Listeria monocytogenes encodes a functional ESX-1 secretion system whose expression is detrimental to in vivo infection

Bacterial pathogenicity deeply depends on the ability to secrete virulence factors that bind specific targets on host cells and manipulate host responses. The Gram-positive bacterium Listeria monocytogenes is a human foodborne pathogen that remains a seriThis work was supported for the DC lab by national funds through FCT - Fundacao para a Ciencia e a Tecnologia/MEC Ministerio da Educacao e Ciencia and co-funded by FEDER funds within the partnership agreement PT2020 related with the research unit number

Distinct Functional Roles of β-Tubulin Isotypes in Microtubule Arrays of Tetrahymena thermophila, a Model Single-Celled Organism

<div><h3>Background</h3><p>The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, <em>Tetrahymena thermophila</em>. <em>Tetrahymena</em> forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of <em>Tetrahymena</em> demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (<em>BTU1</em> and <em>BTU2</em>) encode the canonical β-tubulin, BTU2, and six genes (<em>BLT1-6</em>) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.</p> <h3>Methodology/Principal Findings</h3><p>With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins <em>in vivo</em>, we transformed <em>Tetrahymena</em> with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.</p> <h3>Conclusion/Significance</h3><p>We conclude that <em>Tetrahymena</em> uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.</p> </div

Adaptation and Preadaptation of Salmonella enterica to Bile

Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS− mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10−6 and 10−7 per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations

Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Nursing-Related Smartphone Activities in the Italian Nursing Population: A Descriptive Study

In this study, we describe smartphone-related activities for nurses' work and nonwork purposes; analyzed the differences between smartphone use and nurses' age, gender, and working environment; and observed the influences that personal digital devices have on nurses' performance. We conducted a cross-sectional and correlational study. A convenience sample of nurses was recruited, composed of 256 nurses, mostly women (74.6%) younger than 30 years of age (59%). This study showed that the most frequent work-related smartphone activity was searching for work-related drug references. Smartphones also helped nurses reduce work-related stress and improve unit cohesion and teamwork. Younger nurses and male nurses were more likely than female nurses to use their digital devices during healthcare activities. In addition, 42% of nurses reported that they were distracted by the use of smartphones. If smartphones are used properly, they are certainly helpful tools for improving patient safety and enabling communication among healthcare staff. Otherwise, smartphones can constitute an important source of distraction and endanger patient safet