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To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL) effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR) model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone.

Protein–protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas‐mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome‐wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases. Synopsis The response of 17,991 protein interactions to Fas‐mediated apoptosis is monitored using quantitative proteomics (PCP‐SILAC). Initiation of apoptosis leads to dramatic changes in the interactions of caspase targets, independently of caspase cleavage. Quantitative proteomics is used to analyze mitochondrial and cytosolic interactomes in response to apoptosis initiation. The dynamic interactions of 17,991 interactions among 2,779 proteins are measured. Interactome alterations occur before and independently of caspase activity. Graphical Abstract The response of 17,991 protein interactions to Fas‐mediated apoptosis is monitored using quantitative proteomics (PCP‐SILAC). Initiation of apoptosis leads to dramatic changes in the interactions of caspase targets, independently of caspase cleavage.

3‐phosphorylated phosphoinositides (3‐PtdIns) orchestrate endocytic trafficking pathways exploited by intracellular pathogens such as Salmonella to gain entry into the cell. To infect the host, Salmonellae subvert its normal macropinocytic activity, manipulating the process to generate an intracellular replicative niche. Disruption of the PtdIns(5) kinase, PIKfyve, be it by interfering mutant, siRNA‐mediated knockdown or pharmacological means, inhibits the intracellular replication of Salmonella enterica serovar typhimurium in epithelial cells. Monitoring the dynamics of macropinocytosis by time‐lapse 3D (4D) videomicroscopy revealed a new and essential role for PI(3,5)P 2 in macropinosome‐late endosome/lysosome fusion, which is distinct from that of the small GTPase Rab7. This PI(3,5)P 2 ‐dependent step is required for the proper maturation of the Salmonella ‐containing vacuole (SCV) through the formation of Salmonella ‐induced filaments (SIFs) and for the engagement of the Salmonella pathogenicity island 2‐encoded type 3 secretion system (SPI2‐T3SS). Finally, although inhibition of PIKfyve in macrophages did inhibit Salmonella replication, it also appears to disrupt the macrophage's bactericidal response.

Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes disease in mice that resembles human typhoid. Typhoid pathogenesis consists of distinct phases in the intestine and a subsequent systemic phase in which bacteria replicate in macrophages of the liver and spleen. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is a major virulence factor contributing to the systemic phase of typhoid pathogenesis. Understanding how pathogens regulate virulence mechanisms in response to the environment, including different host tissues, is key to our understanding of pathogenesis. A recombinase-based in vivo expression technology system was developed to assess SPI-2 expression during murine typhoid. SPI-2 expression was detectable at very early times in bacteria that were resident in the lumen of the ileum and was independent of active bacterial invasion of the epithelium. We also provide direct evidence for the regulation of SPI-2 by the Salmonella transcription factors ompR and ssrB in vivo. Together these results demonstrate that SPI-2 expression precedes penetration of the intestinal epithelium. This induction of expression precedes any documented SPI-2-dependent phases of typhoid and may be involved in preparing Salmonella to successfully resist the antimicrobial environment encountered within macrophages.

Although O157:H7 Shiga toxin–producing Escherichia coli (STEC) are the predominant cause of hemolytic-uremic syndrome (HUS) in the world, non-O157:H7 serotypes are a medically important cause of HUS that are underdetected by current diagnostic approaches. Because Shiga toxin is necessary but not sufficient to cause HUS, identifying the virulence determinants that predict severe disease after non-O157 STEC infection is of paramount importance. Disease caused by O157:H7 STEC has been associated with a 26-gene pathogenicity island known as O island (OI) 122. To assess the public-health significance of this pathogenicity island, we examined the association between OI122 genes and outbreaks and HUS after non-O157 STEC infection. We found that a subset of OI122 genes is independently associated with outbreaks and HUS after infection with non-O157 STEC. The presence of multiple virulence genes in non-O157 serotypes strengthened this association, which suggests that the additive effects of a variable repertoire of virulence genes contribute to disease severity. In vivo, Citrobacter rodentium mutants lacking outbreak- and HUS-associated genes were deficient for virulence in mice; in particular, nleB mutant bacteria were unable to cause mortality in mice. The present study shows that virulence genes associated epidemiologically with outbreaks and HUS after non-O157 STEC infection are pivotal to the initiation, progression, and outcome of in vivo disease

The evolution of pathogens presents a paradox. Pathogenic species are often absolutely dependent on their host species for their propagation through evolutionary time, yet the pathogenic lifestyle requires that the host be damaged during this dependence. It is clear that pathogenic strategies are successful in evolutionary terms because a diverse array of pathogens exists in nature. Pathogens also evolve using a broad range of molecular mechanisms to acquire and modulate existing virulence traits in order to achieve this success. Detailing the benefit of enhanced selection derived through virulence and understanding the mechanisms through which virulence evolves are important to understanding the natural world and both have implications for human health.

The transcription factors HilA and SsrB activate expression of two type III secretion systems (T3SSs) and cognate effectors that reprogram host cell functions to benefit infecting Salmonella in the host. These transcription factors, the secretion systems, and the effectors are all encoded by horizontally acquired genes. Using quantitative proteomics, we quantified the abundance of 2,149 proteins from hilA or ssrB Salmonella in vitro . Our results suggest that the HilA regulon does not extend significantly beyond proteins known to be involved in direct interactions with intestinal epithelium. On the other hand, SsrB influences the expression of a diverse range of proteins, many of which are ancestral to the acquisition of ssrB . In addition to the known regulon of T3SS-related proteins, we show that, through SodCI and bacterioferritin, SsrB controls resistance to reactive oxygen species and that SsrB down-regulates flagella and motility. This indicates that SsrB-controlled proteins not only redirect host cell membrane traffic to establish a supportive niche within host cells but also have adapted to the chemistry and physical constraints of that niche. IMPORTANCE Expression of T3SSs typically requires a transcription factor that is linked in a genomic island. Studies of the targets of HilA and SsrB have focused on almost exclusively on T3SS substrates that are either linked or encoded in distinct genomic islands. By broadening our focus, we found that the regulon of SsrB extended considerably beyond T3SS-2 and its substrates, while that of HilA did not. That at least two SsrB-regulated processes streamline existence in the intracellular niche afforded by T3SS-2 seems to be a predictable outcome of evolution and natural selection. However, and importantly, these are the first such functions to be implicated as being SsrB dependent. The concept of T3SS-associated transcription factors coordinating manipulations of host cells together with distinct bacterial processes for increased efficiency has unrealized implications for numerous host-pathogen systems. Expression of T3SSs typically requires a transcription factor that is linked in a genomic island. Studies of the targets of HilA and SsrB have focused on almost exclusively on T3SS substrates that are either linked or encoded in distinct genomic islands. By broadening our focus, we found that the regulon of SsrB extended considerably beyond T3SS-2 and its substrates, while that of HilA did not. That at least two SsrB-regulated processes streamline existence in the intracellular niche afforded by T3SS-2 seems to be a predictable outcome of evolution and natural selection. However, and importantly, these are the first such functions to be implicated as being SsrB dependent. The concept of T3SS-associated transcription factors coordinating manipulations of host cells together with distinct bacterial processes for increased efficiency has unrealized implications for numerous host-pathogen systems.

Salmonella enterica relies on a type III secretion system encoded in Salmonella pathogenicity island-2 (SPI-2) to survive and replicate within macrophages at systemic sites during typhoid. SPI-2 virulence is induced upon entry into macrophages, but the mechanisms of SPI-2 gene control in vivo remain unclear, particularly with regard to negative regulators that control the contextual activation of SPI-2. Here, we identified and characterized YdgT as a negative modulator of the SPI-2 pathogenicity island and established that this negative regulation is central to systemic patho-genesis because ydgT mutants overexpressing typhoid virulence genes were ultimately attenuated during infection. ydgT mutants displayed a biphasic virulence phenotype during in vivo competitive infections that consisted of an early \"gain-of-virulence\" dependent on SPI-2 activation, followed by attenuation later in infection indicating that proper contextual regulation of SPI-2 by YdgT is necessary for full virulence during systemic colonization. These data suggest that overexpression of virulence-associated type III secretion genes can have an adverse effect on bacterial pathogenesis in vivo.

Burkholderia pseudomallei is a recognized biothreat agent and the causative agent of melioidosis. This Gram-negative bacterium exists as a soil saprophyte in melioidosis-endemic areas of the world and accounts for 20% of community-acquired septicaemias in northeastern Thailand where half of those affected die. Here we report the complete genome of B. pseudomallei, which is composed of two chromosomes of 4.07 megabase pairs and 3.17 megabase pairs, showing significant functional partitioning of genes between them. The large chromosome encodes many of the core functions associated with central metabolism and cell growth, whereas the small chromosome carries more accessory functions associated with adaptation and survival in different niches. Genomic comparisons with closely and more distantly related bacteria revealed a greater level of gene order conservation and a greater number of orthologous genes on the large chromosome, suggesting that the two replicons have distinct evolutionary origins. A striking feature of the genome was the presence of 16 genomic islands (GIs) that together made up 6.1% of the genome. Further analysis revealed these islands to be variably present in a collection of invasive and soil isolates but entirely absent from the clonally related organism B. mallei. We propose that variable horizontal gene acquisition by B. pseudomallei is an important feature of recent genetic evolution and that this has resulted in a genetically diverse pathogenic species.