Biochemical Characterization of a Regulatory Cascade Controlling Transcription of the Pseudomonas aeruginosa Type III Secretion System

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a primary cause of pneumonia and urinary tract infections in intensive care units (1, 2). Among the most vulnerable individuals are those with immunodeficiency, cystic fibrosis, or severe burns or who require mechanical ventilation. A major virulence determinant of P. aeruginosa is a type III secretion system (T3SS). 2 The T3SS encodes a multiprotein complex, called an injectisome, which functions by injecting or translocating effector proteins directly into the cytoplasm of eukaryotic host cells. The T3SS of P. aeruginosa is used to translocate four known effectors that subvert signal transduction pathways to inhibit phagocytosis and elicit cytotoxicity toward host cells (3, 4).Expression of the P. aeruginosa T3SS is highly regulated and under the direct transcriptional control of ExsA, a member of the AraC family of transcriptional activators (5). Environmental signals, including contact of P. aeruginosa with host cells and Ca 2ϩ -limiting growth conditions, induce ExsA-dependent transcription of the T3SS (6, 7). Although the mechanism of induction by host cell contact is unclear, Ca 2ϩ depletion regulates T3SS gene transcription by inducing type III secretory activity (8). Transcription of T3SS genes is intimately coupled to type III secretory activity through a regulatory cascade that governs ExsA activity. This regulatory cascade consists of three interacting proteins (ExsD, ExsC, and ExsE). ExsD functions as an anti-activator by directly binding to and inhibiting ExsA-dependent transcription (8). ExsC functions as an anti-anti-activator by binding to and antagonizing ExsD activity (9). Finally, ExsE is an inhibitor of ExsC activity and a secreted substrate of the T3SS (10, 11). Dasgupta et al. (9) have proposed the following model to account for transcriptional induction of T3SS genes by Ca 2ϩ depletion. Under Ca 2ϩ replete conditions, ExsE is retained in the cytoplasm because of the lack of type III secretory activity and forms a complex with ExsC. The sequestration of ExsC by ExsE allows for the binding of the anti-activator ExsD to ExsA, thereby blocking transcription. In response to Ca 2ϩ depletion, however, ExsE is secreted into the extracellular milieu. The corresponding decrease in intracellular ExsE levels releases ExsC, which then sequesters ExsD and thereby makes ExsA available to activate transcription of T3SS genes.A common feature of proteins secreted by the T3SS is the requirement for chaperone activity (12). ExsC has biochemical characteristics of a type III-specific chaperone and is required for ExsE stability within the cytoplasm and for ExsE secretion (9, 10). T3SS-specific chaperones fall into one of three classes based on substrate specificity. Class I, II, and III chaperones facilitate secretion of effectors, components of the transloca-

ExsD Inhibits Expression of thePseudomonas aeruginosaType III Secretion System by Disrupting ExsA Self-Association and DNA Binding Activity

“…1). This finding is consistent with the fact that the binding affinity of ExsC is greater for ExsE than ExsD (37). Additionally, our data suggest that the affinity of ExsD is greater for ExsC than for ExsA.…”

“…In either case, the outcome would promote formation of the inhibitory ExsD·ExsA complex. A previous study demonstrated that addition of ExsE to the ExsD·ExsC complex results in the release of ExsD (37). We also found that incubation of ExsA His and the ExsD·ExsC His complex with ExsE His resulted in the release of free ExsD.…”

“…ExsA His , ExsC His , and ExsE His were purified by Ni 2ϩ -affinity chromatography as previously described (2,9,30,37). It should be noted that native ExsD is insoluble when expressed in E. coli and that neither amino-nor carboxy-terminal histidine-tagged forms of ExsD complement an exsD mutant (data not shown).…”

“…ExsE is a secreted regulator that prevents ExsC from associating with ExsD (27,30). The current model proposes that the nonpermissive condition for T3SS gene expression (i.e., highCa 2ϩ growth conditions) inhibits ExsA-dependent transcription because the binding equilibrium favors formation of the inhibitory ExsD·ExsA and ExsC·ExsE complexes (29,30,37). Inducing conditions (i.e., low calcium or contact of P. aeruginosa with host cells) activate type III secretory activity, resulting in secretion and/or translocation of ExsE.…”

Comparison of the Structure, Regulation and Functions between Type Three and Type Six Secretion System in Gram-Negative Bacteria

“…It consists of different levels, transcriptional, post-transcriptional, and post-translational. The expression of effector protein is regulated by the major transcriptional activator ExsA in response to environmental signals [21]. These signals include direct contact with host cell, and low calcium.…”