Regulation of Pseudomonas aeruginosa ptxR by Vfr.

Pseudomonas aeruginosa PtxR enhances the expression of the exotoxin A gene toxA. The expression of ptxR itself, which occurs from two promoters (P1 and P2), is not completely understood. We have recently demonstrated that the ptxR upstream region contains potential binding sites for multiple regulators, including the virulence factor regulator Vfr. In this study, we identified within the ptxR upstream region, a 25 bp sequence to which Vfr specifically binds. The sequence is located 20-44 (32.5) bp 5' of the ptxR P2 promoter, and overlaps a potential binding site for the iron-starvation sigma factor PvdS. We also show that, throughout the growth cycle, deletion of vfr reduces ptxR expression from the P2 promoter in the P. aeruginosa strain PAO1 by four- to eightfold, but does not affect ptxR expression from P1. Further, loss of Vfr eliminates the PtxR-induced enhancement in the synthesis of exotoxin A and the metalloproteinase LasB. Our results suggest that Vfr modulates toxA and lasB expression in PAO1 through PtxR. A model defining the relationships between these different genes is presented.

Characterization of DNA-binding specificity and analysis of binding sites of the Pseudomonas aeruginosa global regulator, Vfr, a homologue of the Escherichia coli cAMP receptor protein.

Vfr, a global regulator of Pseudomonas aeruginosa virulence factors, is a homologue of the Escherichia coli cAMP receptor protein, CRP. Vfr is 91% similar to CRP and maintains many residues important for CRP to bind cAMP, bind DNA, and interact with RNA polymerase at target promoters. While vfr can complement an E. coli crp mutant in beta-galactosidase production, tryptophanase production and catabolite repression, crp can only complement a subset of Vfr-dependent phenotypes in P. aeruginosa. Using specific CRP binding site mutations, it is shown that Vfr requires the same nucleotides as CRP for optimal transcriptional activity from the E. coli lac promoter. In contrast, CRP did not bind Vfr target sequences in the promoters of the toxA and regA genes. Footprinting analysis revealed Vfr protected sequences upstream of toxA, regA, and the quorum sensing regulator lasR, that are similar to but significantly divergent from the CRP consensus binding sequence, and Vfr causes similar DNA bending to CRP in bound target sequences. Using a preliminary Vfr consensus binding sequence deduced from the Vfr-protected sites, Vfr target sequences were identified upstream of the virulence-associated genes plcN, plcHR, pbpG, prpL and algD, and in the vfr/orfX, argH/fimS, pilM/ponA intergenic regions. From these sequences the Vfr consensus binding sequence, 5'-ANWWTGNGAWNY : AGWTCACAT-3', was formulated. This study suggests that Vfr shares many of the same functions as CRP, but has specialized functions, at least in terms of DNA target sequence binding, required for regulation of a subset of genes in its regulon.

Molecular characterization of Staphylococcus intermedius carriage by healthy dogs and comparison of antimicrobial susceptibility patterns to isolates from dogs with pyoderma.

We conducted an epidemiological study of Staphylococcus intermedius using arbitrarily primed PCR (AP-PCR) and antibiograms. One hundred and twenty-five S. intermedius isolates were recovered from the oral cavity and/or cranial hair coat of healthy dogs enrolled in a pet therapy program. Commensal S. intermedius was cultured from 32% of the oral cavity cultures and 13% of the cranial hair coat cultures. We characterized the colonization of the dogs as transient, intermittent, or persistent. For dogs characterized as persistently colonized, 73% of the isolates came from the oral cavity. These isolates were also genotyped by AP-PCR. A single major AP-PCR type was observed in 91% of the dogs (n=22); minor variations were frequently observed in these major types. Antibiograms of these commensal isolates were compared to antibiograms from 97 historical clinical isolates (1988-1992) obtained from cases of canine pyoderma. Resistance was most often observed to penicillin (64% and 55%) and tetracycline (38% and 38%) among the commensal and clinical isolates, respectively. The commensal isolates were significantly less resistant to erythromycin, clarithromycin, clindamycin, and trimethoprim/sulfamethoxazole. Our data suggests that differences in both genotype and antimicrobial susceptibility phenotypes exist among S. intermedius strains isolated from different anatomic sites from the same dog and supports the opportunistic nature of S. intermedius in canine infections.

Longitudinal development of mucoid Pseudomonas aeruginosa infection and lung disease progression in children with cystic fibrosis.

CONTEXT: Although Pseudomonas aeruginosa is the most common virulent respiratory pathogen in cystic fibrosis (CF), the longitudinal development of P aeruginosa infection and its effect on antibody responses and lung disease progression in children with CF remain unclear. OBJECTIVE: To prospectively examine the epidemiology of P aeruginosa infection and its impact on CF pulmonary morbidity. DESIGN, SETTING, AND PATIENTS: We prospectively evaluated 56 CF patients at 2 CF centers in Madison and Milwaukee, Wis, from birth up to age 16 years between April 15, 1985, and April 15, 2004, with diagnoses made through the Wisconsin CF Neonatal Screening Project. MAIN OUTCOME MEASURES: Timing of nonmucoid P aeruginosa and mucoid P aeruginosa acquisition was assessed by first positive result. Longitudinal development from no P aeruginosa to nonmucoid P aeruginosa and from nonmucoid P aeruginosa to mucoid P aeruginosa was examined. Outcome measurements included antibody titers, respiratory symptoms, quantitative chest radiography, and pulmonary function tests. RESULTS: Sixteen patients (29%) acquired nonmucoid P aeruginosa in the first 6 months of life. The age-specific prevalence of mucoid P aeruginosa increased markedly from age 4 to 16 years. Nonmucoid and mucoid P aeruginosa were acquired at median ages of 1.0 and 13.0 years, respectively. In contrast with the short transition time from no P aeruginosa to nonmucoid P aeruginosa, the transition time from nonmucoid to mucoid P aeruginosa was relatively long (median, 10.9 years) and could be slightly extended by brief/low anti-P aeruginosa antibiotic treatment. Antibody titers increased with both transitions, but the deterioration in cough scores, chest radiograph scores, and pulmonary function correlated best with transition from nonmucoid to mucoid P aeruginosa. CONCLUSIONS: Early prevention and detection of nonmucoid and mucoid P aeruginosa are critical because of early acquisition and prevalence. There is a window of opportunity for suppression and possible eradication (by aggressive anti-P aeruginosa treatment) of initial nonmucoid P aeruginosa. Mucoid P aeruginosa plays a much greater role in CF lung disease progression than nonmucoid P aeruginosa. Antibody titers, cough scores, and chest radiographs are early signs of nonmucoid P aeruginosa and especially mucoid P aeruginosa stages.

fleQ, the gene encoding the major flagellar regulator of Pseudomonas aeruginosa, is sigma70 dependent and is downregulated by Vfr, a homolog of Escherichia coli cyclic AMP receptor protein.

The flagellar transcriptional regulator FleQ appears to be the highest-level regulator in the hierarchical regulatory cascade of flagellar biogenesis in Pseudomonas aeruginosa. Except for the posttranslational downregulation of FleQ activity by FleN, an antiactivator, not much is known about the regulation of the fleQ gene or its gene product. Some FleQ homologs in other bacterial species either are positively regulated by another regulator (e.g., CtrA, the master regulator regulating FlbD in Caulobacter crescentus) or are expressed from a sigma70-dependent promoter (e.g., FlgR of Helicobacter pylori). In this study we demonstrated that Vfr, an Escherichia coli CRP homolog known to function as an activator for various genes, including lasR, regA, and toxA, in P. aeruginosa, is capable of repressing fleQ transcription by binding to its consensus sequence in the fleQ promoter. In a DNase I footprint assay, purified Vfr protected the sequence 5'-AATTGACTAATCGTTCACATTTG-3'. When this putative Vfr binding site in the fleQ promoter was mutated, Vfr was unable to bind the fleQ promoter fragment and did not repress fleQ transcription effectively. Primer extension analysis of the fleQ transcript revealed two transcriptional start sites, t1 and t2, that map within the Vfr binding site. A putative -10 region (TAAAAT) for the t2 transcript, with a five-of-six match with the E. coli sigma70 binding consensus, overlaps with one end of the Vfr binding site. A 4-bp mutation and an 8-bp mutation in this -10 region markedly reduced the activity of the fleQ promoter. The same mutations led to the disappearance of the 203-nucleotide fleQ transcript in an in vitro transcription assay. Vfr probably represses fleQ transcription by binding to the Vfr binding site in the fleQ promoter and preventing the sigma factor from binding to the -10 region to initiate transcription.

Respiratory infections with Pseudomonas aeruginosa in children with cystic fibrosis: early detection by serology and assessment of risk factors.

CONTEXT: Patients with cystic fibrosis (CF) are susceptible to lower respiratory tract infections with Pseudomonas aeruginosa and typically acquire this organism in early childhood. Once P aeruginosa infection is established, eradication may be impossible, and progressive lung disease often aggravates morbidity and mortality risks. The ability to diagnose CF by genetic testing at birth makes it possible to determine the temporal sequence of events that result in P aeruginosa-associated pulmonary infections. OBJECTIVE: To evaluate the longitudinal relationship between the production of an antibody response against P aeruginosa and clinical factors associated with P aeruginosa pulmonary infections in patients with CF diagnosed in early life. DESIGN, SETTING, AND PATIENTS: Serum samples and oropharyngeal cultures (protocol cultures) were obtained at 6-month intervals from April 15, 1985, to April 15, 2000 (or for up to 180 months depending on their enrollment date) from 68 patients at 2 centers in Madison and Milwaukee, Wis, diagnosed through the Wisconsin CF Neonatal Screening Project, a longitudinal cohort study. Additional cultures were obtained at examining physicians' discretion (all cultures). MAIN OUTCOME MEASURES: Time to serum IgG, IgA, and IgM antibody titer of at least 1:256 against P aeruginosa, assessed by enzyme-linked immunosorbent assay using cell lysate, exotoxin A, and elastase as antigens; time to organism isolation from respiratory samples; time to Wisconsin Cystic Fibrosis Radiograph (WCXR) score of 5 or more. RESULTS: The median time to an antibody titer of at least 1:256 was 17.8, 24.2, and 70.9 months for cell lysate, exotoxin A, and elastase, respectively. The rise of anti-cell lysate and anti-exotoxin A titers to 1:256 or more occurred a mean of 11.9 (P<.001) and 5.6 (P =.04) months, respectively, before the isolation of P aeruginosa for all cultures and 18.2 (P<.001) and 11.9 (P =.006) months, respectively, before protocol cultures. There was no significant difference between the rise of anti-cell lysate and anti-exotoxin A titer and a WCXR score of 5 or more (P =.24 and.32, respectively). Treatment with long-term, non-Pseudomonas oral antibiotics and integration of CF infants with older, chronically infected patients were associated with a significantly increased risk of P aeruginosa pulmonary infection. CONCLUSIONS: In CF patients diagnosed through neonatal screening, P aeruginosa pulmonary infections occurred 6 to 12 months before the organism was isolated from respiratory secretions. The longitudinal monitoring of P aeruginosa antibody titers, in concert with WCXR score, should facilitate diagnosis and treatment of P aeruginosa pulmonary infections in young children with CF.

Effect of vfr mutation on global gene expression and catabolite repression control of Pseudomonas aeruginosa.

Vfr of Pseudomonas aeruginosa is 91% similar to the cAMP receptor protein (CRP) of Escherichia coli. Based on the high degree of sequence homology between the two proteins, the question arose whether Vfr had a global regulatory effect on gene expression for P. aeruginosa as CRP did for E. coli. This report provides two-dimensional polyacrylamide gel electrophoretic evidence that Vfr is a global regulator of gene expression in P. aeruginosa. In a vfr101::aacC1 null mutant, at least 43 protein spots were absent or decreased when compared to the proteome pattern of the parent strain. In contrast, 17 protein spots were absent or decreased in the parent strain when compared to the vfr101::aacC1 mutant. Thus, a mutation in vfr affected production of at least 60 proteins in P. aeruginosa. In addition, the question whether Vfr and CRP shared similar mechanistic characteristics was addressed. To ascertain whether Vfr, like CRP, can bind cAMP, Vfr and CRP were purified to homogeneity and their apparent dissociation constants (K(d)) for binding to cAMP were determined. The K(d) values were 1.6 microM for Vfr and 0.4 microM for CRP, suggesting that these proteins have a similar affinity for cAMP. Previously the authors had demonstrated that Vfr could complement a crp mutation and modulate catabolite repression in E. coli. This study presents evidence that Vfr binds to the E. coli lac promoter and that this binding requires the presence of cAMP. Finally, the possible involvement of Vfr in catabolite repression control in P. aeruginosa was investigated. It was found that succinate repressed production of mannitol dehydrogenase, glucose-6-phosphate dehydrogenase, amidase and urocanase both in the parent and in two vfr null mutants. This implied that catabolite repression control was not affected by the vfr null mutation. In support of this, the cloned vfr gene failed to complement a mutation in the P. aeruginosa crc gene. Thus, although Vfr is structurally similar to CRP, and is a global regulator of gene expression in P. aeruginosa, Vfr is not required for catabolite repression control in this bacterium.