Isabelle Rosinski-Chupin

The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome.

Clustered regularly interspaced short palindromic repeats (CRISPR) confer immunity against mobile genetic elements (MGEs) in prokaryotes. Streptococcus agalactiae, a leading cause of neonatal infections contains in its genome two CRISPR/Cas systems. We show that type 1‐C CRISPR2 is present in few strains but type 2‐A CRISPR1 is ubiquitous. Comparative sequence analysis of the CRISPR1 spacer content of 351 S. agalactiae strains revealed that it is extremely diverse due to the acquisition of new spacers, spacer duplications and spacer deletions that witness the dynamics of this system. The spacer content profile mirrors the S. agalactiae population structure. Transfer of a conjugative transposon targeted by CRISPR1 selected for spacer rearrangements, suggesting that deletions and duplications pre‐exist in the population. The comparison of protospacers located within MGE or the core genome and protospacer‐associated motif‐shuffling demonstrated that the GG motif is sufficient to discriminate self and non‐self and for spacer selection and integration. Strikingly more than 40% of the 949 different CRISPR1 spacers identified target MGEs found in S. agalactiae genomes. We thus propose that the S. agalactiae type II‐A CRISPR1/Cas system modulates the cohabitation of the species with its mobilome, as such contributing to the diversity of MGEs in the population.

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

SAGE analysis of mosquito salivary gland transcriptomes during Plasmodium invasion.

Invasion of the vector salivary glands by Plasmodium is a critical step for malaria transmission. To describe salivary gland cellular responses to sporozoite invasion, we have undertaken the analysis of Anopheles gambiae salivary gland transcriptome using Serial Analysis of Gene Expression (SAGE). Statistical analysis of the more than 160 000 sequenced tags generated from four libraries, two from glands infected by Plasmodium berghei, two from glands of controls, revealed that at least 57 Anopheles genes are differentially expressed in infected salivary glands. Among the 37 immune‐related genes identified by SAGE tags, four (Defensin1, GNBP, Serpin6 and Cecropin2) were found to be upregulated during salivary gland invasion, while five genes encoding small secreted proteins display induction patterns strongly reminiscent of that of Cecropin2. Invasion by Plasmodium has also an impact on the expression of genes involved in transport, lipid and energy metabolism, suggesting that the sporozoite may exploit the metabolism of its host. In contrast, protein composition of saliva is predicted to be only slightly modified after infection. This study, which is the first transcriptome analysis of the salivary gland response to Plasmodium infection, provides a basis for a better understanding of Plasmodium/Anopheles salivary gland interactions.

Localization of mRNAs of two androgen-dependent proteins, SMR1 and SMR2, by in situ hybridization reveals sexual differences in acinar cells of rat submandibular gland.

Androgen-dependent sexual differences in the granular convoluted tubules of mouse and rat submandibular glands (SMG) have been extensively reported. We studied two major androgen-dependent mRNAs of the rat SMG encoding proteins named SMR1 and SMR2. To determine which cell type in the SMG is responsible for synthesis of these mRNAs, we performed in situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. We show that SMR1 and SMR2 mRNAs are synthesized in the acinar cells of the SMG. A clear difference in SMR1 and SMR2 mRNA levels in male and female is demonstrated. During the course of this study we also confirmed the acinar localization of mRNAs encoding the glutamine/glutamic acid-rich proteins (GRP) of rat SMG. Our data are the first clear evidence of androgen-dependent sexual differences in acinar cells of rat submandibular gland.

Serial Analysis of Gene Expression in Plasmodium berghei salivary gland sporozoites

BackgroundThe invasion of Anopheles salivary glands by Plasmodium sporozoites is an essential step for transmission of the parasite to the vertebrate host. Salivary gland sporozoites undergo a developmental programme to express genes required for their journey from the site of the mosquito bite to the liver and subsequent invasion of, and development within, hepatocytes. A Serial Analysis of Gene Expression was performed on Anopheles gambiae salivary glands infected or not with Plasmodium berghei and we report here the analysis of the Plasmodium sporozoite transcriptome.ResultsAnnotation of 530 tag sequences homologous to Plasmodium berghei genomic sequences identified 123 genes expressed in salivary gland sporozoites and these genes were classified according to their transcript abundance. A subset of these genes was further studied by quantitative PCR to determine their expression profiles. This revealed that sporozoites modulate their RNA amounts not only between the midgut and salivary glands, but also during their storage within the latter. Among the 123 genes, the expression of 66 is described for the first time in sporozoites of rodent Plasmodium species.ConclusionThese novel sporozoite expressed genes, especially those expressed at high levels in salivary gland sporozoites, are likely to play a role in Plasmodium infectivity in the mammalian host.

Single nucleotide resolution RNA-seq uncovers new regulatory mechanisms in the opportunistic pathogen Streptococcus agalactiae

BackgroundStreptococcus agalactiae, or Group B Streptococcus, is a leading cause of neonatal infections and an increasing cause of infections in adults with underlying diseases. In an effort to reconstruct the transcriptional networks involved in S. agalactiae physiology and pathogenesis, we performed an extensive and robust characterization of its transcriptome through a combination of differential RNA-sequencing in eight different growth conditions or genetic backgrounds and strand-specific RNA-sequencing.ResultsOur study identified 1,210 transcription start sites (TSSs) and 655 transcript ends as well as 39 riboswitches and cis-regulatory regions, 39 cis-antisense non-coding RNAs and 47 small RNAs potentially acting in trans. Among these putative regulatory RNAs, ten were differentially expressed in response to an acid stress and two riboswitches sensed directly or indirectly the pH modification. Strikingly, 15% of the TSSs identified were associated with the incorporation of pseudo-templated nucleotides, showing that reiterative transcription is a pervasive process in S. agalactiae. In particular, 40% of the TSSs upstream genes involved in nucleotide metabolism show reiterative transcription potentially regulating gene expression, as exemplified for pyrG and thyA encoding the CTP synthase and the thymidylate synthase respectively.ConclusionsThis comprehensive map of the transcriptome at the single nucleotide resolution led to the discovery of new regulatory mechanisms in S. agalactiae. It also provides the basis for in depth analyses of transcriptional networks in S. agalactiae and of the regulatory role of reiterative transcription following variations of intra-cellular nucleotide pools.

Acinar Cells Are Target Cells for Androgens in Mouse Submandibular Glands

The variable coding sequence (VCS) multigene family encodes diverse salivary proteins, such as the SMR1 prohormone and the PR-VB1 proline-rich protein in the rat. In situ hybridization was used to study the cell-specific expression of two new mouse VCS genes, Vcs1 and Vcs2. We show that the Vcs1 transcripts, which code for a proline-rich protein, MSG1, are highly abundant in male and female parotid glands, in which they are specifically detected in acinar cells. No expression was seen in the submandibular or sublingual glands. In contrast, Vcs2 transcripts were found only in the acinar cells of the submandibular glands (SMGs) of male mice, in which they are expressed in response to androgens. Expression was found to be heterogeneous within acinar structures. No Vcs2 transcripts were detected in the SMGs of females or castrated males by Northern blot, RNase protection, or in situ hybridization. Androgen administration to females or castrated males induced expression at a level comparable to that of intact males. The Vcs2 gene is the first example of a mouse androgen-regulated gene that is expressed in SMG acinar cells. This result, in addition to our previous observation on SMR1 expression in rats, demonstrates that both acinar cells and granular convoluted tubule (GCT) cells are target cells for androgen action in rodent SMG.

Substantial N diversity is generated in T cell receptor α genes at birth despite low levels of terminal deoxynucleotidyl transferase expression in mouse thymus

N region diversity in antigen receptors is a developmentally regulated process in B and T lymphocytes, which correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). To precisely determine the onset of TdT gene activation during T cell differentiation and thymic ontogeny, TdT expression was directly detected at the cellular level by in situ hybridization and TdT function was assessed by analyzing the distribution of N additions in α and β TCR genes at early stages of development. Even though TdT transcripts were undetectable at birth, substantial N additions were observed in VαJα junctions and 3 days later in VβDβJβ junctions, indicating that TdT expression could be induced in immature thymocytes much earlier than expected. Indeed low TdT expression level was found in TN3/4 and DP from fetal day 17, suggesting that the onset of TdT expression occurs simultaneously in both populations and may depend on microenvironmental cues. Moreover significant increase in the proportion of thymocytes expressing high levels of TdT mRNA during the first week after birth without a similar increase in the level of N diversity suggests that TdT expression and TdT function in the generation of N diversity are not strictly correlated.

Androgen regulation of SMR2 gene Expression in rat submandibular gland: Evidence for a graded but not a binary response

Expression of SMR2, a member of the gene family encoding salivary glutamine/glutamic acid-rich proteins, is regulated by androgens in rat submandibular gland acinar cells. To further characterize SMR2 regulation, we analyzed SMR2 expression during submandibular gland postnatal development and rat puberty at both a global and a single-cell level. Using in situ detection of mature and primary SMR2 transcripts, we show that SMR2 expression is heterogeneous among acinar cells. However, only one cell population with various amounts of mRNAs can be defined. The number of high-expressing cells increases in males during puberty and in females up to 6 weeks of age, suggesting that some factor in addition to acinar differentiation might be important for SMR2 expression in female rats. Involvement of the β-adrenergic system in regulating SMR2 expression was tested in rats exposed daily to isoproterenol for 4 days. Under these conditions we found an increase in SMR2 expression in female rats, associated with an increase in SMR2 mRNA levels in most acinar cells. This suggests that a signaling cascade, elicited by β-adrenergic stimuli, might act in concert with androgens to regulate SMR2 expression.

Parallel Evolution of Group B Streptococcus Hypervirulent Clonal Complex 17 Unveils New Pathoadaptive Mutations

The incidence of group B Streptococcus (GBS) neonatal disease continues to be a significant cause of concern worldwide. Strains belonging to clonal complex 17 (CC17) are the most frequently responsible for GBS infections in neonates, especially among late-onset disease cases. Therefore, we undertook the largest genomic study of GBS CC17 strains to date to decipher the genetic bases of their remarkable colonization and infection ability. We show that crucial functions involved in different steps of the colonization or infection process of GBS are distinctly mutated during the adaptation of CC17 to the human host. In particular, our results implicate the CovRS two-component regulator of virulence in the differentiation between carriage- and disease-associated isolates. Not only does this work raise important implications for the ongoing development of a vaccine against GBS but might also drive the discovery of key functions for GBS adaptation and pathogenesis that have been overlooked until now. ABSTRACT Group B Streptococcus (GBS) is a commensal of the gastrointestinal and genitourinary tracts, while a prevailing cause of neonatal disease worldwide. Of the various clonal complexes (CCs), CC17 is overrepresented in GBS-infected newborns for reasons that are still largely unknown. Here, we report a comprehensive genomic analysis of 626 CC17 isolates collected worldwide, identifying the genetic traits behind their successful adaptation to humans and the underlying differences between carriage and clinical strains. Comparative analysis with 923 GBS genomes belonging to CC1, CC19, and CC23 revealed that the evolution of CC17 is distinct from that of other human-adapted lineages and recurrently targets functions related to nucleotide and amino acid metabolism, cell adhesion, regulation, and immune evasion. We show that the most distinctive features of disease-specific CC17 isolates were frequent mutations in the virulence-associated CovS and Stk1 kinases, underscoring the crucial role of the entire CovRS regulatory pathway in modulating the pathogenicity of GBS. Importantly, parallel and convergent evolution of major components of the bacterial cell envelope, such as the capsule biosynthesis operon, the pilus, and Rib, reflects adaptation to host immune pressures and should be taken into account in the ongoing development of a GBS vaccine. The presence of recurrent targets of evolution not previously implicated in virulence also opens the way for uncovering new functions involved in host colonization and GBS pathogenesis. IMPORTANCE The incidence of group B Streptococcus (GBS) neonatal disease continues to be a significant cause of concern worldwide. Strains belonging to clonal complex 17 (CC17) are the most frequently responsible for GBS infections in neonates, especially among late-onset disease cases. Therefore, we undertook the largest genomic study of GBS CC17 strains to date to decipher the genetic bases of their remarkable colonization and infection ability. We show that crucial functions involved in different steps of the colonization or infection process of GBS are distinctly mutated during the adaptation of CC17 to the human host. In particular, our results implicate the CovRS two-component regulator of virulence in the differentiation between carriage- and disease-associated isolates. Not only does this work raise important implications for the ongoing development of a vaccine against GBS but might also drive the discovery of key functions for GBS adaptation and pathogenesis that have been overlooked until now. Author Video: An author video summary of this article is available.