microLife最新文献

Vibrio cholerae is a major human pathogen causing the diarrheal disease, cholera. Regulation of virulence in V. cholerae is a multifaceted process involving gene expression changes at the transcriptional and post-transcriptional level. Whereas various transcription factors have been reported to modulate virulence in V. cholerae, small regulatory RNAs (sRNAs) have now been established to also participate in virulence control and the regulation of virulence-associated processes, such as biofilm formation, quorum sensing, stress response, and metabolism. In most cases, these sRNAs act by base-pairing with multiple target transcripts and this process typically requires the aid of an RNA-binding protein, such as the widely conserved Hfq protein. This review article summarizes the functional roles of sRNAs in V. cholerae, their underlying mechanisms of gene expression control, and how sRNAs partner with transcription factors to modulate complex regulatory programs. In addition, we will discuss regulatory principles discovered in V. cholerae that not only apply to other Vibrio species, but further extend into the large field of RNA-mediated gene expression control in bacteria.

The outer membrane (OM) protects Gram-negative bacteria from harsh environmental conditions and provides intrinsic resistance to many antimicrobial compounds. The asymmetric OM is characterized by phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. Previous reports suggested an involvement of the signaling nucleotide ppGpp in cell envelope homeostasis in Escherichia coli. Here, we investigated the effect of ppGpp on OM biosynthesis. We found that ppGpp inhibits the activity of LpxA, the first enzyme of LPS biosynthesis, in a fluorometric in vitro assay. Moreover, overproduction of LpxA resulted in elongated cells and shedding of outer membrane vesicles (OMVs) with altered LPS content. These effects were markedly stronger in a ppGpp-deficient background. We further show that RnhB, an RNase H isoenzyme, binds ppGpp, interacts with LpxA, and modulates its activity. Overall, our study uncovered new regulatory players in the early steps of LPS biosynthesis, an essential process with many implications in the physiology and susceptibility to antibiotics of Gram-negative commensals and pathogens.

Cyclic di-AMP is an emerging second messenger that is synthesized by many archaea and bacteria, including the Gram-positive pathogenic bacterium Listeria monocytogenes. Listeria monocytogenes played a crucial role in elucidating the essential function of c-di-AMP, thereby becoming a model system for studying c-di-AMP metabolism and the influence of the nucleotide on cell physiology. c-di-AMP is synthesized by a diadenylate cyclase and degraded by two phosphodiesterases. To date, eight c-di-AMP receptor proteins have been identified in L. monocytogenes, including one that indirectly controls the uptake of osmotically active peptides and thus the cellular turgor. The functions of two c-di-AMP-receptor proteins still need to be elucidated. Here, we provide an overview of c-di-AMP signalling in L. monocytogenes and highlight the main differences compared to the other established model systems in which c-di-AMP metabolism is investigated. Moreover, we discuss the most important questions that need to be answered to fully understand the role of c-di-AMP in osmoregulation and in the control of central metabolism.

In September 2022, an international summer course entitled 'The new microbiology' took place in Greece, on the island of Spetses. The organizers aimed to highlight the spectacular advances and the renaissance occurring in Microbiology, driven by developments in genomics, proteomics, imaging techniques, and bioinformatics. Combinations of these advances allow for single cell analyses, rapid and relatively inexpensive metagenomic and transcriptomic data analyses and comparisons, visualization of previously unsuspected mechanisms, and large-scale studies. A 'New Microbiology' is emerging which allows studies that address the critical roles of microbes in health and disease, in humans, animals, and the environment. The concept of one health is now transforming microbiology. The goal of the course was to discuss all these topics with members of the new generation of microbiologists all of whom were highly motivated and fully receptive.

The Lpl proteins represent a class of lipoproteins that was first described in the opportunistic bacterial pathogen Staphylococcus aureus, where they contribute to pathogenicity by enhancing F-actin levels of host epithelial cells and thereby increasing S. aureus internalization. The model Lpl protein, Lpl1 was shown to interact with the human heat shock proteins Hsp90α and Hsp90ß, suggesting that this interaction may trigger all observed activities. Here we synthesized Lpl1-derived peptides of different lengths and identified two overlapping peptides, namely, L13 and L15, which interacted with Hsp90α. Unlike Lpl1, the two peptides not only decreased F-actin levels and S. aureus internalization in epithelial cells but they also decreased phagocytosis by human CD14⁺ monocytes. The well-known Hsp90 inhibitor, geldanamycin, showed a similar effect. The peptides not only interacted directly with Hsp90α, but also with the mother protein Lpl1. While L15 and L13 significantly decreased lethality of S. aureus bacteremia in an insect model, geldanamycin did not. In a mouse bacteremia model L15 was found to significantly decreased weight loss and lethality. Although the molecular bases of the L15 effect is still elusive, in vitro data indicate that simultaneous treatment of host immune cells with L15 or L13 and S. aureus significantly increase IL-6 production. L15 and L13 represent not antibiotics but they cause a significant reduction in virulence of multidrug-resistant S. aureus strains in in vivo models. In this capacity, they can be an important drug alone or additive with other agents.

Bacterial extracellular vesicles (EVs) are natural lipidic nanoparticles implicated in intercellular communication. Although EV research focused mainly on pathogens, the interest in probiotic-derived EVs is now rising. One example is Propionibacterium freudenreichii, which produces EVs with anti-inflammatory effects on human epithelial cells. Our previous study with P. freudenreichii showed that EVs purified by size exclusion chromatography (SEC) displayed variations in protein content according to bacterial growth conditions. Considering these content variations, we hypothesized that a comparative proteomic analysis of EVs recovered in different conditions would elucidate whether a representative vesicular proteome existed, possibly providing a robust proteome dataset for further analysis. Therefore, P. freudenreichii was grown in two culture media, and EVs were purified by sucrose density gradient ultracentrifugation (UC). Microscopic and size characterization confirmed EV purification, while shotgun proteomics unveiled that they carried a diverse set of proteins. A comparative analysis of the protein content of UC- and SEC-derived EVs, isolated from cultures either in UF (cow milk ultrafiltrate medium) or YEL (laboratory yeast extract lactate medium), showed that EVs from all these conditions shared 308 proteins. This EV core proteome was notably enriched in proteins related to immunomodulation. Moreover, it showed distinctive features, including highly interacting proteins, compositional biases for some specific amino acids, and other biochemical parameters. Overall, this work broadens the toolset for the purification of P. freudenreichii-derived EVs, identifies a representative vesicular proteome, and enumerates conserved features in vesicular proteins. These results hold the potential for providing candidate biomarkers of purification quality, and insights into the mechanisms of EV biogenesis and cargo sorting.

Eukaryotes have historically been studied as parasites, but recent evidence suggests they may be indicators of a healthy gut ecosystem. Here, we describe the eukaryome along the gastrointestinal tract of children aged 2-5 years and test for associations with clinical factors such as anaemia, intestinal inflammation, chronic undernutrition, and age. Children were enrolled from December 2016 to May 2018 in Bangui, Central African Republic and Antananarivo, Madagascar. We analyzed a total of 1104 samples representing 212 gastric, 187 duodenal, and 705 fecal samples using a metabarcoding approach targeting the full ITS2 region for fungi, and the V4 hypervariable region of the 18S rRNA gene for the overall eukaryome. Roughly, half of all fecal samples showed microeukaryotic reads. We find high intersubject variability, only a handful of taxa that are likely residents of the gastrointestinal tract, and frequent co-occurrence of eukaryotes within an individual. We also find that the eukaryome differs between the stomach, duodenum, and feces and is strongly influenced by country of origin. Our data show trends towards higher levels of Fusarium equiseti, a mycotoxin producing fungus, and lower levels of the protist Blastocystis in stunted children compared to nonstunted controls. Overall, the eukaryome is poorly correlated with clinical variables. Our study is of one of the largest cohorts analyzing the human intestinal eukaryome to date and the first to compare the eukaryome across different compartments of the gastrointestinal tract. Our results highlight the importance of studying populations across the world to uncover common features of the eukaryome in health.

Site-2-proteases are a class of intramembrane proteases involved in regulated intramembrane proteolysis. Regulated intramembrane proteolysis is a highly conserved signaling mechanism that commonly involves sequential digestion of an anti-sigma factor by a site-1- and site-2-protease in response to external stimuli, resulting in an adaptive transcriptional response. Variation of this signaling cascade continues to emerge as the role of site-2-proteases in bacteria continues to be explored. Site-2-proteases are highly conserved among bacteria and play a key role in multiple processes, including iron uptake, stress response, and pheromone production. Additionally, an increasing number of site-2-proteases have been found to play a pivotal role in the virulence properties of multiple human pathogens, such as alginate production in Pseudomonas aeruginosa, toxin production in Vibrio cholerae, resistance to lysozyme in enterococci and antimicrobials in several Bacillus spp, and cell-envelope lipid composition in Mycobacterium tuberculosis. The prominent role of site-2-proteases in bacterial pathogenicity highlights the potential of site-2-proteases as novel targets for therapeutic intervention. In this review, we summarize the role of site-2-proteases in bacterial physiology and virulence, as well as evaluate the therapeutic potential of site-2-proteases.