Integrated Regulation of the Type III Secretion System and Other Virulence Determinants in Ralstonia solanacearum

In many plant and animal bacterial pathogens, the Type III secretion system (TTSS) that directly translocates effector proteins into the eukaryotic host cells is essential for the development of disease. In all species studied, the transcription of the TTSS and most of its effector substrates is tightly regulated by a succession of consecutively activated regulators. However, the whole genetic programme driven by these regulatory cascades is still unknown, especially in bacterial plant pathogens. Here, we have characterised the programme triggered by HrpG, a host-responsive regulator of the TTSS activation cascade in the plant pathogen Ralstonia solanacearum. We show through genome-wide expression analysis that, in addition to the TTSS, HrpG controls the expression of a previously undescribed TTSS-independent pathway that includes a number of other virulence determinants and genes likely involved in adaptation to life in the host. Functional studies revealed that this second pathway co-ordinates the bacterial production of plant cell wall-degrading enzymes, exopolysaccharide, and the phytohormones ethylene and auxin. We provide experimental evidence that these activities contribute to pathogenicity. We also show that the ethylene produced by R. solanacearum is able to modulate the expression of host genes and can therefore interfere with the signalling of plant defence responses. These results provide a new, integrated view of plant bacterial pathogenicity, where a common regulator activates synchronously upon infection the TTSS, other virulence determinants and a number of adaptive functions, which act co-operatively to cause disease

Développement d'anticorps monoclonaux humains de type IgA dirigés contre la partie C-terminale de la protéine d'enveloppe gp41 du VIH-1

La transmission du Virus de l Immunodéficience Humaine (VIH) par voie sexuelle représente le mode majoritaire de contamination (80%) (UNAIDS). Ce mode de contamination implique le passage du virus à travers les muqueuses et une interaction avec les cellules épithéliales et les cellules immunitaires présentes au sein de ces muqueuses (cellules dendritiques, macrophages ou lymphocytes). Les muqueuses représentent le principal site d'exposition de l organisme aux antigènes de l environnement. Les SIgA (IgA sécrétoires) présentes dans la lumière de ces muqueuses représentent la première ligne de défense immunitaire contre l infection et la colonisation des muqueuses. Les IgA sont capables d interagir avec les glycoprotéines (gp) exprimées à la surface du VIH et de bloquer l infection et/ou la transcytose à travers l épithélium muqueux. Nous avons pu étudier la prévalence des SIgA anti-gp41 et plus précisément anti-MPER présentes dans la salive parotidienne de personnes Exposées au VIH Séronégatives (ESN) et leur rôle dans l inhibition de l infection par le virus in vitro. Nous avons pu démontrer que ces sujets présentaient un taux plus important de SIgA anti-MPER neutralisantes. Ce premier travail nous a permis de valider la gp41 comme immunogène d intérêt pour la génération de SIgA neutralisantes. Nous avons pu générer des IgA1 dans un modèle murin a1Kl chimérique capable de produire des anticorps IgA1 humanisés. L immunisation de ces souris a permis la production de 6 anticorps monoclonaux spécifiques de la région MPER capables de reconnaître des épitopes conformationnels élargis, correspondant aux épitopes reconnus par le 2F5 et le 4E10. Les IgA1 présentaient de fortes capacités neutralisantes pour différentes souches de laboratoire et de souches primaires du VIH. Les études de caractérisation des fonctions antivirales de ces anticorps permettront de mieux définir le mode d action de ces anticorps. A notre connaissance, ces IgA1 neutralisantes anti-MPER sont les premières décrites à ce jour dans la littérature. De par leur faible immunogénicité et leur faible autoréactivité, ces anticorps peuvent facilement être intégrés dans des approches thérapeutiques locales ou par sérothérapie passive pour la protection après administration de SHIV dans des modèles animaux comme le macaque. L ensemble de mes travaux de thèse ont confirmé l intérêt thérapeutique potentiel des SIgA dans la lutte contre le VIH et notamment celles dirigées contre la partie gp41 de l enveloppeSexual transmission of the Human Immunodeficiency Virus is the major mode of contamination (80%) for this pathogen (UNAIDS). This mode of transmission involves a passage of the virus though the mucosa and an interaction with epithelial cells and immune cells present in the mucosa (dendritic cells, macrophages and lymphocytes). Mucosa represents the major site of exposure for the organism to environmental antigens. The IgA expressed in the lumen of mucosa are the first line of immune defence against infection and colonization of mucosa. IgA are able to interact with glycoproteins (gp) expressed on the surface of HIV and prevent infection and/or block epithelial transcytosis. In this study we have investigated the prevalence of SIgA anti-MPER present in the parotid saliva of Exposed to HIV but Seronegative individuals (ESN). This study has allowed us to validate gp41 as an immunogen of interest for the generation of neutralizing IgA. IgA1 were generated in a chimeric mice model a1Kl that produced humanized IgA1 type antibodies. Immunizations of these mice has led to the elicitation of six monoclonal antibodies specific to the MPER region able to recognize extended conformational neutralizing epitopes of 2F5 and 4E10, two broadly neutralizing monoclonal antibodies specific to MPER. Elicited IgA1 have potent neutralizing properties for both laboratory and primary HIV strains. Characterization studies of the antiviral functions of these antibodies will further define the mode of action of these antibodies. To our knowledge, these anti-MPER humanized monoclonal neutralizing IgA1 antibodies are the first of this type described to date in the literature. By their low immunogenicity and autoreactivity, these antibodies can be easily integrated into local therapeutic approaches or passive serotherapy for protection in animal models such as the macaque challenged with SHIV. All the results of my PhD work confirm the great interest of gp41-specific SIgA as therapeutic agents against HIVST ETIENNE-Bib. électronique (422189901) / SudocSudocFranceF

Immunoglobulin-A and the pathogenesis of schistosomal glomerulopathy

Immunoglobin-A and the pathogenesis of schistosomal glomerulopathy. Several observations suggest that the evolution of schistosomal glomerulopathy into clinically overt and progressive disease may involve pathogenetic mechanisms other than simple glomerular deposition of parasitic antigens. In a previous study, IgA was suggested tobe a mediator of late glomerular lesions in this disease. This issue is further addressed in this work. The study includes 32 patients with hepatosplenic schistosomiasis, of whom 16 had overt glomerular involvement, along with four control groups: (a) 15 healthy volunteers; (b) 15 patients with simple intestinal mansoniasis; (c) 17 patients with non-schistosomal chronic liver disease; and (d) 21 subjects with primary nephrotic syndrome not associated with schistosomiasis. Routine assessment was done for all subjects including confirmatory tests for schistosomal infection, liver and renal function tests, hepatitis viral markers and abdominal ultrasonography. The total serum concentrations of IgG, IgM, IgA were measured, as well astheir respective circulating immune complexes, rheumatoid factors, anti-gliadin- and anti-DNA-antibodies. Liver and renal biopsies were obtained from the relevant groups and studied by light microscopy. Renal biopsies were also examined by immunofluorescence. Patients with simple intestinal schistosomiasis had a significant increase in IgM antigliadin antibodies. Those complicated with hepatosplenic involvement also had a significant increase in the mean IgG anti-gliadin antibodies, IgG rheumatoid factor and IgM anti-DNA activity. Cases further complicated by overt glomerular disease showed a distinct IgA predominance, mainly expressed in the serum anti-gliadin antibody pool and anti-DNA activity. This profile was essentially similar to that observed in control cirrhotics. There was a significant increase in the frequency of IgA glomerular deposits in renal biopsies obtained from patients with overt schistosomal glomerulopathy, in contrast to control nephrotics. The deposits were mainly mesangial, but were also encountered in subendothelial, subepithelial and peritubular locations. Their frequency was significantly higher with more advanced lesions as seen by light microscopy. The relevance of these data is discussed, leading to the following conclusions: (a) serum IgA-anti-gliadin and -anti-DNA antibodies, and glomerular IgA deposits are markers of significant renal involvement in patients with hepatosplenic schistosomiasis, (b) IgA may be involved in the pathogenesis of advanced glomerular pathology when superimposed on parasite-induced lesions, (c) There is a significant increase in serum auto-reactivity in hepatosplenic schistosomiasis, which may also have pathogentic implications, (d) Increased production by the inflammatory bowel lesions, impaired clearance by the fibrotic livers and probable switching of immunoglobulin synthesis are suggested to explain the observed IgA predominance in those who develop renal complications

Specific Antibody Production by Blood B Cells is Retained in Late Stage Drug-naïve HIV-infected Africans

Unseparated peripheral blood mononuclear cells (PBMCs) obtained from drug-naïve African individuals living in a context of multi-infections and presenting with high viral load (VL), were cultured in vitro and tested for their ability to produce antibodies (Abs) reacting with HIV-1 antigens. Within these PBMCs, circulating B cells were differentiated in vitro and produced IgG Abs against not only ENV, but also GAG and POL proteins. Under similar experimental conditions, HAART treated patients produced Abs to ENV proteins only. The in vitro antibody production by drug-naïve individuals' PBMCs depended on exogenous cytokines (IL-2 and IL-10) but neither on the re-stimulation of reactive cells in cultures by purified HIV-1-gp 160 antigen nor on the re-engagement of CD40 surface molecules. Further, it was not abrogated by the addition of various monoclonal Abs (mAbs) to co-stimulatory molecules. This suggests that the in vitro antibody production by drug-naïve individuals' PBMCs resulted from the maturation of already envelope and core antigen-primed, differentiated B cells, presumably pre-plasma cells, which are not known to circulate at homeostasy. As in vitro produced Abs retained the capacity of binding antigen and forming complexes, this study provides pre-clinical support for functional humoral responses despite major HIV- and other tropical pathogen-induced B cell perturbations

Identification of Germinal Center B Cells in Blood from HIV-infected Drug-naive Individuals in Central Africa

To better understand the pathophysiology of B cell populations—the precursors of antibody secreting cells—during chronic human immunodeficiency virus (HIV) infection, we examined the phenotype of circulating B cells in newly diagnosed Africans. We found that all African individuals displayed low levels of naive B cells and of memory-type CD27+ B cells, and high levels of differentiated B cells. On the other hand, HIV-infected African patients had a population of germinal center B cells (i.e. CD20+, sIgM-, sIgD+, CD77+, CD138±), which are generally restricted to lymph nodes and do not circulate unless the lymph node architecture is altered. The first observations could be linked to the tropical environment whereas the presence of germinal center B cells may be attributable to chronic exposure to HIV as it is not observed in HIV-negative African controls and HAART treated HIV-infected Europeans. It may impact the management of HIV infection in countries with limited access to HIV drugs and urges consideration for implementation of therapeutic vaccines

Metabolic Adaptation of Ralstonia solanacearum during Plant Infection: A Methionine Biosynthesis Case Study

MetE and MetH are two distinct enzymes that catalyze a similar biochemical reaction during the last step of methionine biosynthesis, MetH being a cobalamin-dependent enzyme whereas MetE activity is cobalamin-independent. In this work, we show that the last step of methionine synthesis in the plant pathogen Ralstonia solanacearum is under the transcriptional control of the master pathogenicity regulator HrpG. This control is exerted essentially on metE expression through the intermediate regulator MetR. Expression of metE is strongly and specifically induced in the presence of plant cells in a hrpG- and metR-dependent manner. metE and metR mutants are not auxotrophic for methionine and not affected for growth inside the plant but produce significantly reduced disease symptoms on tomato whereas disruption of metH has no impact on pathogenicity. The finding that the pathogen preferentially induces metE expression rather than metH in the presence of plant cells is indicative of a probable metabolic adaptation to physiological host conditions since this induction of metE occurs in an environment in which cobalamin, the required co-factor for MetH, is absent. It also shows that MetE and MetH are not functionally redundant and are deployed during specific stages of the bacteria lifecycle, the expression of metE and metH being controlled by multiple and distinct signals

Tissue Compartment Analysis for Biomarker Discovery by Gene Expression Profiling

BACKGROUND:Although high throughput technologies for gene profiling are reliable tools, sample/tissue heterogeneity limits their outcomes when applied to identify molecular markers. Indeed, inter-sample differences in cell composition contribute to scatter the data, preventing detection of small but relevant changes in gene expression level. To date, attempts to circumvent this difficulty were based on isolation of the different cell structures constituting biological samples. As an alternate approach, we developed a tissue compartment analysis (TCA) method to assess the cell composition of tissue samples, and applied it to standardize data and to identify biomarkers. METHODOLOGY/PRINCIPAL FINDINGS:TCA is based on the comparison of mRNA expression levels of specific markers of the different constitutive structures in pure isolated structures, on the one hand, and in the whole sample on the other. TCA method was here developed with human kidney samples, as an example of highly heterogeneous organ. It was validated by comparison of the data with those obtained by histo-morphometry. TCA demonstrated the extreme variety of composition of kidney samples, with abundance of specific structures varying from 5 to 95% of the whole sample. TCA permitted to accurately standardize gene expression level amongst >100 kidney biopsies, and to identify otherwise imperceptible molecular disease markers. CONCLUSIONS/SIGNIFICANCE:Because TCA does not require specific preparation of sample, it can be applied to all existing tissue or cDNA libraries or to published data sets, inasmuch specific operational compartments markers are available. In human, where the small size of tissue samples collected in clinical practice accounts for high structural diversity, TCA is well suited for the identification of molecular markers of diseases, and the follow up of identified markers in single patients for diagnosis/prognosis and evaluation of therapy efficiency. In laboratory animals, TCA will interestingly be applied to central nervous system where tissue heterogeneity is a limiting factor