Genetic analysis of exoenzyme S expression by Pseudomonas aeruginosa

Goranson, Joanne

doi:10.1016/0378-1097(95)00479-3

Cited by 15 publications

(23 citation statements)

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“…This observation can be reinterpreted in the light of our findings that the exsA mutant appears to be defective in type III secretion and fails also to secrete the 73, 40, and 32 kDa proteins. In addition, it has been reported that the presence of ExoT (but not ExoS) correlates with cytotoxicity (Frank et al, 1996). We failed to identify any transposon insertions into the exoT gene; this may reflect the fact that our screen was not exhaustive.…”

Section: Discussionmentioning

confidence: 79%

“…PA103, the parental strain, is cytotoxic when added to MDCK cells (Liu, 1966) and virulent in an animal model of the early events of acute pneumonia (Kudoh et al, 1994). It is a hypopiliated, non-flagellated strain that produces phospholipase C, alkaline protease, large amounts of exotoxin A, and ExoT but no ExoS (Goranson and Frank, 1996;Yahr et al, 1996a) or elastase (Liu, 1966). P. aeruginosa strain 388, which initially served as a non-cytotoxic control, is a non-isogenic strain that is non-cytotoxic in the MDCK model and avirulent in the animal model (our unpublished results).…”

Section: Methodsmentioning

confidence: 99%

“…P. aeruginosa strain 388, which initially served as a non-cytotoxic control, is a non-isogenic strain that is non-cytotoxic in the MDCK model and avirulent in the animal model (our unpublished results). It produces no detectable exotoxin A but is a hyperproducer of ExoS and also produces small amounts of ExoT (Bjorn et al, 1979;Goranson and Frank, 1996;Sokol et al, 1981;Yahr et al, 1996a). P. aeruginosa strains were routinely cultured in LB broth or VBM medium (Vogel and Bonner, 1956) with antibiotics as needed.…”

Section: Methodsmentioning

confidence: 99%

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Identification of Pseudomonas aeruginosa genes required for epithelial cell injury

Kang¹,

Hauser²,

Apodaca³

et al. 1997

Molecular Microbiology

115

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SummaryWe have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa-induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon-tagged non-cytotoxic mutants of a cytotoxic and lung-virulent strain of P. aeruginosa (PA103). The transposon-insertion site was determined by using an inverse polymerase chain reaction followed by DNA-sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pili, based on their resistance to phage PO4 and/or loss of twitching motility (twt ¹ ). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III proteinsecretion apparatus appears to be involved in this process.

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Section: Discussionmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Identification of Pseudomonas aeruginosa genes required for epithelial cell injury

Kang¹,

Hauser²,

Apodaca³

et al. 1997

Molecular Microbiology

115

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“…ExsA is a transcriptional regulatory protein that co-ordinately regulates several loci involved in the synthesis and secretion of extracellular proteins, postulated to be involved in the virulence of Pseudomonas aeruginosa (reviewed in Goranson and Frank, 1996). Loci transcriptionally activated by ExsA include regulatory (exsC, the B region and exsA), secretory (orf1, exsD /pscB-L) and structural components (exoS, exoT ) of the exoenzyme S regulon Hovey and Frank, 1995;Yahr et al, 1995;1996a,b).…”

Section: Introductionmentioning

confidence: 99%

ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury

Finck-Barbançon

Goranson

Zhu

et al. 1997

Molecular Microbiology

476

530

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SummaryThe production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non-covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non-cytotoxic derivative PA103 exsA::⍀ and several cytotoxic and non-cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70-kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70-kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non-cytotoxic strains or PA103 exsA::⍀. To clone the gene encoding this potential cytotoxin we used Tn5 Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU :: Tn5 Tc, which no longer expressed the 70-kDa extracellular protein but maintained expression of ExoT. PA103 exoU ::Tn5 Tc was non-cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU ::Tn5 Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co-ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.

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“…the phagocytic activities of macrophages [14,15]. The translocation of ExoU, which has patatin-like phospholipase A 2 activity, is correlated with acute cytotoxicity in vitro and lung damage, sepsis, and mortality in animal models [7,[16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

Construction and Characteristics of a Recombinant Single- Chain Antibody Fragment against Bacterial Type III Secretion

Sawa¹,

Kainuma²,

Moriyama³

et al. 2018

Antibody Engineering

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Pseudomonas aeruginosa, a Gram-negative pathogen, causes life-threatening infections. Lung injury and the development of sepsis depend largely on expression of the virulence genes associated with the type III secretion system of this bacterium. The type III secretion system functions as a molecular syringe to deliver type III secretory toxins directly into the cytosol of eukaryotic cells and also acts to inhibit innate immune mechanisms, thereby preventing bacterial clearance. Antibodies against PcrV, the cap structure in the translocational needle of type III secretory apparatus of P. aeruginosa, block toxin translocation of the type III secretion system. We have been investigating the therapeutic use of a recombinant anti-PcrV single-chain antibody. In this chapter, as a preliminary step toward an antibody-based immunotherapy against bacterial infections, we summarize our experience of constructing a recombinant single-chain antibody (called scFv166), in which the heavy (V H ) and light chain (V L ) variable regions of the anti-PcrV monoclonal IgG are joined by a lexible peptide linker. The practical methodologies used to make recombinant scFv166 against a bacterial protein component are described in detail.

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Genetic analysis of exoenzyme S expression by Pseudomonas aeruginosa

Cited by 15 publications

References 0 publications

Identification of Pseudomonas aeruginosa genes required for epithelial cell injury

Identification of Pseudomonas aeruginosa genes required for epithelial cell injury

ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury

Construction and Characteristics of a Recombinant Single- Chain Antibody Fragment against Bacterial Type III Secretion

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