The Vibrio parahaemolyticus type III effector VopS is implicated in cell rounding and the collapse of the actin cytoskeleton by inhibiting Rho guanosine triphosphatases (GTPases). We found that VopS could act to covalently modify a conserved threonine residue on Rho, Rac, and Cdc42 with adenosine 5'-monophosphate (AMP). The resulting AMPylation prevented the interaction of Rho GTPases with downstream effectors, thereby inhibiting actin assembly in the infected cell. Eukaryotic proteins were also directly modified with AMP, potentially expanding the repertoire of posttranslational modifications for molecular signaling.

BackgroundThe Vibrio parahaemolyticus type III secreted effector VopS contains a fic domain that covalently modifies Rho GTPase threonine with AMP to inhibit downstream signaling events in host cells. The VopS fic domain includes a conserved sequence motif (HPFx[D/E]GN[G/K]R) that contributes to AMPylation. Fic domains are found in a variety of species, including bacteria, a few archaea, and metazoan eukaryotes.Methodology/Principal FindingsWe show that the AMPylation activity extends to a eukaryotic fic domain in Drosophila melanogaster CG9523, and use sequence and structure based computational methods to identify related domains in doc toxins and the type III effector AvrB. The conserved sequence motif that contributes to AMPylation unites fic with doc. Although AvrB lacks this motif, its structure reveals a similar topology to the fic and doc folds. AvrB binds to a peptide fragment of its host virulence target in a similar manner as fic binds peptide substrate. AvrB also orients a phosphate group from a bound ADP ligand near the peptide-binding site and in a similar position as a bound fic phosphate.Conclusions/SignificanceThe demonstrated eukaryotic fic domain AMPylation activity suggests that the VopS effector has exploited a novel host posttranslational modification. Fic domain-related structures give insight to the AMPylation active site and to the VopS fic domain interaction with its host GTPase target. These results suggest that fic, doc, and AvrB stem from a common ancestor that has evolved to AMPylate protein substrates.

Microbial pathogens use a variety of mechanisms to disrupt the actin cytoskeleton during infection. Vibrio parahaemolyticus (V. para) is a Gram-negative bacterium that causes gastroenteritis, and new pandemic strains are emerging throughout the world. Analysis of the V. para genome revealed a type III secretion system effector, VopL, encoding three Wiskott-Aldrich homology 2 domains that are interspersed with three proline-rich motifs. Infection of HeLa cells with V. para induces the formation of long actin fibers in a VopL-dependent manner. Transfection of VopL promotes the assembly of actin stress fibers. In vitro, recombinant VopL potently induces assembly of actin filaments that grow at their barbed ends, independent of eukaryotic factors. Vibrio VopL is predicted to be a bacterial virulence factor that disrupts actin homeostasis during an enteric infection of the host.actin assembly ͉ microbial pathogenesis ͉ virulence ͉ stress fibers ͉ WH2 domains

The bacterial pathogen Vibrio parahaemolyticus utilizes a type III secretion system to cause death of host cells within hours of infection. We report that cell death is completely independent of apoptosis and occurs by a mechanism in which injection of multiple type III effectors causes induction of autophagy, cell rounding, and the subsequent release of cellular contents. Autophagy is detected by the appearance of lipidated light chain 3 (LC3) and by increases in punctae and vacuole formation. Electron microscopy reveals the production of early autophagic vesicles during infection. Consistent with phosphoinositide 3 (PI3) kinase playing a role in autophagy, treatment of infected cells with a PI3 kinase inhibitor attenuates autophagy in infected cells. Because many effectors are injected during a V. parahaemolyticus infection, it is not surprising that the presence of a sole PI3 kinase inhibitor does not prevent inevitable host-cell death. Our studies reveal an infection paradigm whereby an extracellular pathogen uses its type III secretion system to cause at least three parallel events that eventually result in the proinflammatory death of an infected host cell.host-pathogen ͉ type III secretion ͉ apoptosis ͉ effector ͉ inflammatory

Trafficking of proteins and RNA into and out of the nucleus occurs through the nuclear pore complex (NPC). Due to its critical function in many cellular processes, the NPC and transport factors are common targets of several viruses that disrupt key constituents of the machinery to facilitate viral replication. Many viruses such as poliovirus and severe acute respiratory syndrome (SARS) virus inhibit protein import into the nucleus, while viruses such as influenza A virus target and disrupt host mRNA nuclear export. Current evidence indicates that these viruses may employ such strategies to avert the host immune response. Conversely, many viruses co-opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. Since viral proteins interact with key regulators of the host nuclear transport machinery, viruses have served as invaluable tools of discovery that led to the identification of novel constituents of nuclear transport pathways. In addition, this review explores the importance of nucleocytoplasmic trafficking to viral pathogenesis as these studies revealed new antiviral therapeutic strategies and exposed previously unknown cellular mechanisms. Further understanding of nuclear transport pathways will determine whether such therapeutics will be useful treatments for important human pathogens.

MicroRNAs (miRNAs) have been shown to regulate viral infection, but the miRNAs that target intracellular sensors and adaptors of innate immunity have not been fully uncovered. Here we conduct a miRNA mimic screen and validation with miRNA inhibitors in cells infected with vesicular stomatitis virus (VSV) to identify miRNAs that regulate viral-host interactions. We identify miR-576-3p as a robust regulator of infection by VSV and other RNA and DNA viruses. While a miR-576-3p mimic sensitizes cells to viral replication, inhibition of endogenous miR-576-3p prevents infection. miR-576-3p is induced by IRF3 concomitantly with interferon and targets STING, MAVS and TRAF3, which are critical factors for interferon expression. Interestingly, miR-576-3p and its binding sites are primate-specific and miR-576-3p levels are reduced in inflammatory diseases. These findings indicate that induction of miR-576-3p by IRF3 triggers a feedback mechanism to reduce interferon expression and set an antiviral response threshold to likely avoid excessive inflammation.

Recent studies have suggested that adenosine 5′-monophosphate (AMP) post-translational modification of proteins could represent a novel molecular signaling pathway. Mass spectrometric fragmentation characteristics of this modification have not previously been described and studied systematically. In this work, we therefore examined the fragmentation pattern of chemically synthesized peptides containing AMPylated Thr, Ser, and Tyr. The formation of characteristic ions and the influence of collision energy (CE) on the detection of characteristic ions and their relative peak intensity are reported. When peptide with AMPylated Ser/Thr underwent collision induced dissociation (CID), peaks at m/z 348.1, 136.1, and 250.1, fragments with AMP group attached, and fragments consistent with neutral loss of 347 Da were major characteristic ions; fragments consistent with neutral loss of 135 Da or 249 Da were weaker and not always detectable. The observations for Tyr AMPylation followed the same general patterns as those for Ser/Thr modification, with the exception that the ions detected for Tyr AMPylation did not include either the peak at m/z 348.1, or fragments with a mass shift of −347 Da. The results described in this paper highlight a series of diagnostic ions, which can be used not only to confidently identify the AMPylation site based on MS and MS/MS data, but also to selectively scan AMPylated peptides in complex protein mixtures.

Objective: To evaluate the impact of changes to urine testing orderables in computerized physician order entry (CPOE) system on urine culturing practices. Design: Retrospective before (January 2015 to April 2016) and after (May 2016 to August 2017) study. Setting: A 1,250-bed academic tertiary referral center. Patients: Hospitalized adults who had ≥1 urine culture performed during their stay. Intervention: The intervention (implemented in April 2017) consisted of notifications to providers, changes to order sets and inclusion of the new urine culture reflex tests in commonly used order sets. We compared the urine culture rates before and after intervention, adjusting for temporal trends. Results: During the study period, 18,954 inpatients (median age 62 years, 68.8% white and 52.3% female) had 24,569 urine cultures ordered. Twenty-seven percent (n=6642) of the urine cultures were positive. Urine culturing rate decreased significantly in the post-intervention period for any specimen type (38.1 pre-vs. 20.9 per 1000 patient days post-intervention, p<0.001), clean catch (30.0 vs. 18.7, p<0.001) and catheterized urine (7.8 vs. 1.9, p<0.001). Using an interrupted time series model, urine culture rates decreased for all specimen types (p<0.05). Conclusions: Our intervention of changes to order sets and inclusion of the new urine culture reflex tests resulted in a 45% reduction in the urine cultures ordered. CPOE system format plays a vital role in reducing the burden of unnecessary urine cultures and should be implemented in combination with other efforts.