Calcium-binding RTX proteins are equipped with C-terminal secretion signals and translocate from the Ca(2+)-depleted cytosol of Gram-negative bacteria directly into the Ca(2+)-rich external milieu, passing through the "channel-tunnel" ducts of type I secretion systems (T1SSs). Using Bordetella pertussis adenylate cyclase toxin, we solved the structure of an essential C-terminal assembly that caps the RTX domains of RTX family leukotoxins. This is shown to scaffold directional Ca(2+)-dependent folding of the carboxy-proximal RTX repeat blocks into β-rolls. The resulting intramolecular Brownian ratchets then prevent backsliding of translocating RTX proteins in the T1SS conduits and thereby accelerate excretion of very large RTX leukotoxins from bacterial cells by a vectorial "push-ratchet" mechanism. Successive Ca(2+)-dependent and cosecretional acquisition of a functional RTX toxin structure in the course of T1SS-mediated translocation, through RTX domain folding from the C-terminal cap toward the N terminus, sets a paradigm that opens for design of virulence inhibitors of major pathogens.

Intestinal microbiota mediate toxicity of irinotecan (CPT-11) cancer therapies and cause systemic infection after CPT-11-induced loss of barrier function. The intestinal microbiota and their functions are thus potential targets for treatment to mitigate CPT-11 toxicity. However, microbiota changes during CPT-11 therapy remain poorly described. This study analysed changes in intestinal microbiota induced by CPT-11 chemotherapy. Qualitative and quantitative taxonomic analyses, and functional analyses were combined to characterize intestinal microbiota during CPT-11-based chemotherapy, and in presence or absence of oral glutamine, a treatment known to reduce CPT-11 toxicity. In the first set of experiments tumour-bearing rats received a dose-intensive CPT-11 regimen (125 mg kg −1 ×3 days), with or without oral glutamine bolus (0.75 g kg −1 ). In a subsequent more clinically-oriented chemotherapy regimen, rats received two cycles of CPT-11 (50 mg kg −1 ) followed by 5-flurouracil (50 mg kg −1 ). The analysis of fecal samples over time demonstrated that tumours changed the composition of intestinal microbiota, increasing the abundance of clostrridial clusters I, XI, and Enterobacteriaceae . CPT-11 chemotherapy increased cecal Clostridium cluster XI and Enterobacteriaceae , particularly after the dose-intensive therapy. Glutamine treatment prevented the reduced abundance of major bacterial groups after CPT-11 administration; i.e. total bacteria, Clostridium cluster VI, and the Bacteroides -group. Virulence factor/toxin genes of pathogenic Escherichia coli and Clostridium difficile were not detected in the cecal microbiota. In conclusion, both colon cancer implantation and CPT-11-based chemotherapies disrupted the intestinal microbiota. Oral glutamine partially mitigated CPT-11 toxicity and induced temporary changes of the intestinal microbiota.

Bordetella pertussis is the causative agent of whooping cough, a respiratory disease still considered as a major public health threat and for which recent re-emergence has been observed. Constant reshuffling of Bordetella pertussis genome organization was observed during evolution. These rearrangements are essentially mediated by Insertion Sequences (IS), a mobile genetic elements present in more than 230 copies in the genome, which are supposed to be one of the driving forces enabling the pathogen to escape from vaccine-induced immunity.Here we use high-throughput sequencing approaches (RNA-seq and differential RNA-seq), to decipher Bordetella pertussis transcriptome characteristics and to evaluate the impact of IS elements on transcriptome architecture. Transcriptional organization was determined by identification of transcription start sites and revealed also a large variety of non-coding RNAs including sRNAs, leaderless mRNAs or long 3′ and 5′UTR including seven riboswitches. Unusual topological organizations, such as overlapping 5′- or 3′-extremities between oppositely orientated mRNA were also unveiled. The pivotal role of IS elements in the transcriptome architecture and their effect on the transcription of neighboring genes was examined. This effect is mediated by the introduction of IS harbored promoters or by emergence of hybrid promoters. This study revealed that in addition to their impact on genome rearrangements, most of the IS also impact on the expression of their flanking genes. Furthermore, the transcripts produced by IS are strain-specific due to the strain to strain variation in IS copy number and genomic context.

The adenylate cyclase toxin–hemolysin (CyaA) plays a key role in the virulence of Bordetella pertussis. CyaA penetrates complement receptor 3–expressing phagocytes and catalyzes uncontrolled conversion of cytosolic ATP to the key second messenger molecule cAMP. This paralyzes the capacity of neutrophils and macrophages to kill bacteria by complement-dependent oxidative burst and opsonophagocytic mechanisms. We show that cAMP signaling through the protein kinase A (PKA) pathway activates Src homology domain 2 containing protein tyrosine phosphatase (SHP) 1 and suppresses production of bactericidal NO in macrophage cells. Selective activation of PKA by the cell-permeable analog N6-benzoyladenosine-3′,5′-cyclic monophosphate interfered with LPS-induced inducible NO synthase (iNOS) expression in RAW264.7 macrophages, whereas inhibition of PKA by H-89 largely restored the production of iNOS in CyaA-treated murine macrophages. CyaA/cAMP signaling induced SHP phosphatase–dependent dephosphorylation of the c-Fos subunit of the transcription factor AP-1 and thereby inhibited TLR4-triggered induction of iNOS gene expression. Selective small interfering RNA knockdown of SHP-1, but not of the SHP-2 phosphatase, rescued production of TLR-inducible NO in toxin-treated cells. Finally, inhibition of SHP phosphatase activity by NSC87877 abrogated B. pertussis survival inside murine macrophages. These results reveal that an as yet unknown cAMP-activated signaling pathway controls SHP-1 phosphatase activity and may regulate numerous receptor signaling pathways in leukocytes. Hijacking of SHP-1 by CyaA action then enables B. pertussis to evade NO-mediated killing in sentinel cells of innate immunity.