Nancy L. Carty

Pseudomonas aeruginosa Autoinducer Enters and Functions in Mammalian Cells

Quorum sensing (QS) is a cell density-dependent signaling mechanism used by many bacteria to control gene expression. Several recent reports indicate that the signaling molecules (autoinducers) that mediate QS in Pseudomonas aeruginosa may also modulate gene expression in host cells; however, the mechanisms are largely unknown. Here we show that two P. aeruginosa autoinducers, N-3-oxododecanoyl-homoserine lactone and N-butyryl-homoserine lactone, can both enter eukaryotic cells and activate artificial chimeric transcription factors based on their cognate transcriptional activators, LasR and RhlR, respectively. The autoinducers promoted nuclear localization of chimeric proteins containing the full LasR or RhlR coding region, and the LasR-based proteins were capable of activating transcription of a LasR-dependent luciferase gene. Responsiveness to autoinducer required the N-terminal autoinducer-binding domains of LasR and RhlR. Truncated proteins consisting of only the C-terminal helix-turn-helix DNA-binding domains of both proteins attached to a nuclear localization signal efficiently translocated to the nucleus in the absence of autoinducer, and truncated LasR-based proteins functioned as constitutively active transcription factors. Chimeric LasR proteins were only activated by their cognate autoinducer ligand and not by N-butyryl-L-homoserine lactone. These data provide evidence that autoinducer molecules from human pathogens can enter mammalian cells and suggest that autoinducers may influence gene expression in host cells by interacting with and activating as-yet-unidentified endogenous proteins.

PtxR modulates the expression of QS-controlled virulence factors in the Pseudomonas aeruginosa strain PAO1.

The production of several virulence factors by Pseudomonas aeruginosa is regulated through the hierarchical cell‐density dependent quorum sensing (QS) systems las and rhl. A third component of the QS hierarchy, the Pseudomonas quinolone signal PQS, also controls the expression of several genes. We previously described P. aeruginosa PtxR as a transcriptional activator of the exotoxin A gene toxA. Here, we provide evidence that PtxR regulates the production of other virulence factors. Mutation of ptxR in PAO1 increased pyocyanin production. This increase was reduced in the presence of a ptxR plasmid. Throughout the growth cycle, PtxR reduced the expression of the pyocyanin operon phzA1‐G1 but not phzA2‐G2. As pyocyanin production is stringently controlled by QS, we examined the effect of PtxR on QS‐related genes in PAO1. PtxR also reduced the expression of the PQS synthesis operon pqsABCDE. ptxR mutation increased the expression of the rhamnolipid synthesis gene rhlA but decreased lasB expression. The expression of the RhlI synthase gene rhlI and the production of the C4‐HSL autoinducer were increased in the ptxR mutant, while the expression of the LasI synthase gene lasI and the production of 3OC12‐HSL were reduced. These results suggest that PtxR negatively regulates the expression of the rhamnolipid and pyocyanin genes through rhlI and the pqsABCDE operon while it positively regulates the expression of lasB through lasI.

Regulation of Pseudomonas Aeruginosa Exotoxin a Synthesis

Exotoxin A (ETA) is one of the most toxic virulence factors produced by Pseudomonas aeruginosa 72: Both clinical and experimental animal studies indicate clearly the importance of ETA in the pathogenesis of different P. aeruginosa infections. Most P. aeruginosa clinical isolates produce ETA9, 23, 47. In addition, infection with ETA-producing P. aeruginosa strains was shown to be significantly associated with mortality due to P. aeruginosa bacteremia98. Furthermore, survival of patients with P. aeruginosa bacteremia correlated with high ETA antibody titers in those patients98, 99. Examination of patients with cystic fibrosis (CF) that were chronically infected with P. aeruginosa showed the presence of an increased titer of ETA antibody57. In addition, mortality among P. aeruginosa infected CF-patients was associated with increased levels of ETA and LPS antibodies together with the presence of immu necomplexes and complement activation84. Recent analysis of RNA extracted from sputa of CF patients revealed that the ETA gene (toxA) was expressed in the lung of CF patients infected with P. aeruginosa 105.

Role of Vfr in regulating exotoxin A production by Pseudomonas aeruginosa

Pseudomonas aeruginosa exotoxin A (ETA) production depends on the virulence-factor regulator Vfr. Recent evidence indicates that the P. aeruginosa iron-starvation sigma factor PvdS also enhances ETA production through the ETA-regulatory gene regA. Mutants defective in vfr, regA and pvdS, plasmids that overexpress these genes individually and lacZ transcriptional/translational fusion plasmids were utilized to examine the relationship between vfr, regA and pvdS in regulating P. aeruginosa ETA production. ETA concentration and regA expression were reduced significantly in PAODeltavfr, but pvdS expression was not affected. Overexpression of Vfr produced a limited increase in ETA production in PAODeltapvdS, but not PAODeltaregA. Additionally, overexpression of either RegA or PvdS did not enhance ETA production in PAODeltavfr. RT-PCR analysis showed that iron did not affect the accumulation of vfr mRNA in PAO1. These results suggest that: (i) Vfr enhances toxA expression in PAO1 both directly and indirectly through regA, but not through pvdS; (ii) vfr expression is not regulated by iron; and (iii) both Vfr and PvdS cooperate in the presence of RegA to achieve a maximum level of toxA expression.

Identification and partial characterization of an elastolytic protease in the amphibian pathogen Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd) is a fungus that causes chytridiomycosis, a disease that has been implicated as a cause of amphibian population declines worldwide. Infected animals experience hyperkeratosis and sloughing of the epidermis due to penetration of the keratinized tissues by the fungus. These symptoms have led us to postulate that Bd produces proteases that play a role in the infection process. Here, we show that Bd is capable of degrading elastin in vitro, a protein found in the extracellular matrix of the host animal. Elastolytic enzyme activity was partially purified using ion exchange chromatography and size-exclusion filtration from cultures grown in inducing media. The elastolytic activity of the purified fraction had a pH optimum of 8, was strongly inhibited by EDTA and phenylmethylsulfonyl fluoride (PMSF), and was partially inhibited by an elastase-specific inhibitor. This activity was also enhanced by the presence of Mg2+ and Ca2+ but not Zn2+. An antiserum directed against Aspergillus fumigatus serine protease (Alp) was found to react with a polypeptide of approximately 110 kDa from the purified material. Using immunofluorescence, this antiserum was also observed to react with zoospores and sporangia grown on toad skin. These observations suggest that Bd may produce proteases similar to those produced by other pathogenic fungi that are capable of degrading proteins found in the extracellular matrix. The proteolytic activity exhibited in vitro might aid the organism in its ability to colonize and destroy the epidermis of its amphibian host.

The pvc Operon Regulates the Expression of the Pseudomonas aeruginosa Fimbrial Chaperone/Usher Pathway (Cup) Genes

The Pseudomonas aeruginosa fimbrial structures encoded by the cup gene clusters (cupB and cupC) contribute to its attachment to abiotic surfaces and biofilm formation. The P. aeruginosa pvcABCD gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the pvc operon and the cup gene clusters in the P. aeruginosa strain MPAO1. Mutations within the pvc genes compromised biofilm development and significantly reduced the expression of cupB1-6 and cupC1-3, as well as different genes of the cupB/cupC two-component regulatory systems, roc1/roc2. Adjacent to pvc is the transcriptional regulator ptxR. A ptxR mutation in MPAO1 significantly reduced the expression of the pvc genes, the cupB/cupC genes, and the roc1/roc2 genes. Overexpression of the intact chromosomally-encoded pvc operon by a ptxR plasmid significantly enhanced cupB2, cupC2, rocS1, and rocS2 expression and biofilm development. Exogenously added paerucumarin significantly increased the expression of cupB2, cupC2, rocS1 and rocS2 in the pvcA mutant. Our results suggest that pvc influences P. aeruginosa biofilm development through the cup gene clusters in a pathway that involves paerucumarin, PtxR, and different cup regulators.

In Vivo Models of Biofilm Infection

The in vivo biofilm models that have been described use a variety of animals ranging from goats to chinchillas, and mimic diseases including dental caries, endocarditis, pneumonia, keratitis, and otitis media. Representatives of these in vivo models are listed in Table 16.1.