Browsing by Subject "Exotoxin A"
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Item The effect of environmental conditions on exotoxin A regulation and production in Pseudomonas aeruginosa(Texas Tech University, 2006-12) Gaines, Jennifer M.; Hamood, Abdul N.; Faust, Charles; Francisco, Michael J. D. S.; Rolfe, Rial; Kennedy, Ronald C.Cystic fibrosis (CF) is a genetic disease that causes the thick accumulation of mucus in the lung alveoli. The mucus layer provides a suitable environment for the growth of bacteria including the predominant pathogen Pseudomonas aeruginosa. To adapt to the reduced environmental oxygen (EO) and iron-limited conditions within the CF mucus, P. aeruginosa likely alters the production of its virulence factors such as pyoverdine and exotoxin A (ETA). Pyoverdine is a siderophore that competes with the host for free and bound iron and is encoded by the pyoverdine genes such as pvdD. ETA, encoded by toxA, is an ADP-ribosylating enzyme that transfers a NAD+ moiety onto elongation factor 2, which halts host cell protein synthesis. Regulation of toxA expression is highly complex and dependent upon several activators including RegA, PtxR, and PvdS. RegA is considered essential for toxA expression and ETA production. PtxR is required for maximal toxA expression and appears to regulate toxA through regA. PvdS is an alternative sigma factor that positively regulates the pyoverdine genes, including pvdD, and appears to regulate toxA expression through regA and ptxR. We hypothesized that under reduced EO and as part of the adaptation process, P. aeruginosa alters ETA production by a mechanism(s) that involves some or all of the above-described regulators. We examined toxA expression throughout the growth cycle of the P. aeruginosa strain PAO1 under different levels of EO (20 %-0 %) using beta-galactosidase reporter fusions and real time polymerase chain reaction. We also measured the amount of secreted ETA protein by sandwich enzyme linked immunoabsorbent assay. Results showed that toxA transcription and ETA synthesis increased as the level of EO decreased. In contrast, the expression of pvdD and the production of pyoverdine were reduced under lower EO levels. The pattern of regA expression was similar to that of pvdD, but ptxR expression followed the same trend as toxA. These results suggest that reduced EO increases toxA expression in P. aeruginosa by a unique mechanism that is different from the known regulatory circuit described for aerobic conditions. Since PvdS is a key regulator of toxA and pvdD, we examined its role in this phenomenon under aerobic (20 %) shaking and microaerobic (10 %) static conditions. The expression levels of toxA, regA, ptxR, and pvdD were determined throughout the growth cycle of PAO1 and its pvdS isogenic mutant under both conditions. The expression of regA and pvdD were stringently regulated by PvdS under both conditions. However, while toxA expression was modulated by PvdS under both conditions, expression of ptxR was partially regulated by PvdS only under aerobic-shaking conditions. Purified PvdS complexed with core RNA polymerase (RNAP) has been shown to bind to a specific consensus sequence in the promoter regions of the pyoverdine genes including pvdD. The upstream regions of toxA, regA, and ptxR contain the potential binding site for PvdS. Using core-RNAP and purified PvdS, we determined that the PvdS-RNAP holoenzyme specifically bound to the upstream regions of toxA, regA, and ptxR. Results of our studies suggest that (1) reduced EO increases toxA and ptxR expression in P. aeruginosa, (2) PvdS regulates the expression of toxA, regA, and ptxR by directly binding to the upstream regions of these genes, (3) the increase in toxA expression appears to occur through a mechanism that is not related to PvdS or RegA since expression of both regulators is decreased under reduced EO, and (4) PtxR may play a role in toxA regulation under lower levels of EO. This study represents the first report of a potential toxA regulatory mechanism that does not involve the main regulators, RegA and PvdS. Therefore, within the thick mucus of the CF lung, P. aeruginosa may utilize such a mechanism to manipulate the production of individual virulence factors that are controlled by a common regulator.Item The role of Vfr and adenylate cyclase in regulating exotoxin a production by pseudomonas aeruginosa(2008-05) Davinic, Marko; Hamood, Abdul N.; Francisco, Michael J. D. S.; Jeter, Randall M.Exotoxin A (ETA) production by Pseudomonas aeruginosa depends on the virulence factor regulator Vfr, a homologue of the Escherichia coli catabolite repressor protein CRP. In E. coli, cyclic AMP activates CRP. Recent evidence indicates that the P. aeruginosa iron-starvation sigma factor PvdS enhances ETA production through the ETA regulatory gene regA. In this study, we examined the relationship between vfr, pvdS, and regA in regulating ETA production by P. aeruginosa. This was done using mutants defective in vfr, pvdS, regA, or the adenylate cyclase genes cyaA and cyaB (cyaAB); plasmids that individually overexpress these genes; and lacZ transcriptional/translational fusion plasmids. Throughout the growth cycle of PAOÄvfr, ETA concentration and regA expression were significantly reduced but pvdS expression was not affected. The presence of a multicopy vfr plasmid significantly increased ETA production in PAO::pvdS and PAOÄregA, yet overexpression of either regA or pvdS did not enhance ETA production in PAOÄvfr. RT-PCR analysis showed that iron had no effect on the accumulation of vfr mRNA in PAO1. Compared with its parent strain PAK, ETA production by PAKÄcyaAB was significantly reduced. This defect was complemented by a plasmid overexpressing cyaB. Neither regA overexpression nor pvdS overexpression enhanced ETA production by PAKÄcyaAB. However, cyaB overexpression enhanced ETA production in PAOÄpvdS and PAOÄregA. These results suggest that: (1) Vfr may regulate ETA production in P. aeruginosa independently of regA and pvdS; (2) Vfr does not regulate pvdS expression; (3) vfr expression is not regulated by iron; and (4) Vfr activation, by cyclic AMP through CyaB, is important for ETA production.