Identification of type III secreted products of the Pseudomonas aeruginosa exoenzyme S regulon

Abstract

Extracellular protein profiles from wild-type and regulatory or secretory isogenic mutants of the Pseudomonas aeruginosa exoenzyme S regulon were compared to identify proteins coordinately secreted with ExoS. Data from amino-terminal sequence analysis of purified extracellular proteins were combined with data from nucleotide sequence analysis of loci linked to exoenzyme S production. We report the identification of P. aeruginosa homologs to proteins of Yersinia spp. that function as regulators of the low calcium response, regulators of secretion, and mediators of the type III translocation mechanism.Exoenzyme S (ExoS) and ExoT are related extracellular ADP-ribosyltransferases secreted by a type III pathway in Pseudomonas aeruginosa (42,43). In previous studies we identified genes required for the regulation and secretion of ExoS (14,43). ExsA, the central regulator of the exoenzyme S regulon, controls transcription of structural, regulatory, and secretory loci (19,41,43,44). Loci, including pscB-L and pscN, encode homologs of type III secretion components (33, 43). Mutations in either regulatory or secretory loci result in a phenotype characterized by a defect in the production of ExoS, ExoT, and several additional extracellular proteins (15, 43). We postulated that proteins, coordinately regulated and secreted with ExoS, may represent additional virulence determinants.Identification of proteins coordinately secreted with ExoS and ExoT. To identify the proteins coordinately secreted with ExoS and ExoT, wild-type (PAK, PA103, and 388) and isogenic mutant (PAKexsA::⍀, PA103exsA::⍀, and 388exs1::Tn1[pscC]) strains of P. aeruginosa were grown under inducing conditions for exoenzyme S synthesis (medium containing 10 mM nitrilotriacetic acid) (38). Extracellular fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15,24). Major proteins that were common among the three wild-type strains included polypeptides that possessed molecular masses of 32.2, 32.8, 34, and 39 kDa and ExoT (53 kDa) ( Fig. 1; Table 1). ExoS (49 kDa) and a 42-kDa protein were absent from strain PA103. A 72-kDa protein (ExoU) was not produced in strains 388 and PAK. Mutations in either regulatory (exsA::⍀) or secretory (exs1::Tn1[pscC]) loci resulted in a general defect in the production of the set of proteins specific for each strain. Hybridization studies with probes for exoS and exoU confirm that the lack of ExoS synthesis in strain PA103 and the lack of ExoU synthesis in strains 388 and PAK are due to the absence of the corresponding genes (9-11).To determine if the extracellular proteins are related to previously cloned and sequenced members of the ExoS regulon, a protein sequencing approach was used. Proteins in concentrated culture supernatants were separated by SDS-PAGE, transferred to polyvinyl difluoride membranes, and stained with amido black (2). Stained bands corresponding to proteins of 32.2, 32.8, 34, 39 (isolated from the PA103 supernatant), and 42 (isolated from the PAK supernatant) kDa w...

Distribution of type III secretion gene clusters in Burkholderia pseudomallei, B. thailandensis and B. mallei

“…Secretion of ExoS and other proteins by P. aeruginosa can be induced artificially by growth in the presence of the chelator NTA [29]. The present observations indicate that expression of the B. pseudomallei TTS system is detectable during growth in Luria broth at 378C.…”

“…In Pseudomonas aeruginosa, the addition of NTA to growth media induces TTS genes [29]. The initial approach in the present study involved culturing the bacteria at 378C in the presence or absence of NTA.…”

Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of MucoidPseudomonas aeruginosaBiofilms

“…An inverse regulatory relationship was found to exist between alginate production and the extracellular protease LasA (47). However, Ca 2ϩ is known to affect the production and/or stability of certain extracellular proteins of P. aeruginosa (52,75). To determine if Ca 2ϩ addition affects expression and/or stability of extracellular proteins from an alginate-producing strain, we extracted cell supernatants of P. aeruginosa FRD1 cultured in BMM with and without added Ca 2ϩ .…”

“…In P. aeruginosa, secretion and/or stability of certain extracellular proteins is affected by calcium ions. Toxins secreted by the type III secretion system are repressed by calcium (75). The amounts of extracellular elastase (LasB) and LasA, secreted by the type II secretory pathway, increase in the presence of added calcium (52).…”

Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis

“…Central to this study is the fact that P. aeruginosa also possesses a homologous translocase operon (pcrGVHpopBD) (Fig. 1) (Yahr et al, 1997). Like the LcrH chaperone of Yersinia, PcrH is responsible for pre-secretory stabilization and efficient secretion of PopB and PopD by P. aeruginosa (Bröms et al, 2003b).…”