Characterization of a Mycobacterium smegmatis uvrA mutant impaired in dormancy induced by hypoxia and low carbon concentration

<p>Abstract</p> <p>Background</p> <p>The aerobic fast-growing <it>Mycobacterium smegmatis</it>, like its slow-growing pathogenic counterpart Mycobacterium tuberculosis, has the ability to adapt to microaerobiosis by shifting from growth to a non-proliferating or dormant state. The molecular mechanism of dormancy is not fully understood and various hypotheses have been formulated to explain it. In this work, we open new insight in the knowledge of <it>M. smegmatis </it>dormancy, by identifying and characterizing genes involved in this behavior.</p> <p>Results</p> <p>In a library generated by transposon mutagenesis, we searched for <it>M. smegmatis </it>mutants unable to survive a coincident condition of hypoxia and low carbon content, two stress factors supposedly encountered in the host and inducing dormancy in tubercle bacilli. Two mutants were identified that mapped in the <it>uvrA </it>gene, coding for an essential component of the Nucleotide Excision Repair system (NER). The two mutants showed identical phenotypes, although the respective transposon insertions hit different regions of the <it>uvrA </it>gene. The restoration of the <it>uvrA </it>activity in <it>M. smegmatis </it>by complementation with the <it>uvrA </it>gene of <it>M. tuberculosis</it>, confirmed that i) <it>uvrA </it>inactivation was indeed responsible for the inability of <it>M. smegmatis </it>cells to enter or exit dormancy and, therefore, survive hypoxia and presence of low carbon and ii) showed that the respective <it>uvrA </it>genes of <it>M. tuberculosis </it>and <it>M. smegmatis </it>are true orthologs. The rate of survival of wild type, <it>uvrA </it>mutant and complemented strains under conditions of oxidative stress and UV irradiation was determined qualitatively and quantitatively.</p> <p>Conclusions</p> <p>Taken together our results confirm that the mycobacterial NER system is involved in adaptation to various stress conditions and suggest that cells with a compromised DNA repair system have an impaired dormancy behavior.</p

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

Identification of Genes Involved in Neisseria meningitidis Colonization

International audienceNeisseria meningitidis is a worldwide cause of meningitis and septicemia leading at least to 50,000 deaths every year. Nevertheless, N. meningitidis is also a commensal bacterium that asymptomatically colonizes the epithelial cells of the nasopharynx of 10 to 30% of healthy individuals. Occasionally, N. meningitidis crosses the nasopharyngeal barrier and enters the bloodstream. During bacteremia, N. meningitidis may adhere to endothelial cells of brain vessels and invade meninges. To identify the genes required for meningococcal host colonization, we screened a signature-tagged transposon mutagenesis library using an innovative in vitro colonization model in order to identify mutants displaying decreased capacity to colonize human epithelial cells. Approximately 1,600 defined insertion mutants of invasive serogroup C strain NEM8013 were screened. Candidate mutants were tested individually for quantification of bacterial biomass with confocal microscope and COMSTAT software. Five mutants were demonstrated to exhibit significantly decreased colonization ability. The identified genes, including narP and estD, appeared to be involved in adaptation to hypoxic conditions and stress resistance. Interestingly, the genes fadD1, nnrS, and NMV_2034 (encoding a putative thioredoxin), prior to this study, had not been shown to be involved in colonization. Therefore, we provide here insights into the meningococcal functions necessary for the bacterium to adapt to growth on host cells

Genomic analysis of Staphylococcus aureus sequential isolates from lungs of patients with cystic fibrosis

Staphylococcus aureus is the predominant pathogen in children with cystic fibrosis (CF) in France and, around 80 % of them harbored S. aureus in their lungs. This study investigated virulence and antimicrobial resistance-associated genes and within-host evolution polymorphisms in 14 S. aureus persistent clones from 14 chronically infected CF children. For each of the 14 patients, we compared genomes of two isogenic sequential isolates separated by 2-9 years. All isolates were methicillin-sensitive and harbored the immune evasion gene cluster, whereas half of them harbored the enterotoxin gene cluster. Most clones were capsule type 8 (8/14) and accessory gene regulator (agr)-specificity group 1 (9/14). We identified convergent mutations in genes involved in carbohydrate metabolism, cell wall metabolism, genetic information Manuscript Click here to view linked References processing and adhesion, which are likely to play important role in intracellular invasion and persistence. Further explorations relying notably on proteomics will contribute to improve our understanding of the mechanisms at play in the striking long-term persistence ability of S. aureus

SigF Controls Carotenoid Pigment Production and Affects Transformation Efficiency and Hydrogen Peroxide Sensitivity in Mycobacterium smegmatis▿ †

Carotenoids are complex lipids that are known for acting against photodynamic injury and free radicals. We demonstrate here that σF is required for carotenoid pigment production in Mycobacterium smegmatis. We further show that a sigF mutant exhibits a transformation efficiency 104-fold higher than that of the parental strain, suggesting that σF regulates the production of components affecting cell wall permeability. In addition, a sigF mutant showed an increased sensitivity to hydrogen peroxide. An in silico search of the M. smegmatis genome identified a number of SigF consensus sites, including sites upstream of the carotenoid synthesis locus, which explains its SigF regulation

Interrupted coding sequences in Mycobacterium smegmatis: authentic mutations or sequencing errors?

BACKGROUND: In silico analysis has shown that all bacterial genomes contain a low percentage of ORFs with undetected frameshifts and in-frame stop codons. These interrupted coding sequences (ICDSs) may really be present in the organism or may result from misannotation based on sequencing errors. The reality or otherwise of these sequences has major implications for all subsequent functional characterization steps, including module prediction, comparative genomics and high-throughput proteomic projects. RESULTS: We show here, using Mycobacterium smegmatis as a model species, that a significant proportion of these ICDSs result from sequencing errors. We used a resequencing procedure and mass spectrometry analysis to determine the nature of a number of ICDSs in this organism. We found that 28 of the 73 ICDSs investigated correspond to sequencing errors. CONCLUSION: The correction of these errors results in modification of the predicted amino acid sequences of the corresponding proteins and changes in annotation. We suggest that each bacterial ICDS should be investigated individually, to determine its true status and to ensure that the genome sequence is appropriate for comparative genomics analyses

A virulence-associated filamentous bacteriophage of Neisseria meningitidis increases host-cell colonisation.

Neisseria meningitidis is a commensal of human nasopharynx. In some circumstances, this bacteria can invade the bloodstream and, after crossing the blood brain barrier, the meninges. A filamentous phage, designated MDAΦ for Meningococcal Disease Associated, has been associated with invasive disease. In this work we show that the prophage is not associated with a higher virulence during the bloodstream phase of the disease. However, looking at the interaction of N. meningitidis with epithelial cells, a step essential for colonization of the nasopharynx, we demonstrate that the presence of the prophage, via the production of viruses, increases colonization of encapsulated meningococci onto monolayers of epithelial cells. The analysis of the biomass covering the epithelial cells revealed that meningococci are bound to the apical surface of host cells by few layers of heavily piliated bacteria, whereas, in the upper layers, bacteria are non-piliated but surrounded by phage particles which (i) form bundles of filaments, and/or (ii) are in some places associated with bacteria. The latter are likely to correspond to growing bacteriophages during their extrusion through the outer membrane. These data suggest that, as the biomass increases, the loss of piliation in the upper layers of the biomass does not allow type IV pilus bacterial aggregation, but is compensated by a large production of phage particles that promote bacterial aggregation via the formation of bundles of phage filaments linked to the bacterial cell walls. We propose that MDAΦ by increasing bacterial colonization in the mucosa at the site-of-entry, increase the occurrence of diseases

Reactive Oxygen Species-Dependent Innate Immune Mechanisms Control Methicillin-Resistant Staphylococcus aureus Virulence in the Drosophila Larval Model

International audienceVolume 12, Number 37 July 2021ABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCESSupplemental MaterialABSTRACTAntibiotic-resistant Staphylococcus aureus strains constitute a major public health concern worldwide and are responsible for both health care- and community-associated infections. Here, we establish a robust and easy-to-implement model of oral S. aureus infection using Drosophila melanogaster larvae that allowed us to follow the fate of S. aureus at the whole-organism level as well as the host immune responses. Our study demonstrates that S. aureus infection triggers H2O2 production by the host via the Duox enzyme, thereby promoting antimicrobial peptide production through activation of the Toll pathway. Staphylococcal catalase mediates H2O2 neutralization, which not only promotes S. aureus survival but also minimizes the host antimicrobial response, hence reducing bacterial clearance in vivo. We show that while catalase expression is regulated in vitro by the accessory gene regulatory system (Agr) and the general stress response regulator sigma B (SigB), it no longer depends on these two master regulators in vivo. Finally, we confirm the versatility of this model by demonstrating the colonization and host stimulation capabilities of S. aureus strains belonging to different sequence types (CC8 and CC5) as well as of two other bacterial pathogens, Salmonella enterica serovar Typhimurium and Shigella flexneri. Thus, the Drosophila larva can be a general model to follow in vivo the innate host immune responses triggered during infection by human pathogens

Lipopentapeptide induces a strong host humoral response and distinguishes Mycobacterium avium subsp. paratuberculosis from M. avium subsp. avium

Background: Many non-tuberculous mycobacteria synthesize abundant glycopeptidolipids (GPLs). These surface-located GPLs are involved in pathogenicity by interfering with the host immune system. In Mycobacterium avium subsp. avium (Mav), GPLs consist of a lipopeptide core composed of a tetrapeptide O-linked to mono- and oligo-saccharides. The biosynthesis pathway of the simplest GPLs is now relatively well understood and involves probably more than fifteen genes. Whereas it is very obvious that most, if not all, of the Mav isolates produce GPLs, the picture is not as clear for M. avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease in cattle, and several conflicting data have been produced. Methods: Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs. To provide a genomic basis for the synthesis of this compound, the recently published genome sequence of Map was explored using in silico methods. Even though Map produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of Mav. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav and is in agreement with the amino acid content of the L5P. We also showed that the peptidyl moiety of the L5P is a target for a strong specific humoral response in Map infected animals. Conclusions: These genomic and biochemical differences may help to unambiguously distinguish Map from Mav and also from M. bovis, to reclassify related strains of the Map species and to allow the convenient and specific diagnosis of paratuberculosis