Regulation of virulence in Francisella tularensis by small non-coding RNAs

Using a cDNA cloning and sequencing approach we have shown that Francisella tularensis expresses homologues of several small RNAs
(sRNAs) that are well-conserved among diverse bacteria. We have also discovered two abundant putative sRNAs that share no sequence similarity or conserved genomic context with any previously annotated regulatory transcripts. Deletion of either of these two loci led to significant changes in the expression of several mRNAs that likely include the cognate target(s) of these sRNAs. Deletion of these sRNAs did not, however, significantly alter F. tularensis growth under various stress conditions in vitro, its replication in murine cells, or its ability to induce disease in a mouse model of F. tularensis infection

Identification of small RNAs in Francisella tularensis

Background: Regulation of bacterial gene expression by small RNAs (sRNAs) have proved to be important for many biological processes. Francisella tularensis is a highly pathogenic Gram-negative bacterium that causes the disease tularaemia in humans and animals. Relatively little is known about the regulatory networks existing in this organism that allows it to survive in a wide array of environments and no sRNA regulators have been identified so far. Results: We have used a combination of experimental assays and in silico prediction to identify sRNAs in F. tularensis strain LVS. Using a cDNA cloning and sequencing approach we have shown that F. tularensis expresses homologues of several sRNAs that are well-conserved among diverse bacteria. We have also discovered two abundant putative sRNAs that share no sequence similarity or conserved genomic context with any previously annotated regulatory transcripts. Deletion of either of these two loci led to significant changes in the expression of several mRNAs that likely include the cognate target(s) of these sRNAs. Deletion of these sRNAs did not, however, significantly alter F. tularensis growth under various stress conditions in vitro, its replication in murine cells, or its ability to induce disease in a mouse model of F. tularensis infection. We also conducted a genome-wide in silico search for intergenic loci that suggests F. tularensis encodes several other sRNAs in addition to the sRNAs found in our experimental screen. Conclusion: Our findings suggest that F. tularensis encodes a significant number of non-coding regulatory RNAs, including members of well conserved families of structural and housekeeping RNAs and other poorly conserved transcripts that may have evolved more recently to help F. tularensis deal with the unique and diverse set of environments with which it must contend

Detecting the molecular scars of evolution in the Mycobacterium tuberculosis complex by analyzing interrupted coding sequences

<p>Abstract</p> <p>Background</p> <p>Computer-assisted analyses have shown that all bacterial genomes contain a small percentage of open reading frames with a frameshift or in-frame stop codon We report here a comparative analysis of these interrupted coding sequences (ICDSs) in six isolates of <it>M. tuberculosis</it>, two of <it>M. bovis </it>and one of <it>M. africanum </it>and question their phenotypic impact and evolutionary significance.</p> <p>Results</p> <p>ICDSs were classified as "common to all strains" or "strain-specific". Common ICDSs are believed to result from mutations acquired before the divergence of the species, whereas strain-specific ICDSs were acquired after this divergence. Comparative analyses of these ICDSs therefore define the molecular signature of a particular strain, phylogenetic lineage or species, which may be useful for inferring phenotypic traits such as virulence and molecular relationships. For instance, <it>in silico </it>analysis of the W-Beijing lineage of <it>M. tuberculosis</it>, an emergent family involved in several outbreaks, is readily distinguishable from other phyla by its smaller number of common ICDSs, including at least one known to be associated with virulence. Our observation was confirmed through the sequencing analysis of ICDSs in a panel of 21 clinical <it>M. tuberculosis </it>strains. This analysis further illustrates the divergence of the W-Beijing lineage from other phyla in terms of the number of full-length ORFs not containing a frameshift. We further show that ICDS formation is not associated with the presence of a mutated promoter, and suggest that promoter extinction is not the main cause of pseudogene formation.</p> <p>Conclusion</p> <p>The correlation between ICDSs, function and phenotypes could have important evolutionary implications. This study provides population geneticists with a list of targets, which could undergo selective pressure and thus alters relationships between the various lineages of <it>M. tuberculosis </it>strains and their host. This approach could be applied to any closely related bacterial strains or species for which several genome sequences are available.</p

Organised Genome Dynamics in the Escherichia coli Species Results in Highly Diverse Adaptive Paths

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the ∼18,000 families of orthologous genes, we found ∼2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome

A cell-contact-regulated operon is involved in genetic variability in Neisseria meningitidis.

International audienceThe ability of Neisseria meningitidis to establish efficient interaction with host cells is crucial for its survival. We recently demonstrated that an entire operon containing genes NMA1802 to NMA1806 was overexpressed during the early stage of the colonization process. In this work, we investigated whether upregulation of the expression of this operon facilitated the ability of N. meningitidis to adapt to growth on host cells. Using a strain displaying an inducible operon, we demonstrated that the NMA1802-NMA1806 cell-contact-regulated operon could potentially improve the adaptability of meningococcus during growth on the cell surface through enhanced generation of variants

How Protective are Antibodies to SARS-CoV-2, the Main Weapon of the B-Cell Response?

International audienceSince the beginning of the Coronavirus disease (COVID)-19 pandemic in December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for more than 600 million infections and 6.5 million deaths worldwide. Given the persistence of SARS-CoV-2 and its ability to develop new variants, the implementation of an effective and long-term herd immunity appears to be crucial to overcome the pandemic. While a vast field of research has focused on the role of humoral immunity against SARS-CoV-2, a growing body of evidence suggest that antibodies alone only confer a partial protection against infection of reinfection which could be of high importance regarding the strategic development goals (SDG) of the United Nations (UN) and in particular UN SDG3 that aims towards the realization of good health and well being on a global scale in the context of the COVID-19 pandemic.In this review, we highlight the role of humoral immunity in the host defense against SARS-CoV-2, with a focus on highly neutralizing antibodies. We summarize the results of the main clinical trials leading to an overall disappointing efficacy of convalescent plasma therapy, variable results of monoclonal neutralizing antibodies in patients with COVID-19 but outstanding results for the mRNA based vaccines against SARS-CoV-2. Finally, we advocate that beyond antibody responses, the development of a robust cellular immunity against SARS-CoV-2 after infection or vaccination is of utmost importance for promoting immune memory and limiting disease severity, especially in case of (re)-infection by variant viruses

Identification of a Novel Small RNA Modulating <em>Francisella tularensis</em> Pathogenicity

<div><p><em>Francisella tularensis</em> is a highly virulent bacterium responsible for the zoonotic disease tularemia. It is a facultative intracellular pathogen that replicates in the cytoplasm of host cells, particularly in macrophages. Here we show that <em>F. tularensis</em> live vaccine strain (LVS) expresses a novel small RNA (sRNA), which modulates the virulence capacities of the bacterium. When this sRNA, designated FtrC (for <em><u>F</u>rancisella <u>t</u>ularensis</em><u>R</u>NA <u>C</u>), is expressed at high levels, <em>F. tularensis</em> replicates in macrophages less efficiently than the wild-type parent strain. Similarly, high expression of FtrC reduces the number of viable bacteria recovered from the spleen and liver of infected mice. Our data demonstrate that expression of gene <em>FTL_1293</em> is regulated by FtrC. Furthermore, we show by <em>in vitro</em> gel shift assays that FtrC interacts specifically with <em>FTL_1293</em> mRNA and that this happens independently of the RNA chaperone Hfq. Remarkably, FtrC interacts only with full-length <em>FTL_1293</em> mRNA. These results, combined with a bioinformatic analysis, indicate that FtrC interacts with the central region of the mRNA and hence does not act by sterically hindering access of the ribosome to the mRNA. We further show that gene <em>FTL_1293</em> is not required for <em>F. tularensis</em> virulence <em>in vitro</em> or <em>in vivo</em>, which indicates that another unidentified FtrC target modulates the virulence capacity of the bacterium.</p> </div

Sequence, structure and expression of <i>ftrC</i>.

<p>(A) Sequence of <i>ftrC</i> in different <i>F. tulrensis</i> subspecies. LVS: <i>F. tularensis</i> subsp. <i>holarctica</i> strain LVS; SCHU: <i>F. tularensis</i> subsp. <i>tularensis</i> strain SCHU S4; media: <i>F. tularensis</i> subsp. <i>mediasiatica</i> strain FSC147; U112: <i>F. tularensis</i> subsp. <i>novicida</i> strain U112; philo: <i>F. philomiragia</i> strain ATCC 25071. The numbers after U112 indicate the four different copies of <i>ftrC</i>. Nucleotides that are conserved in all strains are marked with an * and those that differ from the LVS sequence are shown in red. (B) Predicted secondary structure of FtrC (using mFold). (C) Northern blot showing FtrC expression in wild-type LVS bacteria (lane 1) and in a strain overexpressing FtrC, LVS/p<i>ftrC</i>+ (lane 3). Lack of signal in lane 2 (LVSΔ<i>ftrC</i>) confirms absence of FtrC in the deletion strain. Size markers are indicated in nt at right. (D) Northern blot showing the stability of FtrC in LVS and LVSΔ<i>hfq</i> strains. Total RNA was isolated at different times (0–30 min, indicated at top) after addition of rifampicin and same amount of each sample was loaded onto gel. The estimated half-lifes (in min) are indicated. 5S RNA served as loading control.</p