Caractérisation du système BAC : vers l'hypothèse d'un système Psp original chez Pseudomonas aeruginosa.

The emergence and spread of bacteria that are multi-resistant to antibiotics has become a real public health concern. In particular, P. aeruginosa is one of the major human opportunistic pathogens for which it is urgent to develop new therapeutic strategies. P. aeruginosa is a microorganism that preferentially targets immuno-compromised individuals. Thus, patients suffering from cystic fibrosis, cancer, AIDS, or hospitalized for a long period are subjects at high risk of P. aeruginosa infection. In these patients, these infections are associated with high morbidity and mortality, due to the persistence and resistance of bacteria colonizing host tissues most often within biofilms.My thesis work is part of the objective of discovering new targets for the design of future innovative treatments. In this manuscript I describe the latest elements of characterization of the BAC system (for “Biofilm Associated Cluster”), which allow us to consider it as an original Psp system. This system is thus made up of 6 proteins encoded by the bacABCDEF operon. The expression of this operon would occur in response to stimuli and would be dependent on the sigma factor σ54 and the Gac/Rsm two-components system. The structure of BacA (protein presenting a DUF2170 domain), newly elucidated, is homologous to that of T3SS chaperone proteins suggesting that BacA is involved in the regulation of bacABCDEF expression. The BacB protein is a structural homolog of the PspA and Vipp1 proteins, suggesting that the BAC system is involved in membrane protection under the action of a yet unknown extra-cytoplasmic stress. The BacD protein appears to be a flotillin, a “scaffolding” protein known to reside in functional membrane microdomains, also called lipid rafts.L’émergence et la propagation des bactéries multirésistantes aux antibiotiques est devenue une problématique majeure de santé publique. En particulier, P. aeruginosa fait partie des pathogènes opportunistes humains pour lesquels il devient urgent de développer de nouvelles stratégies thérapeutiques. P. aeruginosa est un germe qui cible préférentiellement les individus dont le système immunitaire est affaibli. Ainsi, les patients atteints de mucoviscidose, d’un cancer, du SIDA, ou hospitalisés sur une longue durée sont des sujets à haut risque d’infection à P. aeruginosa. Chez ces patients, ces infections sont associées à une forte morbidité et mortalité, en raison de la persistance et de la résistance des bactéries colonisant les tissus de l’hôte sous forme de biofilms.Mes travaux de thèse s’inscrivent dans l’objectif de découvrir de nouvelles cibles pour le design de futurs traitements novateurs. Je décris dans ce manuscrit les derniers éléments de caractérisation du système BAC (pour « Biofilm Associated Cluster »), qui nous permettent de l’envisager comme un système Psp original. Ce système est ainsi constitué de 6 protéines codées par l’opéron bacABCDEF. L’expression de cet opéron se ferait en réponse à des stimuli et serait sous la dépendance du facteur sigma σ54 et du système à deux composants Gac/Rsm. La structure de BacA (protéine présentant un domaine DUF2170), nouvellement élucidée, est homologue à celle de protéines chaperonnes du SST3 suggérant que BacA serait impliquée dans la régulation de l’expression de bacABCDEF. La protéine BacB est quant à elle un homologue structural des protéines PspA et Vipp1 supposant que le système BAC serait impliqué dans la protection membranaire sous l’action d’un stress extra-cytoplasmique encore inconnu. La protéine BacD semble être une flotilline, protéine « échafaudage » connue pour résider dans les microdomaines fonctionnels, aussi appelés lipid rafts

Characterization of the BAC system : towards the hypothesis of an original Psp system in P. aeruginosa

L’émergence et la propagation des bactéries multirésistantes aux antibiotiques est devenue une problématique majeure de santé publique. En particulier, P. aeruginosa fait partie des pathogènes opportunistes humains pour lesquels il devient urgent de développer de nouvelles stratégies thérapeutiques. P. aeruginosa est un germe qui cible préférentiellement les individus dont le système immunitaire est affaibli. Ainsi, les patients atteints de mucoviscidose, d’un cancer, du SIDA, ou hospitalisés sur une longue durée sont des sujets à haut risque d’infection à P. aeruginosa. Chez ces patients, ces infections sont associées à une forte morbidité et mortalité, en raison de la persistance et de la résistance des bactéries colonisant les tissus de l’hôte sous forme de biofilms.Mes travaux de thèse s’inscrivent dans l’objectif de découvrir de nouvelles cibles pour le design de futurs traitements novateurs. Je décris dans ce manuscrit les derniers éléments de caractérisation du système BAC (pour « Biofilm Associated Cluster »), qui nous permettent de l’envisager comme un système Psp original. Ce système est ainsi constitué de 6 protéines codées par l’opéron bacABCDEF. L’expression de cet opéron se ferait en réponse à des stimuli et serait sous la dépendance du facteur sigma σ54 et du système à deux composants Gac/Rsm. La structure de BacA (protéine présentant un domaine DUF2170), nouvellement élucidée, est homologue à celle de protéines chaperonnes du SST3 suggérant que BacA serait impliquée dans la régulation de l’expression de bacABCDEF. La protéine BacB est quant à elle un homologue structural des protéines PspA et Vipp1 supposant que le système BAC serait impliqué dans la protection membranaire sous l’action d’un stress extra-cytoplasmique encore inconnu. La protéine BacD semble être une flotilline, protéine « échafaudage » connue pour résider dans les microdomaines fonctionnels, aussi appelés lipid rafts.The emergence and spread of bacteria that are multi-resistant to antibiotics has become a real public health concern. In particular, P. aeruginosa is one of the major human opportunistic pathogens for which it is urgent to develop new therapeutic strategies. P. aeruginosa is a microorganism that preferentially targets immuno-compromised individuals. Thus, patients suffering from cystic fibrosis, cancer, AIDS, or hospitalized for a long period are subjects at high risk of P. aeruginosa infection. In these patients, these infections are associated with high morbidity and mortality, due to the persistence and resistance of bacteria colonizing host tissues most often within biofilms.My thesis work is part of the objective of discovering new targets for the design of future innovative treatments. In this manuscript I describe the latest elements of characterization of the BAC system (for “Biofilm Associated Cluster”), which allow us to consider it as an original Psp system. This system is thus made up of 6 proteins encoded by the bacABCDEF operon. The expression of this operon would occur in response to stimuli and would be dependent on the sigma factor σ54 and the Gac/Rsm two-components system. The structure of BacA (protein presenting a DUF2170 domain), newly elucidated, is homologous to that of T3SS chaperone proteins suggesting that BacA is involved in the regulation of bacABCDEF expression. The BacB protein is a structural homolog of the PspA and Vipp1 proteins, suggesting that the BAC system is involved in membrane protection under the action of a yet unknown extra-cytoplasmic stress. The BacD protein appears to be a flotillin, a “scaffolding” protein known to reside in functional membrane microdomains, also called lipid rafts

The Biofilm Resistance: Many Responses but Still Many Questions

International audienceMicrobial biofilms (i.e., bacterial and fungal) are often very deleterious due to their exceptional tolerance to treatments, in particular to conventional antimicrobials therapy. The processes involved in this low efficacy of antimicrobials on adherent cells have been widely studied in the last decades. It is now accepted that this tolerance of sessile cells is multiparametric. It involves different mechanisms, physico-chemical and biological. A major characteristic of biofilm microorganisms is that they are subjected to very deleterious environmental conditions which induce in them a large number of stress response mechanisms, some of which are obviously involved in this tolerance. On the other hand, few resistance systems, specifically expressed by sessile cells, have been described so far. Moreover, the fact that most natural and clinical biofilms are polymicrobial raises questions about the relevance of some observations obtained on monocultures

BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa

International audiencePreviously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes ( pa3732 to pa3729 ) that we named bac for “Biofilm Associated Cluster.” The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins

Image_2_BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.pdf

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for “Biofilm Associated Cluster.” The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.</p

BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for “Biofilm Associated Cluster.” The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins

Table_4_BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.pdf

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for “Biofilm Associated Cluster.” The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.</p

Image_3_BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.pdf

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for “Biofilm Associated Cluster.” The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.</p

Image_4_BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.pdf

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for “Biofilm Associated Cluster.” The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.</p

Table_3_BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.pdf

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for “Biofilm Associated Cluster.” The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.</p