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ABSTRACTSalmonella entericaserovar Typhimurium, an intracellular pathogen and leading cause of food-borne illness, encodes a plethora of virulence effectors. Salmonellavirulence factors are translocated into host cells and manipulate host cellular activities, providing a more hospitable environment for bacterial proliferation. In this study, we report a new set of virulence factors that is translocated into the host cytoplasm via bacterial outer membrane vesicles (OMV). PagK (or PagK1), PagJ, and STM2585A (or PagK2) are small proteins composed of ∼70 amino acids and have high sequence homology to each other (>85% identity). Salmonellalacking all three homologues was attenuated for virulence in a mouse infection model, suggesting at least partial functional redundancy among the homologues. While each homologue was translocated into the macrophage cytoplasm, their translocation was independent of all three Salmonellagene-encoded type III secretion systems (T3SSs)–Salmonellapathogenicity island 1 (SPI-1) T3SS, SPI-2 T3SS, and the flagellar system. Selected methods, including direct microscopy, demonstrated that the PagK-homologous proteins were secreted through OMV, which were enriched with lipopolysaccharide (LPS) and outer membrane proteins. Vesicles produced by intracellular bacteria also contained lysosome-associated membrane protein 1 (LAMP1), suggesting the possibility of OMV convergence with host cellular components during intracellular trafficking. This study identified novel Salmonellavirulence factors secreted via OMV and demonstrated that OMV can function as a vehicle to transfer virulence determinants to the cytoplasm of the infected host cell.

ABSTRACTIn this review, we provide an overview of the methods employed in four recent studies that described novel methods for computational prediction of secreted effectors from type III and IV secretion systems in Gram-negative bacteria. We present the results of these studies in terms of performance at accurately predicting secreted effectors and similarities found between secretion signals that may reflect biologically relevant features for recognition. We discuss the Web-based tools for secreted effector prediction described in these studies and announce the availability of our tool, the SIEVE server (http://www.sysbep.org/sieve). Finally, we assess the accuracies of the three type III effector prediction methods on a small set of proteins not known prior to the development of these tools that we recently discovered and validated using both experimental and computational approaches. Our comparison shows that all methods use similar approaches and, in general, arrive at similar conclusions. We discuss the possibility of an order-dependent motif in the secretion signal, which was a point of disagreement in the studies. Our results show that there may be classes of effectors in which the signal has a loosely defined motif and others in which secretion is dependent only on compositional biases. Computational prediction of secreted effectors from protein sequences represents an important step toward better understanding the interaction between pathogens and hosts.

Salmonella enterica serovar Typhimurium is an intracellular pathogen and a main cause of food-borne illness. In this study, a quantitative PCR (qPCR)-based competitive index (CI) method was developed to simultaneously compare the growth of multiple Salmonella strains. This method was applied to a mixture of 17 Salmonella mutants lacking regulator genes, and their survival ratios were compared based on expression of natural resistance-associated macrophage protein 1 (Nramp1). Nramp1, as a major host innate immune component, controls the intracellular replication of pathogens. Deletion strains containing unique DNA barcodes in place of regulator genes were mixed with the parental control, and the bacteria were inoculated into congenic mice differing only at Nramp1. Most of the deletion strains were outcompeted by wild-type bacteria in either mouse strain, and the lack of Nramp1 didn't increase the tested strain/parent control replication ratios. When the same collection of mutants was tested in congenic mouse-derived primary macrophages, a major Nramp1-expressing cell type, six strains (himD, phoP/phoQ, rpoE, rpoS, ompR/envZ, and hfq strains) grew better in Nramp1–/–than in Nramp1+/+macrophages, suggesting that these six regulators may play roles in overcoming Nramp1-mediated bactericidal activity in primary macrophages. The discrepancy in survival of macrophages and that of mice suggests either that there are differences in macrophage populations or that other cell types expressing Nramp1 control Salmonella proliferation in the host. The method described allows competitive infection analysis to be carried out on complex mixtures of bacteria and provides high reproducibility from independent biological replicates.

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 (ssaL) 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 ssaL 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.

ABSTRACTSalmonella entericaserovar Typhimurium is an intracellular pathogen and a main cause of food-borne illness. In this study, a quantitative PCR (qPCR)-based competitive index (CI) method was developed to simultaneously compare the growth of multiple Salmonellastrains. This method was applied to a mixture of 17 Salmonellamutants lacking regulator genes, and their survival ratios were compared based on expression of natural resistance-associated macrophage protein 1 (Nramp1). Nramp1, as a major host innate immune component, controls the intracellular replication of pathogens. Deletion strains containing unique DNA barcodes in place of regulator genes were mixed with the parental control, and the bacteria were inoculated into congenic mice differing only at Nramp1. Most of the deletion strains were outcompeted by wild-type bacteria in either mouse strain, and the lack of Nramp1 didn't increase the tested strain/parent control replication ratios. When the same collection of mutants was tested in congenic mouse-derived primary macrophages, a major Nramp1-expressing cell type, six strains (ΔhimD, ΔphoP/phoQ, ΔrpoE, ΔrpoS, ΔompR/envZ, and Δhfqstrains) grew better in Nramp1−/−than in Nramp1+/+macrophages, suggesting that these six regulators may play roles in overcoming Nramp1-mediated bactericidal activity in primary macrophages. The discrepancy in survival of macrophages and that of mice suggests either that there are differences in macrophage populations or that other cell types expressing Nramp1 control Salmonellaproliferation in the host. The method described allows competitive infection analysis to be carried out on complex mixtures of bacteria and provides high reproducibility from independent biological replicates.

ABSTRACTSalmonella entericaserovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonellapathogenicity 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 ΔssaLmutant, 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 Salmonellavirulence.

ABSTRACTFrancisella tularensisis the bacterial pathogen that causes tularemia in humans and a number of animals. To date, there is no approved vaccine for this widespread and life-threatening disease. The goal of this study was to identify F. tularensismutants that can be used in the development of a live attenuated vaccine. We screened F. novicidatransposon mutants to identify mutants that exhibited reduced growth in mouse macrophages, as these cells are the preferred host cells of Francisellaand an essential component of the innate immune system. This approach yielded 16 F. novicidamutants that were 100-fold more attenuated for virulence in a mouse model than the wild-type parental strain. These mutants were then tested to determine their abilities to protect mice against challenge with high doses of wild-type bacteria. Five of the 16 attenuated mutants (with mutations corresponding to dsbB, FTT0742, pdpB, fumA, and carBin the F. tularensisSCHU S4 strain) provided mice with protection against challenge with high doses (>8 × 105CFU) of wild-type F. novicida. We believe that these findings will be of use in the design of a vaccine against tularemia.

ABSTRACTA common theme in bacterial pathogenesis is the secretion of bacterial products that modify cellular functions to overcome host defenses. Gram-negative bacterial pathogens use type III secretion systems (TTSSs) to inject effector proteins into host cells. The genes encoding the structural components of the type III secretion apparatus are conserved among bacterial species and can be identified by sequence homology. In contrast, the sequences of secreted effector proteins are less conserved and are therefore difficult to identify. A strategy was developed to identify virulence factors secreted by Salmonella entericaserovar Typhimurium into the host cell cytoplasm. We constructed a transposon, which we refer to as mini-Tn5-cycler, to generate translational fusions between Salmonellachromosomal genes and a fragment of the calmodulin-dependent adenylate cyclase gene derived from Bordetella pertussis(cyaA′). In-frame fusions to bacterial proteins that are secreted into the eukaryotic cell cytoplasm were identified by high levels of cyclic AMP in infected cells. The assay was sufficiently sensitive that a single secreted fusion could be identified among several hundred that were not secreted. This approach identified three new effectors as well as seven that have been previously characterized. A deletion of one of the new effectors, steA(Salmonellatranslocated effector A), attenuated virulence. In addition, SteA localizes to the trans-Golgi network in both transfected and infected cells. This approach has identified new secreted effector proteins in Salmonellaand will likely be useful for other organisms, even those in which genetic manipulation is more difficult.

ABSTRACTSalmonella entericaserovar Typhimurium utilizes macrophages to disseminate from the intestine to deeper tissues within the body. While S. entericaserovar Typhimurium has been shown to kill its host macrophage, it can persist intracellularly beyond 18 h postinfection. To identify factors involved in late stages of infection, we screened a transposon library made in S. entericaserovar Typhimurium for the ability to persist in J774 macrophages at 24 h postinfection. Through this screen, we identified a gene, sciS, found to be homologous to icmFin Legionella pneumophila. icmF, which is required for intracellular multiplication, is conserved in several gram-negative pathogens, and its homolog appears to have been acquired horizontally in S. entericaserovar Typhimurium. We found that an sciSmutant displayed increased intracellular numbers in J774 macrophages when compared to the wild-type strain at 24 h postinfection. sciSwas maximally transcribed at 27 h postinfection and is repressed by SsrB, an activator of genes required for promoting intracellular survival. Finally, we demonstrate that an sciSmutant is hypervirulent in mice when administered intragastrically. Taken together, these data indicate a role for SciS in controlling intracellular bacterial levels at later stages of infection and attenuating virulence in a murine host

ABSTRACTThe enteric pathogen Salmonella entericaserotype Typhimurium induces apoptosis in infected macrophages. This process is rapid, specific, and depends on the type III protein secretion system encoded within Salmonellapathogenicity island 1 (SPI1). Here, we demonstrate that serotype Typhimurium can activate programmed macrophage cell death independently of SPI1. SPI1 independent induction of apoptosis in infected macrophages is observed as early as 12 to 13 h postinfection, even in the absence of intracellular bacterial replication. Delayed activation of programmed macrophage cell death is not observed with serotype Typhimurium strains mutated inompRor SPI2. Even though SPI2 mutants have a defect in intracellular proliferation, our results indicate that long-term intracellular survival and growth are not required for delayed macrophage killing per se, since Salmonellamutants that are severely defective in intracellular growth still induce delayed apoptosis. Inactivation of genes required for either rapid or delayed induction of apoptosis results in a conditional noncytotoxic phenotype, whereas simultaneous inactivation of genes required for both rapid and delayed induction of apoptosis renders serotype Typhimurium noncytotoxic under all conditions tested. Our hypothesis is that differential activation of programmed macrophage cell death by serotype Typhimurium occurs under discrete physiological conditions at distinct locations within an infected host.

ABSTRACTSalmonella typhimuriuminfection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimuriumgenes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1,000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimuriumgenome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonellaspp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of theSalmonellagenome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.

ABSTRACTA panel of Salmonella typhimurium14028s mutants, which were previously shown to be highly attenuated in the BALB/c mouse model of infection, were analyzed for their potential as liveSalmonellaoral-vaccine candidates. A prototypicalaroAmutant was chosen as a basis of comparison. From the panel of mutants initially chosen for this study, three mutants with comparable levels of attenuation elicited higherSalmonella-specific serum immunoglobulin G (IgG) and/or mucosal secretory-IgA antibody titers than the aroAvaccine strain. The three mutants, CL288, CL401, and CL554, also elicited a better protective immune response than the aroAcontrol strain, after a single oral dose of 1 × 109to 2 × 109bacteria.

ABSTRACTAdhesion is an important initial step during bacterial colonization of the intestinal mucosa. However, mutations in the Salmonella typhimuriumfimbrial operons lpf, pef, orfimonly moderately alter mouse virulence. The respective adhesins may thus play only a minor role during infection or S. typhimuriummay encode alternative virulence factors that can functionally compensate for their loss. To address this question, we constructed mutations in all four known fimbrial operons of S. typhimurium: fim,lpf, pef, and agf. A mutation in the agfBgene resulted in a threefold increase in the oral 50% lethal dose (LD50) of S. typhimuriumfor mice. In contrast, an S. typhimuriumstrain carrying mutations in all four fimbrial operons (quadruple mutant) had a 26-fold increased oral LD50. The quadruple mutant, but not theagfBmutant, was recovered in reduced numbers from murine fecal pellets, suggesting that a reduced ability to colonize the intestinal lumen contributed to its attenuation. These data are evidence for a synergistic action of fimbrial operons during colonization of the mouse intestine and the development of murine typhoid fever.