Modulation of host phosphatidylinositol phosphates by salmonella effector protein SOPB

Salmonella spp. are gram negative bacteria capable of infecting a number of eukaryotic hosts. In humans, Salmonella infection can range anywhere from acute gastroenteritis to typhoid fever which can oftentimes be fatal.Salmonella are facultative intracellular pathogens that have acquired the ability to enter non-phagocytic cells such as those lining the intestinal epithelium. Uptake into epithelial cells is mediated by the Salmonellapathogenicity island 1 (SPI1) encoded type III secretion system (T3SS), a needle-like complex composed of over 20 proteins that translocates effector proteins directly into the host cell cytosol. Salmonella possess a second type III secretion system encoded on Salmonella pathogenicity island 2 (SPI2) that secretes effector proteins involved in Salmonella containing vacuole (SCV) formation, maturation, and intracellular survival. ^ Entrance into epithelial cells is modulated by effector proteins SopE, SopE2 and SopB. SopE and SopE2 having 69% sequence identity are known to be guanine nucleotide exchange factors (GEFs). SopE is a GEF capable of activating both Rac1 and Cdc42 while SopE2 activates Cdc42 only. SopB is a known phosphatidylinositol phosphate phosphatase possessing both 4- and 5-phosphatase activities promoting membrane ruffling and invasion. The mechanisms through which SopB utilizes its phosphatase activities to mediate membrane ruffling and invasion are still unclear. Previous research has demonstrated that SopB is responsible for the production of PtdIns(3)P at the Salmonella induced ruffles, but the mechanism through which SopB acts is still not understood. This work will directly link the phosphatase activity of SopB, demonstrating the requirement of both the 4- phosphatase and 5-phosphatase activities, to ruffle formation and invasion. We found that the 5-phosphatase activity is responsible for generating PtdIns(3,4)P2 which recruits host SNX9, a protein involved in actin modulation to the plasma membrane. The 4-phosphatase activity of SopB is solely responsible for the hydrolysis of host PtdIns(3,4)P2 into PtdIns(3)P which accumulates around the ruffles and becomes incorporated into the forming SCV membrane. Either activity alone does not result in ruffling or invasion, but when acting in conjunction with one another, the 4-phosphatase and 5-phosphatase activities of SopB lead to SNX9- mediated ruffling and Salmonella invasion

Association of ARRDC3 and NFIA variants with bovine congestive heart failure in feedlot cattle [version 2; peer review: 1 approved, 2 approved with reservations]

Background Bovine congestive heart failure (BCHF) has become increasingly prevalent among feedlot cattle in the Western Great Plains of North America with up to 7% mortality in affected herds. BCHF is an untreatable complex condition involving pulmonary hypertension that culminates in right ventricular failure and death. Genes associated with BCHF in feedlot cattle have not been previously identified. Our aim was to search for genomic regions associated with this disease. Methods A retrospective, matched case-control design with 102 clinical BCHF cases and their unaffected pen mates was used in a genome-wide association study. Paired nominal data from approximately 560,000 filtered single nucleotide polymorphisms (SNPs) were analyzed with McNemar’s test. Results Two independent genomic regions were identified as having the most significant association with BCHF: the arrestin domain-containing protein 3 gene (ARRDC3), and the nuclear factor IA gene (NFIA, mid-p-values, 1x10−8 and 2x10 −7, respectively). Animals with two copies of risk alleles at either gene were approximately eight-fold more likely to have BCHF than their matched pen mates with either one or zero risk alleles at both genes (CI 95 = 3-17). Further, animals with two copies of risk alleles at both genes were 28-fold more likely to have BCHF than all others (p-value = 1×10−7, CI95 = 4-206). A missense variant in ARRDC3 (C182Y) represents a potential functional variant since the C182 codon is conserved among all other jawed vertebrate species observed. A two-SNP test with markers in both genes showed 29% of 273 BCHF cases had homozygous risk genotypes in both genes, compared to 2.5% in 198 similar unaffected feedlot cattle. This and other DNA tests may be useful for identifying feedlot animals with the highest risk for BCHF in the environments described here. Conclusions Although pathogenic roles for variants in the ARRDC3 and NFIA genes are unknown, their discovery facilitates classifying animals by genetic risk and allows cattle producers to make informed decisions for selective breeding and animal health management

The EHEC Type III Effector NleL Is an E3 Ubiquitin Ligase That Modulates Pedestal Formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.National Institutes of Health (U.S.) (grant AI078092)National Institutes of Health (U.S.) (grant AI068655

Modulation of host phosphatidylinositol phosphates by salmonella effector protein SOPB

Salmonella spp. are gram negative bacteria capable of infecting a number of eukaryotic hosts. In humans, Salmonella infection can range anywhere from acute gastroenteritis to typhoid fever which can oftentimes be fatal. Salmonella are facultative intracellular pathogens that have acquired the ability to enter non-phagocytic cells such as those lining the intestinal epithelium. Uptake into epithelial cells is mediated by the Salmonella pathogenicity island 1 (SPI1) encoded type III secretion system (T3SS), a needle-like complex composed of over 20 proteins that translocates effector proteins directly into the host cell cytosol. Salmonella possess a second type III secretion system encoded on Salmonella pathogenicity island 2 (SPI2) that secretes effector proteins involved in Salmonella containing vacuole (SCV) formation, maturation, and intracellular survival. Entrance into epithelial cells is modulated by effector proteins SopE, SopE2 and SopB. SopE and SopE2 having 69% sequence identity are known to be guanine nucleotide exchange factors (GEFs). SopE is a GEF capable of activating both Rac1 and Cdc42 while SopE2 activates Cdc42 only. SopB is a known phosphatidylinositol phosphate phosphatase possessing both 4- and 5-phosphatase activities promoting membrane ruffling and invasion. The mechanisms through which SopB utilizes its phosphatase activities to mediate membrane ruffling and invasion are still unclear. Previous research has demonstrated that SopB is responsible for the production of PtdIns(3)P at the Salmonella induced ruffles, but the mechanism through which SopB acts is still not understood. This work will directly link the phosphatase activity of SopB, demonstrating the requirement of both the 4- phosphatase and 5-phosphatase activities, to ruffle formation and invasion. We found that the 5-phosphatase activity is responsible for generating PtdIns(3,4)P2 which recruits host SNX9, a protein involved in actin modulation to the plasma membrane. The 4-phosphatase activity of SopB is solely responsible for the hydrolysis of host PtdIns(3,4)P2 into PtdIns(3)P which accumulates around the ruffles and becomes incorporated into the forming SCV membrane. Either activity alone does not result in ruffling or invasion, but when acting in conjunction with one another, the 4-phosphatase and 5-phosphatase activities of SopB lead to SNX9- mediated ruffling and Salmonella invasion

Contribution of <i>nleL</i> to <i>C. rodentium</i> virulence.

<p>(<b>A</b>) <i>C. rodentium</i> NleL is an E3 ubiquitin ligase. Combinations of ATP, ubiquitin, E1, UbcH5a and GST-NleL or GST-NleL^C753S were incubated at 35°C for 90 min, and the Western blot was performed using polyclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom). (<b>B)</b> Fecal bacteria shedding of the wild-type strain (DBS130), the <i>nleL</i> null mutant strain, the <i>nleL</i> mutant strain expressing the wild-type NleL, or the <i>nleL</i> mutant strain complemented with the catalytically-dead NleL^C753S in <i>C. rodentium</i>. (<b>C</b>) Virulence of <i>C. rodentium</i> strains as indicated by weight of the mice post-inoculation. The percent weight change of the mice over the 13 days post-inoculation was measured. The error bars indicate standard errors. Mice infected with <i>nleL</i> deletion strain (DBS792) had significantly reduced weight loss when compared to the wild type (DBS130). This could be partially rescued by plasmid expressing the full length NleL (DBS793) but not by plasmid expressing the catalytically inactive NleL^C753S mutant (DBS794). (<b>D</b>) Contribution of <i>nleL</i> to mouse colitis caused by <i>C. rodentium</i>. Pathology of mouse colon by different <i>C. rodentium</i> strains, as indicated by histologic activity index (sum of lesion scores). At day 13 post-inoculation colon tissue was scored for lesions: inflammation, edema, epithelial defects, crypt atrophy, hyperplasia, and dysplasia. Mice infected with <i>nleL</i> deletion strain showed significantly reduced lesions when compared to the wild type (P<0.05). This could be rescued by plasmid expressing the full length NleL but not by plasmid expressing the catalytically inactive NleL^C753S mutant (P<0.01).</p

Secretion of Tir and translocation of NleL into HeLa cells.

<p>(<b>A</b>) The expression and secretion levels of Tir in either the wild-type EHEC or the mutant EHEC expressing the chromosomal catalytically-dead mutant NleL^C753A was examined by Western blot. (<b>B</b>) Intracellular cAMP levels are an indication of the translocation of CyaA fusion proteins in EHEC WT (ZP250) and EHEC TTSS deficient mutant (<i>escF</i>; ZP2251) strains. Cells were infected for 4 hrs, and the adenylate cyclase activity was determined. The data were from three independent experiments, with standard deviations shown as error bars. cAMP values are presented as pmol per milligram of total cellular protein. The expression levels of the NleL-CyaA fusions were found to be similar by Western blot as shown on the bottom panel.</p

The E3 ligase activity of EHEC NleL down modulates EPEC pedestal formation.

<p>HeLa cells were infected at a multiplicity of infection of 100 with wild type EPEC, or EPEC harboring plasmid expressing wild-type EHEC NleL or NleL^C753A. Cells were infected for 4 hours. (<b>A</b>) Bacteria were visualized by staining with an anti-EPEC LPS antibody (green). Actin was detected with a Texas Red Phalloidin (red). (<b>B</b>) The number of micro-clusters of pedestals on HeLa cells were counted and were grouped into clusters having 1–10 pedestals, 11–20 pedestals and >20 pedestals. Quantitative analysis includes three independent experiments. A minimum of 300 cells were counted from each experiment with standard deviation shown as error bars.</p

Self-ubiquitination of GST-NleL.

<p>(<b>A</b>) NleL-mediated self-ubiquitination requires ATP, ubiquitin, E1 and E2. Combinations of ATP, ubiquitin, E1, UbcH5a and GST-NleL^59–782 were incubated at 35°C for 90 min, and the Western blot was performed using polyclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom). (<b>B</b>) NleL C753 residue is required for its E3 ubiquitin ligase activity. Reactions containing ubiquitin, E1 and UbcH5a were incubated with the wild type GST-NleL, GST-NleL^C688S or GST-NleL^C753S or GST-NleL^C753A at 35°C for 90 min. The Western blot was performed using monoclonal anti-ubiquitin antibodies (top) or anti-GST antibodies (bottom).</p