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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 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.

The pathogen Salmonella entericais known to cause both food poisoning and typhoid fever. Because of the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g.contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study S. entericaserovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal environment (within which Salmonellaare known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified and quantified 5,163 peptides originating from 682 proteins, and the data clearly indicated that compared with Salmonellacultured in the rich medium, cells cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonellafrom the same cultures were also analyzed using traditional, bottom-up proteomic methods, and when the peptidomics and proteomics data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonellaare discussed.

Salmonella entericaserovar Typhimurium (also known as Salmonella typhimurium) is a facultative intracellular pathogen that causes ∼8,000 reported cases of acute gastroenteritis and diarrhea each year in the United States. Although many successful physiological, biochemical, and genetic approaches have been taken to determine the key virulence determinants encoded by this organism, the sheer number of uncharacterized reading frames observed within the S. entericagenome suggests that many more virulence factors remain to be discovered. We used a liquid chromatography-mass spectrometry-based “bottom-up” proteomic approach to generate a more complete picture of the gene products that S. typhimuriumsynthesizes under typical laboratory conditions as well as in culture media that are known to induce expression of virulence genes. When grown to logarithmic phase in rich medium, S. typhimuriumis known to express many genes that are required for invasion of epithelial cells. Conversely stationary phase cultures of S. typhimuriumexpress genes that are needed for both systemic infection and growth within infected macrophages. Lastly bacteria grown in an acidic, magnesium-depleted minimal medium (MgM) designed to mimic the phagocytic vacuole have been shown to up-regulate virulence gene expression. Initial comparisons of protein abundances from bacteria grown under each of these conditions indicated that the majority of proteins do not change significantly. However, we observed subsets of proteins whose expression was largely restricted to one of the three culture conditions. For example, cells grown in MgM had a higher abundance of Mg2+transport proteins than found in other growth conditions. A second more virulent S. typhimuriumstrain (14028) was also cultured under these same growth conditions, and the results were directly compared with those obtained for strain LT2. This comparison offered a unique opportunity to contrast protein populations in these closely related bacteria. Among a number of proteins displaying a higher abundance in strain 14028 were the products of the pduoperon, which encodes enzymes required for propanediol utilization. These pduoperon proteins were validated in culture and during macrophage infection. Our work provides further support for earlier observations that suggest pdugene expression contributes to S. typhimuriumpathogenesis.

Pseudomonas aeruginosaelastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-termP. aeruginosacultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosastrain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa(i.e.elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.

ABSTRACTKaposi sarcoma (KS) herpesvirus (KSHV) infection of endothelial cells (EC) is associated with strong induction of heme oxygenase-1(HO-1), a stress-inducible host gene that encodes the rate-limiting enzyme responsible for heme catabolism. KS is an angioproliferative tumor characterized by the proliferation of KSHV-infected spindle cells, and HO-1 is highly expressed in such cells. HO-1 converts the pro-oxidant, proinflammatory heme molecule into metabolites with antioxidant, anti-inflammatory, and proliferative activities. Previously published work has shown that KSHV-infected EC in vitroproliferate in response to free heme in a HO-1-dependent manner, thus implicating virus-enhanced HO-1 activity in KS tumorigenesis. The present study investigated the molecular mechanisms underlying KSHV induction of HO-1 in lymphatic EC (LEC), which are the likely spindle cell precursors. In a time course analysis of KSHV-infected cells, HO-1 expression displays biphasic kinetics characterized by an early transient induction that is followed by a more sustained upregulation coincident with the establishment of viral latency. A viral microRNA miR-K12-11 deletion mutant of KSHV was found to be defective for induction of HO-1 during latency. A potential mechanism for this phenotype was provided by BACH1, a cellular HO-1transcriptional repressor targeted by miR-K12-11. In fact, in KSHV-infected LEC, the BACH1message level is reduced, BACH1 subcellular localization is altered, and miR-K12-11 mediates the inverse regulation of HO-1and BACH1during viral latency. Interestingly, the data indicate that neither miR-K12-11 nor de novoKSHV gene expression is required for the burst of HO-1 expression observed at early times postinfection, which suggests that additional virion components promote this phenotype.IMPORTANCEWhile the mechanisms underlying KSHV induction of HO-1 remain unknown, the cellular mechanisms that regulate HO-1 expression have been extensively investigated in the context of basal and pathophysiological states. The detoxifying action of HO-1 is critical for the protection of cells exposed to high heme levels. KS spindle cells are erythrophagocytic and contain erythrocyte ghosts. Erythrocyte degeneration leads to the localized release of heme, creating oxidative stress that may be further exacerbated by environmental or other cofactors. Our previous work showed that KSHV-infected cells proliferate in response to heme and that this occurs in a HO-1-dependent manner. We therefore hypothesize that KSHV induction of HO-1 contributes to KS tumor development via heme metabolism and propose that HO-1 be evaluated as a therapeutic target for KS. Our present work, which aimed to understand the mechanisms whereby KSHV induces HO-1, will be important for the design and implementation of such a strategy.