Bacteria swim by means of rotating flagella that are powered by ion influx through membrane-spanning motor complexes. Escherichia coli and related species harness a chemosensory and signal transduction machinery that governs the direction of flagellar rotation and allows them to navigate in chemical gradients. Here, we show that Escherichia coli can also fine-tune its swimming speed with the help of a molecular brake (YcgR) that, upon binding of the nucleotide second messenger cyclic di-GMP, interacts with the motor protein MotA to curb flagellar motor output. Swimming velocity is controlled by the synergistic action of at least five signaling proteins that adjust the cellular concentration of cyclic di-GMP. Activation of this network and the resulting deceleration coincide with nutrient depletion and might represent an adaptation to starvation. These experiments demonstrate that bacteria can modulate flagellar motor output and thus swimming velocity in response to environmental cues.

During infection by invasive bacteria, epithelial cells contribute to innate immunity via the local secretion of inflammatory cytokines. These are directly produced by infected cells or by uninfected bystanders via connexin-dependent cell-cell communication. However, the cellular pathways underlying this process remain largely unknown. Here we perform a genome-wide RNA interference screen and identify TIFA and TRAF6 as central players of Shigella flexneri and Salmonella typhimurium-induced interleukin-8 expression. We show that threonine 9 and the forkhead-associated domain of TIFA are necessary for the oligomerization of TIFA in both infected and bystander cells. Subsequently, this process triggers TRAF6 oligomerization and NF-κB activation. We demonstrate that TIFA/TRAF6-dependent cytokine expression is induced by the bacterial metabolite heptose-1,7-bisphosphate (HBP). In addition, we identify alpha-kinase 1 (ALPK1) as the critical kinase responsible for TIFA oligomerization and IL-8 expression in response to infection with S. flexneri and S. typhimurium but also to Neisseria meningitidis. Altogether, these results clearly show that ALPK1 is a master regulator of innate immunity against both invasive and extracellular gram-negative bacteria.

A close relationship exists between regional myocardial blood flow (RMBF) and function during acute coronary inflow restriction (perfusion-contraction matching). However, the relationship of flow and function during coronary microvascular obstruction is unknown. In 12 anesthetized dogs, the left circumflex coronary artery was perfused from an extracorporeal circuit. After control measurements, 3,000 microspheres (42 micrometer diameter) per milliliter per minute inflow were injected to cause a microembolism (ME, n = 6). With unchanged systemic hemodynamics and RMBF, posterior systolic wall thickening (PWT) decreased from 19.8 +/- 1.9% SD at control to 13.3 +/- 4.0, 10.3 +/- 3.8, and 6.9 +/- 4.7% (P < 0.05 vs. control) at 1, 4, and 8 h, respectively. For comparison, inflow was progressively reduced to match PWT to that of the ME group at 1, 4, and 8 h (stenosis, STE, n = 6). RMBF in the STE group was reduced in proportion to PWT. Infarct size was not different among groups (6.5 +/- 4.5 vs. 3.4 +/- 3.2%). However, the number of leukocytes infiltrating the area at risk was significantly greater in the ME group than in the STE group. Coronary microembolization results in perfusion-contraction mismatch and is associated with an inflammatory response.

The enteroinvasive bacterium Shigella flexneri uses multiple secreted effector proteins to downregulate interleukin-8 (IL-8) expression in infected epithelial cells. Yet, massive IL-8 secretion is observed in Shigellosis. Here we report a host mechanism of cell-cell communication that circumvents the effector proteins and strongly amplifies IL-8 expression during bacterial infection. By monitoring proinflammatory signals at the single-cell level, we found that the activation of the transcription factor NF-κB and the MAP kinases JNK, ERK, and p38 rapidly propagated from infected to uninfected adjacent cells, leading to IL-8 production by uninfected bystander cells. Bystander IL-8 production was also observed during Listeria monocytogenes and Salmonella typhimurium infection. This response could be triggered by recognition of peptidoglycan and is mediated by gap junctions. Thus, we have identified a mechanism of cell-cell communication that amplifies innate immunity against bacterial infection by rapidly spreading proinflammatory signals via gap junctions to yet uninfected cells.

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BackgroundLarge-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries.ResultsWe show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied.ConclusionsOur results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1162) contains supplementary material, which is available to authorized users.

The enteroinvasive bacterium Shigella flexneri invades the intestinal epithelium of humans. During infection, several injected effector proteins promote bacterial internalization, and interfere with multiple host cell responses. To obtain a systems-level overview of host signaling during infection, we analyzed the global dynamics of protein phosphorylation by liquid chromatography-tandem MS and identified several hundred of proteins undergoing a phosphorylation change during the first hours of infection. Functional bioinformatic analysis revealed that they were mostly related to the cytoskeleton, transcription, signal transduction, and cell cycle. Fuzzy c-means clustering identified six temporal profiles of phosphorylation and a functional module composed of ATM-phosphorylated proteins related to genotoxic stress. Pathway enrichment analysis defined mTOR as the most overrepresented pathway. We showed that mTOR complex 1 and 2 were required for S6 kinase and AKT activation, respectively. Comparison with a published phosphoproteome of Salmonella typhimuriuminfected cells revealed a large subset of coregulated phosphoproteins. Finally, we showed that S. flexneri effector OspF affected the phosphorylation of several hundred proteins, thereby demonstrating the wide-reaching impact of a single bacterial effector on the host signaling network. Molecular & Cellular

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