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Journal of Clinical Microbiology, August 2004, p. 3857-3860, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3857-3860.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

V-Antigen Genotype and Phenotype Analyses of Clinical Isolates of Pseudomonas aeruginosa

Leonard R. Allmond,¹ Temitayo Ajayi,^2,3 Kiyoshi Moriyama,¹ Jeanine P. Wiener-Kronish,^1,2,3 and Teiji Sawa^1**

Department of Anesthesia and Perioperative Care,¹ Cardiovascular Research Institute,² Department of Medicine, University of California, San Francisco, California 94143³

Received 20 February 2004/ Returned for modification 13 April 2004/ Accepted 7 May 2004

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The pcrV genotype was analyzed in clinical isolates of Pseudomonas aeruginosa which showed a negative phenotype for secretion of V-antigen PcrV. The suppression of PcrV secretion in these isolates was due not to a lack of the pcrV gene but rather to suppression of PcrV expression.

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Pseudomonas aeruginosa possesses PcrV, a functional homolog of the Yersinia V-antigen LcrV (18). Both PcrV and LcrV have been shown to be involved in the translocational process of their type III secretion systems, and blocking antibodies against these proteins prevents translocation (1, 2, 12, 14, 15). Immunization against PcrV in animal models of P. aeruginosa pneumonia significantly increases survival (7, 14). Production of PcrV is more frequently found in P. aeruginosa strains isolated from acutely ill patients than in strains causing chronic infection (8, 13). Many P. aeruginosa isolates from chronically infected patients do not produce PcrV and show decreased virulence compared to isolates that express PcrV (13). These findings suggest that either phenotypic or genotypic variations in the PcrV proteins in clinical isolates may be associated with the clinical backgrounds of the patients.

This study attempts to answer the question of whether the lack of PcrV secretion in isolates from patients is due to a negative pcrV genotype or to suppression of either secretion or expression of PcrV in pcrV genotype-positive isolates. Clinical isolates that showed a negative phenotype for secretion of PcrV in a previous study were reanalyzed (13). Twenty clinical isolates, each derived from a separate patient and characterized as unique by tests of clonality, were analyzed (Table 1); clonality was determined by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) typing methods as previously reported (Table 2) (8, 10, 11). A laboratory strain, PA103, and a clinical isolate, PA1027, that have positive phenotypes for PcrV secretion were used as controls for analysis.

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TABLE 1. P. aeruginosa isolates analyzed in this studya

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TABLE 2. DNA sequences of PCR primers

Analysis of pcrV loci in clinical isolates of P. aeruginosa. To determine genotypes, Southern blot hybridization was performed with genomic DNA extracted from the isolates and with the digoxigenin-labeled DNA probe generated by PCR and designed to amplify the entire pcrV coding region from PAO1 chromosomal DNA (Table 2) (16). The presence of a pcrV homolog was detected in all strains that were characterized as PcrV phenotype negative (data not shown). Next, we confirmed the amplification of the pcrV genes of the isolates by PCR (Table 2). Appropriately sized DNA bands corresponding to pcrV were successfully detected for all isolates tested (Fig. 1). We cloned and sequenced the amplified DNA fragments and confirmed that they were all from pcrV. The predicted PcrV amino acid sequences for the six isolates showing a negative phenotype of PcrV secretion had no significant differences from the sequence for PAO1 (Table 3). Next, by PCR (Table 2), we cloned the promoter region of the pcrGVH-popBD operons in the isolates PA1065 and PA1079, which were isolated from cystic fibrosis patients and showed suppression of PcrV secretion. When compared to the sequence from PAO1, sequences from these two clinical strains showed no mutations in the binding site (ACAAAAA) of the transcriptional activator ExsA and no changes in the gene structure of the operon (data not shown) (9).

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FIG. 1. Detection of pcrV in clinical isolates of P. aeruginosa by PCR. pcrV genes were amplified by PCR using genomic DNA from clinical isolates of P. aeruginosa as template DNA and a specific primer set outside of the coding region (Table 2). The PCR products were analyzed by 0.9% agarose gel electrophoresis and ethidium bromide staining. PCR genotyping analysis showed that all isolates possessed pcrV. The figure shows data for 20 clinical isolates and a laboratory strain, PA103.

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TABLE 3. Variations in PcrV amino acid sequences from clinical isolates

Expression of PcrV in P. aeruginosa isolates. We examined expression and secretion of PcrV in several clinical isolates cultured in Ca²⁺-chelating deferrated tryptic soy broth medium. After 8 h of culture, the bacterial cell-associated fractions were separated from the culture medium by centrifugation and the secreted protein in the culture medium was precipitated by the addition of saturated ammonium sulfate. PcrV in the culture medium and in the cell-associated fractions was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an immunoblot assay. We used a rabbit polyclonal anti-PcrV immunoglobulin G that recognizes the PcrV proteins of both PA103 and PAO1 and a highly sensitive chemiluminescence substrate for horseradish peroxidase (13, 15) (Fig. 2). PcrV was detected in both the secreted and the cell-associated fractions of PA103 and PA1027. Small quantities of PcrV were secreted by three strains, PA1111, PA2009, and PA1034. PcrV had not been detected by previous dot blot assays of these four strains (13), but we could detect secretion with the immunoblot assay used here. PcrV was not detected in the cell-associated fractions from these four strains. Similarly, we repeated the immunoblot analyses several times under the same conditions and detected variable but positive secretion responses for strains PA1060, PA1065, and PA1079, which previously had been characterized as PcrV phenotype negative. However, as far as intensities, the detected levels of PcrV were significantly lower than those in PcrV-positive strains PA103 and PA1027.

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FIG. 2. Phenotype analysis of PcrV expression and secretion in clinical isolates of P. aeruginosa. Immunoblot analysis by a highly sensitive chemiluminescence method was used to determine the phenotype of PcrV secretion in selected P. aeruginosa isolates. PcrV proteins from P. aeruginosa PA103 (a laboratory strain) and PA1027 (a clinical isolate from an acutely infected patient) were used as positive controls for detection. Isolates were cultured in Luria-Bertani medium overnight, and the next day, 10% of the volume of the overnight culture was transferred into Ca2+-chelating deferrated tryptic soy broth medium and cultured for 8 h at 32°C. (Top panels) PcrV secreted into the inducing culture medium. (Bottom panels) PcrV in cell-associated fractions. PcrV was recognized as a 34-kDa band.

In vivo PcrV expression. We performed an infection study using rats to examine whether the expression of PcrV in the P. aeruginosa isolates from chronically infected patients remains suppressed in vivo. Experimental protocols were approved by the Committee on Animal Research, University of California, San Francisco. P. aeruginosa isolates (10⁹ CFU of either PA103, PA1065, or PA1079) were instilled into the lungs of rats, as described previously (5). Four hours after infection, the instilled bacteria were recovered from the infected lungs by bronchoalveolar lavage (BAL) with 5 ml of lactated Ringer's solution, and the pelleted bacteria (10⁷ CFU) were analyzed by immunoblotting for PcrV secretion by using an anti-PcrV monoclonal antibody, mAb 166, which recognizes the PcrV proteins from strains PA103 and PAO1 (7). While PA103 recovered from infected lungs showed PcrV secretion, strains PA1065 and PA1079 did not secrete PcrV in vivo, although PA1065 had shown PcrV secretion in vitro (Fig. 3). This result emphasizes the importance of the environmental conditions for bacterial secretion.

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FIG. 3. In vivo PcrV expression in clinical isolates of P. aeruginosa. Immunoblot analysis by a chemiluminescence method was used to determine the expression of PcrV in P. aeruginosa isolates instilled in mice. Bacteria were recovered by BAL 4 h after instillation into the lungs. The bacterial density among the samples was adjusted by quantitative culture of BAL fluid on agar plates. The secreted protein from P. aeruginosa PA103 (a laboratory strain) was used as a positive control for PcrV detection. PcrV was recognized as a 34-kDa band. PA1065 and PA1079 were isolates from cystic fibrosis patients and did not express PcrV in vivo.

We confirmed the finding that pcrV was present in all P. aeruginosa isolates tested. This result is analogous to those from a previous study in which popB was found to be present in all isolates (6). In most of the isolates tested in the present study, PcrV was suppressed before the expression level but not before the secretion level. As reported by others, the secretion of PcrV can be suppressed at various levels of type III secretion regulation (3, 4, 17). Our study indicates that the suppression of PcrV secretion in P. aeruginosa isolates was due not to a lack of pcrV but to suppression of PcrV expression.

 
This research was supported by National Institutes of Health grants RO1 HL59239 and AI44101 to J.P.W.-K., a supplement to AI44101 to L.R.A., grants from National Medical Fellowships and AA medical society to L.R.A., and American Lung Association research grant RG004N to T.S.

 
* Corresponding author. Mailing address: 513 Parnassus, S-261, Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA 94143-0542. Phone: (415) 476-6784. Fax: (415) 476-8841. E-mail: teiji{at}itsa.ucsf.edu.

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Journal of Clinical Microbiology, August 2004, p. 3857-3860, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3857-3860.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Allmond, L. R. Articles by Sawa, T. Search for Related Content PubMed PubMed Citation Articles by Allmond, L. R. Articles by Sawa, T.