Regulation of ExoS Production and Secretion by Pseudomonas aeruginosa in Response to Tissue Culture Conditions

Abstract

This study was initiated to characterize the regulation and secretion of ExoS by Pseudomonas aeruginosa during contact with eukaryotic cells. The production of ExoS was monitored by a sensitive ADP-ribosyltransferase activity assay, and specific activities were calculated for supernatant and cell-associated fractions. Time course analysis indicated that ExoS was produced after a lag period, suggesting that induction of the regulon is necessary for the expression of detectable amounts of enzyme activity. Under tissue culture growth conditions, ExoS was induced when P. aeruginosa was in contact with Chinese hamster ovary (CHO) cells or after growth in tissue culture medium with serum. The serum induction of ExoS appeared to result in generalized type III secretion, while induction by contact with CHO cells appeared to result in polarized type III secretion. Mutants in the type III secretory system that express a null phenotype for ExoS production in bacteriological medium produced but did not secrete the enzyme when P. aeruginosa was grown under inducing conditions in tissue culture medium. These results suggest that both induction and secretion of ExoS may differ when the bacteria are exposed to different growth environments. The putative type III translocation proteins and secretion apparatus of P. aeruginosa were required for translocation of bacterial factors that mediate changes in CHO cell morphology during infection.

Biological Effects of Pseudomonas aeruginosa Type III-Secreted Proteins on CHO Cells

“…In previous studies, we have used ExoS ADP-ribosyltransferase activity to measure type III-mediated translocation from P. aeruginosa 388 (expresses ExoS and ExoT) into CHO cells (19). To determine if PA103⌬exoUexoT::Tc could be used to study translocation of individual components, we pretreated CHO cells with cytochalasin D to inhibit the uptake of bacteria.…”

“…Infected CHO cells were lysed with 150 l of distilled water. The lysate was subjected to centrifugation at 14,000 ϫ g (4°C), and a portion of the soluble fraction was retained to perform ADP-ribosyltransferase activity assays (lysate-associated activity) as described previously (19). A duplicate well, not treated with antibiotics, was used to perform viable counts.…”

A translocator‐specific export signal establishes the translocator–effector secretion hierarchy that is important for type III secretion system function

“…The T3SS is a molecular syringe that spans the bacterial envelope. While the apparatus is pre‐assembled, effector secretion is triggered in a cell contact‐dependent manner (Rosqvist et al ., ; Vallis et al ., ; Nordfelth and Wolf‐Watz, ). Delivery of effector proteins into the host cell requires a specialized set of proteins called translocators.…”

Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system

“…Expression of the T3SS genes is finely tuned to environmental conditions. The two most potent inducing signals for T3SS gene expression are contact of P. aeruginosa with host cells and extracellular calcium concentrations in the micromolar range (hereafter referred to as 'low Ca 2+ ') (Frank, 1997;Vallis et al, 1999). Host cell contact and low Ca 2+ also serve as inducing signals for the highly homologous T3SSs of Yersinia sp.…”