A multi-phenotypic imaging screen to identify bacterial effectors by exogenous expression in a HeLa cell line

We present a high-content screen (HCS) for the simultaneous analysis of multiple phenotypes in HeLa cells expressing an autophagy reporter (mcherry-LC3) and one of 209 GFP-fused proteins from the Crohn’s Disease (CD)-associated bacterium, Adherent Invasive E. coli (AIEC) strain LF82. Using automated confocal microscopy and image analysis (CellProfiler), we localised GFP fusions within cells, and monitored their effects upon autophagy (an important innate cellular defence mechanism), cellular and nuclear morphology, and the actin cytoskeleton. This data will provide an atlas for the localisation of 209 AIEC proteins within human cells, as well as a dataset to analyse their effects upon many aspects of host cell morphology. We also describe an open-source, automated, image-analysis workflow to identify bacterial effectors and their roles via the perturbations induced in reporter cell lines when candidate effectors are exogenously expressed

Hepatitis a among men who have sex with men in Barcelona, 1989-2010: insufficient control and need for new approaches

<p>Abstract</p> <p>Background</p> <p>Men who have sex with men (MSM) are a known group at risk for hepatitis A and outbreaks among this group are frequent. In Barcelona, vaccination for MSM has been recommended since 1994. In 1998 a vaccination campaign among preadolescents was implemented and an immunization program in gay bathhouses began in 2004. Objective: to asses the incidence of hepatitis A in adults in Barcelona from 1989 to 2010 and to evaluate the outbreaks among MSM including all genotypes involved.</p> <p>Methods</p> <p>All cases of acute hepatitis A among young adults notified to the Public Health Agency of Barcelona from 1989 to 2010 were included for analyses. We calculated the annual incidence rate and the incidence ratio male-to-female (M:F) as a marker for MSM. Spearman's coefficient was used to evaluate trends. We also evaluated the outbreaks among MSM and compared their characteristics using Chi-squared and ANOVA test. Fragment amplification of the VP1/P2A region was used for genetic analysis.</p> <p>Results</p> <p>The median annual incidence for the period of study was 4.7/100000 among females and 11.7/100000 among males. The rate of hepatitis A for adult woman decreased over time (Spearman' coefficient = -0.63, <it>p </it>= 0.002), whereas there was no decrease for adult men (Spearman' coefficient = 0.097, <it>p </it>= 0.67). During the study period the M:F ratio increased (Spearman' coefficient = 0.73, <it>p </it>< 0.001).</p> <p>Three large outbreaks among MSM were detected. When comparing outbreaks, there was a decrease in the percentage of bathhouse users (from 47% to 19%, <it>p </it>= 0.0001) and sex workers (from 6.5% to 0%) while the percentage of HIV infected individuals did not change significantly (range: 21%-28%, <it>p </it>= 0.36). The isolated strains were closely related to those circulating in Europe.</p> <p>Conclusions</p> <p>Annual incidences remain high among MSM without tendency to decrease. More strategies which effectively reach the whole MSM community are needed.</p

Cardiovascular risk factors are major determinants of thrombotic risk in patients with the lupus anticoagulant

BACKGROUND: Patients with the lupus anticoagulant (LA) are at an increased risk of thrombotic events, which in turn increase the risk of death. Understanding the determinants of thrombotic risk in patients with LA may pave the way towards targeted thromboprophylaxis. In the Vienna Lupus Anticoagulant and Thrombosis Study (LATS), we systematically evaluate risk factors for thrombotic events in patients with LA. METHODS: We followed 150 patients (mean age: 41.3 years, female gender: n = 122 (81.3%), history of thrombosis or pregnancy complications: n = 111 (74.0%)), who tested repeatedly positive for LA until development of thrombosis, death, or censoring. The primary endpoint was a composite of arterial or venous thrombotic events (TEs). RESULTS: During a median follow-up of 9.5 years (range: 12 days–13.6 years) and 1076 person-years, 32 TEs occurred (arterial: n = 16, venous: n = 16; cumulative 10-year TE incidence: 24.3%). A prolonged lupus-sensitive activated partial thromboplastin time (aPTT-LA) (adjusted subdistribution hazard ratio (SHR) = 2.31, 95% CI: 1.07–-5.02), diabetes (adjusted SHR = 4.39, 95% CI: 1.42–13.57), and active smoking (adjusted SHR = 2.31, 95% CI: 1.14–5.02) emerged as independent risk factors of both arterial and venous thrombotic risk. A risk model that includes a prolonged lupus-sensitive aPTT, smoking, and diabetes enabled stratification of LA patients into subgroups with a low, intermediate, and high risk of thrombosis (5-year TE risk of 9.7% (n = 77), 30.9% (n = 51), and 56.8% (n = 22). CONCLUSIONS: Long-term thrombotic risk in patients with LA is clustered within subjects harboring typical cardiovascular risk factors in addition to a prolonged lupus-sensitive aPTT, whereas patients with none of these risk factors represent a large subgroup with a low risk of thrombosis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12916-017-0807-7) contains supplementary material, which is available to authorized users

A ribosome–nascent chain sensor of membrane protein biogenesis in Bacillus subtilis

Proteins in the YidC/Oxa1/Alb3 family have essential functions in membrane protein insertion and folding. Bacillus subtilis encodes two YidC homologs, one that is constitutively expressed (spoIIIJ/yidC1) and a second (yqjG/yidC2) that is induced in spoIIIJ mutants. Regulated induction of yidC2 allows B. subtilis to maintain capacity of the membrane protein insertion pathway. We here show that a gene located upstream of yidC2 (mifM/yqzJ) serves as a sensor of SpoIIIJ activity that regulates yidC2 translation. Decreased SpoIIIJ levels or deletion of the MifM transmembrane domain arrests mifM translation and unfolds an mRNA hairpin that otherwise blocks initiation of yidC2 translation. This regulated translational arrest and yidC2 induction require a specific interaction between the MifM C-terminus and the ribosomal polypeptide exit tunnel. MifM therefore acts as a ribosome–nascent chain complex rather than as a fully synthesized protein. B. subtilis MifM and the previously described secretion monitor SecM in Escherichia coli thereby provide examples of the parallel evolution of two regulatory nascent chains that monitor different protein export pathways by a shared molecular mechanism

MPIase is a glycolipozyme essential for membrane protein integration

Protein integration into biological membranes is a vital cellular event for all organisms. We previously reported an integration factor in the inner membrane of Escherichia coli, named MPIase (membrane protein integrase). Here we show that in contrast to previously identified integration factors that are proteins, MPIase is a glycolipid composed of diacylglycerol and a glycan chain of three acetylated aminosugars linked through pyrophosphate. Hydrolytic removal of the lipid moiety gives a soluble product with higher integration activity than that of the original MPIase. This soluble form of MPIase directly interacts with a newborn membrane protein, maintaining its integration-competent structure and allowing its post-translational integration. MPIase actively drives protein integration following chaperoning membrane proteins. We further demonstrate with anti-MPIase antibodies that MPIase is likely involved in integration in vivo. Collectively, our results suggest that MPIase, essential for membrane protein integration, is to our knowledge the first glycolipid with an enzyme-like activity

Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle

The YidC insertase also integrates multispanning membrane proteins that had been considered to be exclusively SecYEG dependent. Only membrane proteins that require SecA can be inserted only via SecYEG. Targeting to YidC is SRP dependent, and the C-terminus of YidC cross-links to SRP, FtsY, and ribosomal subunits