Deciphering the pneumococcal cell cycle:Identification and characterization of new genes involved in growth and replication

Understanding how pathogenic bacteria grow and divide is essential to better fight them. Streptococcus pneumoniae (pneumococcus) is a bacterium that is usually found in the throats of healthy people, but which is also held responsible for millions of deaths each year. With the constantly increasing resistance to antibiotics and the fact that vaccination programs target only a few types of S. pneumoniae, it is necessary to identify new therapeutic targets. To identify new essential proteins, certain methods can be used to disrupt every gene, but validation of their essentiality can be tedious. For this reason, part of this work consisted in developing a fast and reliable method based on CRISPRi to selectively inactivate each essential gene and study the effect on the pneumococcus. We were thus able to unravel the function of some unknown genes specific to S. pneumoniae. Potential drug targets can be genes involved in the cell cycle. Little is known how S. pneumoniae detects when to start duplicating its DNA each time a new cell is formed. Using our data, we have identified and described a new protein that localizes at the division site and is able to stimulate DNA replication once per cell cycle. Finally, we also described the importance of lipid biosynthesis in the survival and morphogenesis of pneumococcus. These findings broaden our knowledge on the regulation of the pneumococcal cell cycle, which could also be transposed to other pathogenic bacteria, and therefore help define new potential therapeutic targets

The Tyrosine-Autokinase UbK Is Required for Proper Cell Growth and Cell Morphology of Streptococcus pneumoniae

International audienceProtein phosphorylation is a key post-translational modification required for many cellular functions of the bacterial cell. Recently, we identified a new protein-kinase, named UbK, in Bacillus subtilis that belongs to a new family of protein-kinases widespread in bacteria. In this study, we analyze the function of UbK in Streptococcus pneumoniae. We show that UbK displays a tyrosine-kinase activity and autophosphorylates on a unique tyrosine in vivo. To get insights into its cellular role, we constructed a set of pneumococcal ubk mutants. Using conventional and electron microscopy, we show that the ubk deficient strain, as well as an ubk catalytic dead mutant, display both severe cell-growth and cell-morphology defects. The same defects are observed with a mutant mimicking permanent phosphorylation of UbK whereas they are not detected for a mutant mimicking defective autophosphorylation of UbK. Moreover, we find that UbK phosphorylation promotes its ability to hydrolyze ATP. These observations show that the hydrolysis of ATP by UbK serves not only for its autophosphorylation but also for a distinct purpose essential for the optimal cell growth and cell-morphogenesis of the pneumococcus. We thus propose a model in which the autophosphorylation/dephosphorylation of UbK regulates its cellular function through a negative feedback loop

A survey of primary care physician practices in antibiotic prescribing for the treatment of uncomplicated male gonoccocal urethritis

<p>Abstract</p> <p>Background</p> <p>The development of resistance to antimicrobial therapy by <it>Neisseria gonorrhoeae </it>causes on-going problems for individual case management of gonorrhoea. Surveillance data about <it>N. gonorrhoeae </it>have indicated an increase in the incidence of gonorrhoea in France in 2006. As a consequence of the development of antibiotic resistance in <it>N. gonorrhoeae</it>, French guidelines excluded fluoroquinolones as a standard treatment for <it>N. gonorrhoeae</it>. Ceftriaxone became the recommended treatment, associated with azithromycin for <it>Clamydia trachomatis </it>infection. Our aim was to describe the practice patterns of general practitioners (GPs) in managing the antibiotic treatment of patients with symptoms suggestive of uncomplicated male urethritis.</p> <p>Methods</p> <p>We developed a clinical vignette describing a man with typical gonococcal urethritis symptoms to elicit questions about antibiotic treatment. We mailed the electronic questionnaire to a random sample of 1000 French GPs belonging to the <it>Sentinelles </it>Network.</p> <p>Results</p> <p>By the end of the survey period, 350 vignettes were received, yielding a response rate of 35%. Sixty-six GPs (20.2%) prescribed the recommended antibiotics for the simultaneous treatment of <it>N</it>. <it>gonorrhoeae </it>and <it>C. trachomatis </it>infections, while 132 GPs (40.4%) prescribed only non-recommended antibiotics, including ciprofloxacin in 69 cases (21.1%). General practitioners with less than 10 years in practice showed better compliance to guidelines than those with more years in practice (p < 0.05).</p> <p>Conclusions</p> <p>The results suggest a mismatch between the guidelines and the antibiotic treatment of male uncomplicated urethritis by French GPs, mostly among the subgroup of physicians who have been in practice longer. Educational approaches based on practice feedback need to be developed to improve these deficits in the quality of care.</p

A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism

Follicle-Stimulating Hormone Receptor: Advances and Remaining Challenges

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The follicle-stimulating hormone signaling network in gonadal cells.

International audienceFSH (follicle-stimulating hormone) is a master endocrine regulator of somatic cells of the gonads that support gametogenesis. In the male, FSH dictates the Sertoli cell proliferation rate prior to puberty, then maintains their main biological roles as nurturing cells and physical support to spermatogenesis throughout life. In the female, FSH is instrumental in processing the terminal differentiation phase of folliculogenesis that progressively drives the ovarian follicle to ovulation. The biological function of FSH is transduced by a membrane receptor, the FSH receptor (FSHR). Deciphering the molecular bases of the developmental switch in FSH biological activities is required to gain insights into FSH-induced signaling pathways. This quest has led to the identification of a complex interconnected signaling network affected by testicular paracrine factors, implying not only protein post-translational modifications but also regulation by microRNA and chromatin remodeling. In the female, the emphasis is now placed on deciphering the complex functional relationships between the LHCGR (luteinizing hormone/choriogonadotropin hormone receptor) and the FSHR that trigger intertwined signaling networks in the same granulosa cell type. Breakthroughs in the organization and dynamic functioning of the FSH-induced signaling network are expected to identifying novel regulatory processes and therapeutic strategies for infertilities and contraception