Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow–generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.

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Neisseria meningitidis is a cause of meningitis epidemics worldwide and of rapidly progressing fatal septic shock. A crucial step in the pathogenesis of invasive meningococcal infections is the adhesion of bloodborne meningococci to both peripheral and brain endothelia, leading to major vascular dysfunction. Initial adhesion of pathogenic strains to endothelial cells relies on meningococcal type IV pili, but the endothelial receptor for bacterial adhesion remains unknown. Here, we report that the immunoglobulin superfamily member CD147 (also called extracellular matrix metalloproteinase inducer (EMMPRIN) or Basigin) is a critical host receptor for the meningococcal pilus components PilE and PilV. Interfering with this interaction potently inhibited the primary attachment of meningococci to human endothelial cells in vitro and prevented colonization of vessels in human brain tissue explants ex vivo and in humanized mice in vivo. These findings establish the molecular events by which meningococci target human endothelia, and they open new perspectives for treatment and prevention of meningococcus-induced vascular dysfunctions.

Pathogenic Neisseria express type IV pili (tfp), which have been shown to play a central role in the interactions of bacteria with their environment. The regulation of piliation thus constitutes a central element in bacterial life cycle. The PilC proteins are outer membrane-associated proteins that have a key role in tfp biogenesis since PilCnull mutants appear defective for fibre expression. Moreover, tfp are also subjected to retraction, which is under the control of the PilT nucleotide-binding protein.In this work, we bring evidence that fibre retraction involves the translocation of pilin subunits to the cytoplasmic membrane. Furthermore, by engineering meningococcal strains that harbour inducible pilC genes, and with the use of meningococcus-cell interaction as a model for the sequential observation of fibre expression and retraction, we show that the PilC proteins regulate PilTmediated fibre retraction.

We report the case of a cystic fibrosis patient colonized with a smooth-morphotype form of Mycobacterium abscessus who developed acute respiratory failure with the emergence of an isogenic rough (R) variant while he was recovering from peritonitis-induced shock. This report emphasizes the role of R forms in severe M. abscessus infections. (Fig. 1). CASE REPORTIn December 2003, the patient was admitted to the emergency unit of Hôpital Cochin, Assistance Publique-Hôpitaux de Paris, Paris, France, with a colic perforation and diffuse peritonitis secondary to a stercolith. Septic shock with severe hypoxemia occurred on day 1, requiring mechanical ventilation, inotropic adrenaline support, and treatment with combined antibiotics (ceftazidime, vancomycin, tobramycin, and ornidazole), hydrocortisone hemisuccinate, and drotrecogin alpha. Laboratory parameters showed lymphopenia (absolute lymphocyte count, 1,060/mm 3 ) and raised serum transaminase levels (alanine aminotransferase level, 80 IU/liter, three times the upper limit of the normal range). The patient's clinical status slowly improved. Bronchial aspiration performed on day 6 yielded A. fumigatus and a rough (R) variant of M. abscessus (M. abscessus CF01-R). Positive A. fumigatus antigenemia results led to the initiation of antifungal therapy on day 9.Unexpectedly, septic shock recurred on day 11. Severe respiratory failure was present (ratio of the partial pressure of oxygen in arterial blood to the fraction of inspired oxygen [PaO 2 /FiO 2 index], 190), associated with patchy alveolar consolidation (Fig. 2). A surgical lung biopsy (right thoracotomy) was performed on day 12: the biopsy specimen culture was positive for an R-morphotype isolate of M. abscessus as the sole pathogen, with concordant histology showing a granulomatous epithelioid reaction with giant cells in areas of peribronchovascular fibrosis and multiple microabscesses. Antibiotic therapy was shifted empirically to imipenem-cilastin, amikacin, and clarithromycin on day 13. Bronchial aspiration performed on day 13 yielded results similar to those from the lung biopsy, thus confirming the diagnosis of M. abscessus respiratory infection (7). Antibiotic susceptibility testing performed on several isolated strains (Institut Pasteur, Centre National de Références, Paris, France) showed the strains to be susceptible to the prescribed regimen. Bronchoalveolar lavage on day 32 still yielded an R-morphotype isolate of M. abscessus. Nebulized amikacin was added to the systemic anti-M. abscessus therapy on day 56. The patient became apyrexial on day 77, and mechanical ventilation was stopped on day 83. The patient was discharged on day 128 (April 2004) under a regimen of clarithromycin monotherapy, which was maintained until April 2005. Sputum samples remained repeatedly positive for M. abscessus (R form) throughout treatment (Fig. 1) (24) and multilocus sequence typing (data not shown) confirmed the clonal nature of the isolates of different morphotypes (Fig. 3). These results established the persistence ...

SummaryPilus-mediated adherence makes an essential contribution to the pathogenesis of Neisseria meningitidis by allowing the initial localized adherence. Pili are assembled from a protein subunit called pilin. Two proteins, PilC1 and PilC2, are also key elements in the formation of pili as the production of at least one PilC protein is required for pilus assembly. In addition, PilC1 but not PilC2 modulates adhesiveness, most probably by being the adhesin. Recently, both genes have been demonstrated to be controlled by different promoters, pilC2 is expressed from a single transcription starting point (TSP), whereas pilC1 has three TSPs. One of these, PC1.1, corresponds to the unique TSP of pilC2, and two others, PC1.2 and PC1.3, are located in a region upstream of pilC1 but not pilC2. This suggests that both genes may be under the control of separate regulatory pathways. In this work, by engineering pilC1-lacZ and pilC2-lacZ transcriptional fusions, we provide evidence that expression of pilC1, but not that of pilC2, is transiently induced by bacterial cell contact. This induction required viable cells, did not need the presence of pili and relied on the expression of pilC1 from PC1.3. Destruction of this TSP by site-directed mutagenesis did not significantly diminish the piliation level or the basal expression of PilC1, but led to the loss of cell contact-dependent upregulation of pilC1 and to a dramatic decrease in bacterial adhesiveness. Taken together, these data demonstrate that cell contact-dependent upregulation of the transcription of pilC1 at PC1.3 is essential for meningococcal pilusmediated adhesion.

Young cystic fibrosis (CF) patients' airways are mainly colonized by Staphylococcus aureus, while Pseudomonas aeruginosa predominates in adults. However, the mechanisms behind this infection switch are unclear. Here, we show that levels of type-IIA-secreted phospholipase A2 (sPLA2-IIA, a host enzyme with bactericidal activity) increase in expectorations of CF patients in an age-dependent manner. These levels are sufficient to kill S. aureus, with marginal effects on P. aeruginosa strains. P. aeruginosa laboratory strains and isolates from CF patients induce sPLA2-IIA expression in bronchial epithelial cells from CF patients (these cells are a major source of the enzyme). In an animal model of lung infection, P. aeruginosa induces sPLA2-IIA production that favours S. aureus killing. We suggest that sPLA2-IIA induction by P. aeruginosa contributes to S. aureus eradication in CF airways. Our results indicate that a bacterium can eradicate another bacterium by manipulating the host immunity.

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