Functional dissection of translocon proteins of the Salmonella Pathogenicity Island 2-encoded type III secretion system

<p>Abstract</p> <p>Background</p> <p>Type III secretion systems (T3SS) are essential virulence factors of most Gram-negative bacterial pathogens. T3SS deliver effector proteins directly into the cytoplasm of eukaryotic target cells and for this function, the insertion of a subset of T3SS proteins into the target cell membrane is important. These proteins form hetero-oligomeric pores acting as translocon for the delivery of effector proteins. <it>Salmonella enterica </it>is a facultative intracellular pathogen that uses the <it>Salmonella </it>Pathogenicity Island 2 (SPI2)-encoded T3SS to manipulate host cells in order to survive and proliferate within the <it>Salmonella</it>-containing vacuole of host cells. Previous work showed that SPI2-encoded SseB, SseC and SseD act to form the translocon of the SPI2-T3SS.</p> <p>Results</p> <p>Here we investigated the structural requirements of SseB and SseD to form a functional translocon. Based on bioinformatic predictions, deletional analyses of SseB and SseD were performed and the effect on secretion by the T3SS, formation of a translocon, translocation of effector proteins and intracellular replication was investigated. Our data showed that both SseB and SseD are very sensitive towards alterations of the primary structure of the proteins. Although proteins encoded by mutant alleles were still secreted, we observed that all mutations resulted in a loss of function of the SPI2-T3SS.</p> <p>Conclusion</p> <p>These observations indicate that translocon proteins of the SPI2-T3SS are highly evolved towards the formation of multi-subunit complex in the host cell membrane. Structural alterations are not tolerated and abrogate translocon function.</p

ATHENA Research Book

The ATHENA European University is an alliance of nine Higher Education Institutions with the mission of fostering excellence in research and innovation by facilitating international cooperation. The ATHENA acronym stands for Advanced Technologies in Higher Education Alliance. The partner institutions are from France, Germany, Greece, Italy, Lithuania, Portugal, and Slovenia: the University of Orléans, the University of Siegen, the Hellenic Mediterranean University, the Niccolò Cusano University, the Vilnius Gediminas Technical University, the Polytechnic Institute of Porto, and the University of Maribor. In 2022 institutions from Poland and Spain joined the alliance: the Maria Curie-Skłodowska University and the University of Vigo. This research book presents a selection of the ATHENA university partners' research activities. It incorporates peer-reviewed original articles, reprints and student contributions. The ATHENA Research Book provides a platform that promotes joint and interdisciplinary research projects of both advanced and early-career researchers

A rigorous benchmarking of methods for SARS-CoV-2 lineage abundance estimation in wastewater

In light of the continuous transmission and evolution of SARS-CoV-2 coupled with a significant decline in clinical testing, there is a pressing need for scalable, cost-effective, long-term, passive surveillance tools to effectively monitor viral variants circulating in the population. Wastewater genomic surveillance of SARS-CoV-2 has arrived as an alternative to clinical genomic surveillance, allowing to continuously monitor the prevalence of viral lineages in communities of various size at a fraction of the time, cost, and logistic effort and serving as an early warning system for emerging variants, critical for developed communities and especially for underserved ones. Importantly, lineage prevalence estimates obtained with this approach aren't distorted by biases related to clinical testing accessibility and participation. However, the relative performance of bioinformatics methods used to measure relative lineage abundances from wastewater sequencing data is unknown, preventing both the research community and public health authorities from making informed decisions regarding computational tool selection. Here, we perform comprehensive benchmarking of 18 bioinformatics methods for estimating the relative abundance of SARS-CoV-2 (sub)lineages in wastewater by using data from 36 in vitro mixtures of synthetic lineage and sublineage genomes. In addition, we use simulated data from 78 mixtures of lineages and sublineages co-occurring in the clinical setting with proportions mirroring their prevalence ratios observed in real data. Importantly, we investigate how the accuracy of the evaluated methods is impacted by the sequencing technology used, the associated error rate, the read length, read depth, but also by the exposure of the synthetic RNA mixtures to wastewater, with the goal of capturing the effects induced by the wastewater matrix, including RNA fragmentation and degradation.Comment: For correspondence: [email protected]

SARS-CoV-2 Wastewater Genomic Surveillance: Approaches, Challenges, and Opportunities

During the SARS-CoV-2 pandemic, wastewater-based genomic surveillance (WWGS) emerged as an efficient viral surveillance tool that takes into account asymptomatic cases and can identify known and novel mutations and offers the opportunity to assign known virus lineages based on the detected mutations profiles. WWGS can also hint towards novel or cryptic lineages, but it is difficult to clearly identify and define novel lineages from wastewater (WW) alone. While WWGS has significant advantages in monitoring SARS-CoV-2 viral spread, technical challenges remain, including poor sequencing coverage and quality due to viral RNA degradation. As a result, the viral RNAs in wastewater have low concentrations and are often fragmented, making sequencing difficult. WWGS analysis requires advanced computational tools that are yet to be developed and benchmarked. The existing bioinformatics tools used to analyze wastewater sequencing data are often based on previously developed methods for quantifying the expression of transcripts or viral diversity. Those methods were not developed for wastewater sequencing data specifically, and are not optimized to address unique challenges associated with wastewater. While specialized tools for analysis of wastewater sequencing data have also been developed recently, it remains to be seen how they will perform given the ongoing evolution of SARS-CoV-2 and the decline in testing and patient-based genomic surveillance. Here, we discuss opportunities and challenges associated with WWGS, including sample preparation, sequencing technology, and bioinformatics methods.Comment: V Munteanu and M Saldana contributed equally to this work A Smith and S Mangul jointly supervised this work For correspondence: [email protected]

Divergent Roles of Salmonella Pathogenicity Island 2 and Metabolic Traits during Interaction of S. enterica Serovar Typhimurium with Host Cells

The molecular mechanisms of virulence of the gastrointestinal pathogen Salmonella enterica are commonly studied using cell culture models of infection. In this work, we performed a direct comparison of the interaction of S. enterica serovar Typhimurium (S. Typhimurium) with the non-polarized epithelial cell line HeLa, the polarized cell lines CaCo2, T84 and MDCK, and macrophage-like RAW264.7 cells. The ability of S. Typhimurium wild-type and previously characterized auxotrophic mutant strains to enter host cells, survive and proliferate within mammalian cells and deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) was quantified. We found that the entry of S. Typhimurium into polarized cells was much more efficient than entry into non-polarized cells or phagocytic uptake. While SPI2-T3SS dependent intracellular proliferation was observed in HeLa and RAW cells, the intracellular replication in polarized cells was highly restricted and not affected by defective SPI2-T3SS. The contribution of aromatic amino acid metabolism and purine biosynthesis to intracellular proliferation was distinct in the various cell lines investigated. These observations indicate that the virulence phenotypes of S. Typhimurium are significantly affected by the cell culture model applied

The Higgs as a Portal to Plasmon-like Unparticle Excitations

12 LaTeX pages, 2 figures.-- Published in: JHEP04(2008)028.-- Final full-text version available at: http://dx.doi.org/10.1088/1126-6708/2008/04/028.A renormalizable coupling between the Higgs and a scalar unparticle operator O_U of non-integer dimension d_U<2 triggers, after electroweak symmetry breaking, an infrared divergent vacuum expectation value for O_U. Such IR divergence should be tamed before any phenomenological implications of the Higgs-unparticle interplay can be drawn. In this paper we present a novel mechanism to cure that IR divergence through (scale-invariant) unparticle self-interactions, which has properties qualitatively different from the mechanism considered previously. Besides finding a mass gap in the unparticle continuum we also find an unparticle pole reminiscent of a plasmon resonance. Such unparticle features could be explored experimentally through their mixing with the Higgs boson.Work supported in part by the European Commission under the European Union through the Marie Curie Research and Training Networks “Quest for Unification” (MRTN-CT- 2004-503369) and “UniverseNet” (MRTN-CT-2006-035863); by the Spanish Consolider- Ingenio 2010 Programme CPAN (CSD2007-0042); by a Comunidad de Madrid project (P-ESP-00346) and by CICYT, Spain, under contracts FPA 2007-60252 and FPA 2005-02211