Functional analysis of exsC and exsB in regulation of exoenzyme S production by Pseudomonas aeruginosa

Abstract

Expression of ExsC, ExsB, and ExsA (the exoenzyme S trans-regulatory locus) of Pseudomonas aeruginosa was analyzed by using complementation, RNase protection, translational fusion, and T7-directed protein expression analyses. T7 expression analyses in E. coli hosts demonstrated that ExsC, ExsA, and a truncated form of ExsD (a partial open reading frame located 3 of ExsA) were translated; however, a product corresponding to ExsB was undetectable. T7-mediated transcription and translation of the antisense strand resulted in production of a 18.5-kDa product, termed ExsB, which overlapped the predicted ExsB product. In complementation experiments, deletion of the region encoding ExsB and most of ExsB severely reduced exoenzyme S production. Site-specific mutagenesis of the start codons for ExsB and ExsB, however, did not affect exoenzyme S production. RNase protection studies were initiated to examine the hypothesis that RNA encoded within the ExsB/ExsB region exerted a regulatory effect. RNA encoding ExsB was not detectable from chromosomal genes or complementation constructs, indicating that ExsB was not expressed in P. aeruginosa. To determine the pattern of translation, a chloramphenicol acetyltransferase gene (cat) reporter was fused in frame with ExsB and with ExsA in the context of the entire locus or in the absence of the exsB region. These experiments indicated that exsB was not translated but that deletion of the exsB region affected the translation of ExsA-CAT. RNase protection assays further suggested that deletion of exsB resulted in a processing of ExsA mRNA. Our data indicate that the untranslated exsB region of the trans-regulatory locus mRNA mediates either the stability or the translation of exsA. Complementation analysis further suggests that ExsC may play a role in the translation or stability of ExoS.Exoenzyme S is an ADP-ribosyltransferase produced and secreted by the opportunistic pathogen Pseudomonas aeruginosa (3,5,22,34,42). Two immunologically related forms of exoenzyme S with molecular masses of 49 and 53 kDa have been identified (22). The two forms are encoded by separate but coordinately regulated genes (48). Exoenzyme S-mediated ADP-ribosyltransferase activity requires the participation of a eukaryotic protein termed FAS (Factor activating exoenzyme S) (5,8). FAS is a member of the 14-3-3 family of proteins, which regulate the activity and interaction of several eukaryotic proteins (15). Exoenzyme S covalently modifies monomeric vimentin (5, 6) and a variety of small (21-to 25-kDa) GTP-binding proteins of the H-and K-Ras families (5, 7). In burn wound and lung infection models, exoenzyme S production correlates with the dissemination of P. aeruginosa from epithelial colonization sites to the bloodstream (34,35,36). An attractive model that fits these data suggests that exoenzyme S ADP-ribosyltransferase activity disrupts epithelial cell signal transduction pathways, resulting in a breakdown of the epithelial barrier.Genetic studies and complementation analysis of the exoenzyme S-defici...

Control of the Type 3 Secretion System in Vibrio harveyi by Quorum Sensing through Repression of ExsA

“…Therefore, the chromosomal exsB::cat mutation appears to lead to a reduction in T3SS gene expression due to polar effects on exsA because exsB and exsA exist in an operon. The orientation of these two genes is reminiscent of the exsB and exsA genes of Pseudomonas aeruginosa, which have been shown to be expressed in the exsCEBA operon (6,21,44). Although the exsA gene of V. harveyi is 44% identical to its P. aeruginosa exsA homolog, the exsB genes of these organisms are only 13% identical.…”

Mapping of a YscY Binding Domain within the LcrH Chaperone That Is Required for Regulation of Yersinia Type III Secretion

“…It is pertinent that the genome of P. aeruginosa is uniquely large, expanded by the presence of many genes coding for putative regulatory factors (66). This is now reflected by a number of independent studies which connect several of these factors with a complex pattern of type III secretion regulation in P. aeruginosa (13,33,34,40,47,58,59,72,78,79). Therefore, the need for a regulatory complex involving PcrH and Pcr4 is likely bypassed by one or more of these numerous regulatory factors.…”

ExsB Is Required for Correct Assembly of the Pseudomonas aeruginosa Type III Secretion Apparatus in the Bacterial Membrane and Full Virulence In Vivo

“…The first attempts to detect ExsB in P. aeruginosa failed, and this protein was then thought not to be expressed (42). More recently, the use of antibodies raised against this protein allowed the demonstration of its presence in P. aeruginosa outer membrane fractions, and its crystallographic structure was solved (41).…”

“…It was thought for more than a decade that this protein was not produced in P. aeruginosa (42), suggesting an important evolutionary divergence between P. aeruginosa and Yersinia spp. (35).…”