Transcriptional Induction of the Pseudomonas aeruginosa Type III Secretion System by Low Ca ²⁺ and Host Cell Contact Proceeds through Two Distinct Signaling Pathways

Abstract

The opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to intoxicate eukaryotic host cells. Transcription of the T3SS is induced under calcium-limited growth conditions or following intimate contact of P. aeruginosa with host cells. In the present study, we demonstrate that expression of the T3SS is controlled by two distinct regulatory mechanisms and that these mechanisms are differentially activated in a host cell-dependent manner. The first mechanism is dependent upon ExsC, a regulatory protein that couples transcription of the T3SS to the activity of the type III secretion machinery. ExsC is essential for induction of the T3SS under low-calcium-growth conditions and for T3SS-dependent cytotoxicity towards social amoebae, insect cells, and erythrocytes. The second regulatory mechanism functions independently of ExsC and is sufficient to elicit T3SS-dependent cytotoxicity towards certain types of mammalian cells. Although this second pathway (ExsC independent) is sufficient, an exsC mutant demonstrates a lag in the induction of cytotoxicity towards Chinese hamster ovary cells and is attenuated for virulence in a mouse pneumonia model. We propose that the ExsC-dependent pathway is required for full cytotoxicity towards all host cell types tested whereas the ExsC-independent pathway may represent an adaptation that allows P. aeruginosa to increase expression of the T3SS in response to specific types of mammalian cells.

Calcium and Iron Regulate Swarming and Type III Secretion in Vibrio parahaemolyticus

“…Calcium has not been reported as a T3SS inducing signal, although EGTA induces T3SS gene expression in organisms such as P. aeruginosa and Yersinia spp. (8,9). To examine whether the calcium-and EGTA-induced changes in gene expression were meaningful with respect to the virulence potential of the organism, the wild-type strain was grown on plates and harvested under the same conditions used for microarray analysis (i.e., HI, HI EGTA, and HI Ca) and used to infect Chinese hamster ovary (CHO) cells at an MOI of approximately 15.…”

“…In Yersinia pestis, 2.5 mM calcium prevents T3SS secretion, and it does so in part by modulating gene expression and protein activity (7,9,14,56). Similarly to Yersinia strains, secretion of P. aeruginosa T3SS effectors can be induced when calcium is chelated from the medium (8). The absence of calcium may reflect a particular environmental cue and/or mimic an unknown signal generated upon contact with host cells, perhaps by destabilizing the injectisome (reviewed in references 8, 15, and 59).…”

The Pseudomonas aeruginosa Magnesium Transporter MgtE Inhibits Transcription of the Type III Secretion System

“…(ii) ␤-Galactosidase assays. The P exsD-lacZ reporter strains were created using the mini-CTX-P exsD-lacZ construct, and ␤-galactosidase assays for planktonically grown cells were performed as previously reported (11,31). T3SS was stimulated by chelation of calcium with 1 mM EGTA.…”

“…Control of ExsA activity is maintained by alternative sets of complexes, where ExsD binds and sequesters ExsA (ExsD-ExsA), and ExsC can alternatively bind to ExsD or ExsE (ExsC-ExsD or ExsE-ExsC) (51). ExsE, which sits at the top of this cascade, is exported through the T3SS apparatus upon EGTA stimulation or mammalian cell contact, thus freeing ExsC (11,51). ExsC then binds to ExsD (ExsC-ExsD), which in turn liberates ExsA.…”

The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

“…1A) (13,44). Using cytotoxicity as a screen for regulators of T3SS gene expression by transposon mutagenesis, however, is tedious owing to the high rate of insertions that occur within T3SS genes and type IV pilus biosynthetic genes, the latter being required for attachment to host cells (45).…”