Identification of Potential Brucella abortus Virulence Factors Expressed during Macrophage Infection Using Differential Proteomics

ROSET M. S.; ALEFANTIS, T.; GARCÍA FERNANDEZ L.; COMERCI D. J; DELVECCHIO, V. G.

Buenos Aires

Conferencia; 2011 Brucellosis International Research Conference; 2011

Asociación Argentina de Microbiología

Brucella abortus is an intracellular pathogen that replicates within mammalian cells. Virulence of this pathogen is associated with the ability to survive and replicate within professional and non professional phagocytes. To further understand the pathogenesis of Brucella a comprehensive study of its molecular mechanisms of virulence is needed. One way to identify virulence mechanisms is to characterize proteins differentially expressed by the pathogen during its intracellular life, that most likely encode products required for survival within the hostand for progressive infection. Chemical isobaric tagging iTRAQ and 2D Gels were used to study changes in B. abortus protein expression during the intracellular life. Our results revealed several B. abortus proteins that are differentially expressed during intracellular life. Interestingly, some of them were described related to virulence in other bacterial pathogens, suggesting a conserved role in pathogenesis. We also identified proteins that have been already described to be involved in Brucella virulence. Two up-regulated proteins codified by BAB1_1117 and BAB1_1118 genes were identified as cyclophilins. Cyclophilins belong to the group of peptidyl prolyl cistrans isomerases (PPIases) which accelerate the rate-limiting cis-trans or trans-cis conformational changes at Xaa-Pro bonds during protein folding in both eukaryotes and prokaryotes. Spontaneous isomerization of the peptidylproline bond is slow and therefore PPIases accelerate rate-determining steps during protein folding. We constructed a double mutant of these genes by in frame deletion and evaluated their potential role during the infection. We demonstrated that a double mutant displays a reduced virulence in BALB/c mice and defective intracellular replication in HeLa cells. In addition, using the Bordetella pertussis Adenylate Cyclase reporter gene (CyaA) fusion approach we observed that at least one ciclophilin protein is translocated to the host cell citosol in a type IV secretion system dependent manner. These results confirm that intracellular proteomic analysis is a valuable approach for the identification of Brucella genes related to pathogenicity.is an intracellular pathogen that replicates within mammalian cells. Virulence of this pathogen is associated with the ability to survive and replicate within professional and non professional phagocytes. To further understand the pathogenesis of Brucella a comprehensive study of its molecular mechanisms of virulence is needed. One way to identify virulence mechanisms is to characterize proteins differentially expressed by the pathogen during its intracellular life, that most likely encode products required for survival within the hostand for progressive infection. Chemical isobaric tagging iTRAQ and 2D Gels were used to study changes in B. abortus protein expression during the intracellular life. Our results revealed several B. abortus proteins that are differentially expressed during intracellular life. Interestingly, some of them were described related to virulence in other bacterial pathogens, suggesting a conserved role in pathogenesis. We also identified proteins that have been already described to be involved in Brucella virulence. Two up-regulated proteins codified by BAB1_1117 and BAB1_1118 genes were identified as cyclophilins. Cyclophilins belong to the group of peptidyl prolyl cistrans isomerases (PPIases) which accelerate the rate-limiting cis-trans or trans-cis conformational changes at Xaa-Pro bonds during protein folding in both eukaryotes and prokaryotes. Spontaneous isomerization of the peptidylproline bond is slow and therefore PPIases accelerate rate-determining steps during protein folding. We constructed a double mutant of these genes by in frame deletion and evaluated their potential role during the infection. We demonstrated that a double mutant displays a reduced virulence in BALB/c mice and defective intracellular replication in HeLa cells. In addition, using the Bordetella pertussis Adenylate Cyclase reporter gene (CyaA) fusion approach we observed that at least one ciclophilin protein is translocated to the host cell citosol in a type IV secretion system dependent manner. These results confirm that intracellular proteomic analysis is a valuable approach for the identification of Brucella genes related to pathogenicity.