lower at 3 days postinfection (32).Taken together, the experimental evidence from the in vitro studies suggests that S. Typhimurium SPI-1 has an impact on invasion. However, studies investigating the role of S. Enteritidis SPI-1 in vivo are limited. Moreover, research from the murine, bovine, and porcine models of salmonellosis indicates that SPI-1 may play a role in breaching the intestinal epithelial layer during the course of an infection. Nevertheless, little is known about the virulence properties of the S. Enteritidis SPI-1 T3SS in the colonization of chickens. In addition, recent evidence suggests that S. enterica is capable of establishing infection without the presence of SPI-1 (11, 25, 28). The objective of the present study was to investigate the contribution of the S. Enteritidis SPI-1 T3SS in the invasion of polarized Caco-2 cells and chicken intestinal explants and in the colonization of chickens over a period of 4 days postchallenge. Our data indicate that S. Enteritidis SPI-1 is important for invasion in polarized Caco-2 cells and intestinal epithelial cells in vitro. We also show that a ΔSPI-1 mutant strain is not impaired in the cecal colonization of 1-week-old chickens. However, the deletion of the SPI-1 region causes a delay in systemic infection.     

Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens

Salmonella enterica serovar Gallinarum is a host-specific serotype that causes the severe systemic disease fowl typhoid in domestic poultry and a narrow range of other avian species but rarely causes disease in mammalian hosts. Specificity of the disease is primarily at the level of the reticuloendothelial system, but few virulence factors have been described other than the requirement for an 85-kb virulence plasmid. In this work, by making functional mutations in the type III secretion systems (TTSS) encoded by Salmonella pathogenicity island 1 (SPI-1) and SPI-2, we investigated the role of these pathogenicity islands in interactions between Salmonella serovar Gallinarum and avian cells in vitro and the role of these pathogenicity islands in virulence in chickens. The SPI-1 mutant showed decreased invasiveness into avian cells in vitro but was unaffected in its ability to persist within chicken macrophages. In contrast the SPI-2 mutant was fully invasive in nonphagocytic cells but failed to persist in macrophages. In chicken infections the SPI-2 mutant was attenuated while the SPI-1 mutant showed full virulence. In oral infections the SPI-2 mutant was not observed in the spleen or liver, and following intravenous inoculation it was cleared rapidly from these sites. SPI-2 function is required by Salmonella serovar Gallinarum for virulence, primarily through promoting survival within macrophages allowing multiplication within the reticuloendothelial system, but this does not preclude the involvement of SPI-2 in uptake from the gut to the spleen and liver. SPI-1 appears to have little effect on virulence and survival of Salmonella serovar Gallinarum in the host.     

SspA is required for lethal Salmonella enterica serovar Typhimurium infections in calves but is not essential for diarrhea

Salmonella pathogenicity island 1 (SPI-1) encodes virulence determinants, which are important for enteropathogenicity in calves. To determine whether the Salmonella enterica serovar Typhimurium SPI-1 effector proteins SspA and SptP are important for enteropathogenicity, strains lacking these proteins were tested during oral infection of calves. Calves infected with a sptP mutant or its isogenic parent developed diarrhea and lethal morbidity. In contrast, calves infected with an sspA mutant developed diarrhea, which resolved within 10 days but did not result in mortality. The sspA mutant was recovered from bovine intestinal tissues at numbers similar to those obtained for its isogenic parent and caused marked intestinal lesions. Thus, the severity of pathological changes caused by serovar Typhimurium strains or their ability to cause diarrhea were not predictive of their ability to cause lethal morbidity in calves. We conclude that factors other than or in addition to bacterial colonization, intestinal lesions, or electrolyte loss contribute to lethal morbidity in calves infected with serovar Typhimurium.     

Salmonella typhimurium Virulence Genes Are Induced upon Bacterial Invasion into Phagocytic and Nonphagocytic Cells

Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identify Salmonella typhimurium genes that are induced inside Salmonella-containing vacuoles within macrophage and epithelial cells. A promoterless luciferase gene cassette was inserted randomly into the Salmonella chromosome, and the resulting mutants were screened for genes upregulated in intracellular bacteria compared to extracellular bacteria. We identified four genes in S. typhimurium that were upregulated upon bacterial invasion of both phagocytic and nonphagocytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time points (2 to 4 h) postinvasion and continued to be upregulated within host cells at later times (5 to 7 h). One mutant contained an insertion in the ssaR gene, within Salmonella pathogenicity island 2 (SPI-2), which abolished bacterial virulence in a murine typhoid model. Two other mutants contained insertions within SPI-5, one in the sopB/sigD gene and the other in a downstream gene, pipB. The insertions within SPI-5 resulted in the attenuation of S. typhimurium in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the S. typhimurium chromosome, iicA (induced intracellularly A). We detected no effect on virulence as a result of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustrating the requirement for specific gene expression inside mammalian cells and indicating the key role that virulence factor regulation plays in Salmonella pathogenesis.     

Activation of apoptosis by Salmonella pathogenicity island-1 effectors through both intrinsic and extrinsic pathways in Salmonella-infected macrophages

BackgroundSalmonella enterica serovar Typhimurium, a non-typhoidal food-borne pathogen, causes acute enterocolitis, bacteremia, extraintestinal focal infections in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) contribute to invading into host cellular cytosol, residing in Salmonella-containing vacuoles for intracellular survival, and inducing cellular apoptosis. This study aimed to better understand the mechanism underlying apoptosis in Salmonella-infected macrophages.MethodsS. Typhimurium SL1344 was used to evaluate extrinsic and intrinsic apoptosis pathways in THP-1 monocyte-derived macrophages in response to Salmonella infection.ResultsActivated caspase-3-induced apoptosis pathways, including extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, in Salmonella-infected macrophages were verified. THP-1 cells with dysfunction of TLR-4 and TLR-5 and Salmonella SPI-1 and SPI-2 mutants were constructed to identify the roles of the genes associated with programmed cell death in the macrophages. Caspase-3 activation in THP-1 macrophages was induced by Salmonella through TLR-4 and TLR-5 signaling pathways. We also identified that SPI-1 structure protein PrgH and effectors SipB and SipD, but not SPI-2 structure protein SsaV, could induce apoptosis via caspase-3 activation and reduce the secretion of inflammation marker TNF-α in the Salmonella-infected cells. The two effectors also reduced the translocation of the p65 subunit of NF-κB into the nucleus and the expression of TNF-α, and then inflammation was diminished.ConclusionNon-typhoid Salmonella induced apoptosis of macrophages and thereby reduced inflammatory cytokine production through the expression of SPI-1. This mechanism in host–pathogen interaction may explain why Salmonella usually manifests as occult bacteremia with less systemic inflammatory response syndrome in the bloodstream infection of children.     

The Salmonella Typhi hlyE gene plays a role in invasion of cultured epithelial cells and its functional transfer to S. Typhimurium promotes deep organ infection in mice

Comparison of genome sequences of Salmonella enterica serovars Typhi and Typhimurium reveals that S. Typhi has a small 2.3kb genomic island missing in S. Typhimurium, designated Salmonella pathogenicity island 18 (SPI-18), which includes two potential genes. One of these, hlyE, encodes a hemolysin related to the Escherichia coli K12 HlyE hemolysin. PCR assays show that SPI-18 is present in S. Typhi and in many other, but not all, serovars of S. enterica subsp. enterica belonging to the SARB collection. HlyE activity cannot be detected in S. Typhi by means of standard plate assays. Nevertheless, we were able to reveal this activity upon lysis of bacterial cells with phages, in the presence of ampicillin, and in a ompA genetic background, conditions that compromise the integrity of the bacterial envelope. Almost all serovars of the SARB collection shown to cause systemic infections in humans have SPI-18 and hlyE and express an active hemolysin revealed upon bacterial envelope destabilization. S. Typhi hlyE mutants are impaired in invasion of human epithelial cells in vitro, and its heterologous expression in S. Typhimurium improves the colonization of deep organs in mice, demonstrating that the HlyE hemolysin is a new virulence determinant.     

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.