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Infection and Immunity, April 2000, p. 2359-2362, Vol. 68, No. 4
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

In Vivo-Induced Genes in Pseudomonas aeruginosa

Martin Handfield,¹ Dario E. Lehoux,¹ François Sanschagrin,¹ Michael J. Mahan,² Donald E. Woods,³ and Roger C. Levesque^1,*

Microbiologie Moléculaire et Génie des Protéines, Pavillon Charles-Eugène Marchand, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4¹; Department of Microbiology and Infectious Diseases, University of Calgary, Alberta, Canada T2N 4M1³; and Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106²

Received 23 September 1999/Returned for modification 16 November 1999/Accepted 3 January 2000

	ABSTRACT

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In vivo expression technology was used for testing Pseudomonas aeruginosa in the rat lung model of chronic infection and in a mouse model of systemic infection. Three of the eight ivi proteins found showed sequence identity to known virulence factors involved in iron acquisition via an open reading frame (called pvdI) implicated in pyoverdine biosynthesis, membrane biogenesis (FtsY), and adhesion (Hag2).

	TEXT

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Pseudomonas aeruginosa is an opportunistic pathogen important in cystic fibrosis patients, for whom chronic P. aeruginosa infections remain the major cause of acute pneumonia, leading to debilitating lung malfunction and premature death. Although several P. aeruginosa virulence factors have been extensively studied in vitro, less is known about virulence factors during infection. Several approaches have been reported to allow the recovery, identification, and characterization of genes that are expressed in the host (2-4). We have utilized the in vivo expression technology (IVET) purA promoter trap system (5) to identify P. aeruginosa genes that are specifically induced during mucosal and/or systemic infections. Here, we present evidence that the DNA fragments cloned in the promoter trap carry ivi genes in both animal models used.

Generation of chromosomal cointegrated P. aeruginosa PAO909 library. A library of random genomic DNA fragments from P. aeruginosa were cloned to the promoterless purA-lacZY into pIVET1. Genomic DNA fragments from P. aeruginosa strain PAO1 from 1 to 4 kb were size selected, purified, ligated with pIVET1, and electroporated into Escherichia coli DH5pir (strains and plasmids are listed in Table 1). Analysis of 48 recombinant plasmids confirmed that 99% had different P. aeruginosa DNA fragments ranging between 1 and 4 kb (data not shown). This random pool of plasmids was transformed into E. coli SM10pir and transferred by conjugation into the purA mutant P. aeruginosa strain PAO909. The resulting chromosomal cointegrated library was represented by at least 2 × 10⁵ colonies of P. aeruginosa transformants.

View this table: [in this window] [in a new window]   TABLE 1.   Bacterial strains and plasmids used in this study

Selection of P. aeruginosa in vivo-induced genes. The cointegrated PAO909 library was used to infect BALB/c mice weighing 18 to 20 g (a septicemia model) intraperitoneally with 10⁶ to 10⁷ bacteria/mouse and to infect Sprague-Dawley rats intratracheally with 10⁵ bacteria enmeshed into agar beads per lung (a chronic lung infection model [1]). After incubation, bacteria recovered from mouse livers and rat lungs were plated on rich selective medium containing the sensitive chromogenic substrate, 5-bromo-4-chloro-3-indoyl--D-galactopyranoside (X-Gal). A collection of 100 ivi fusions were recovered from infected mouse livers and infected rat lungs (Fig. 1).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Induction of ivi genes is required for survival in the animal. The vertical axis represents the number of CFU recovered from the organ of interest after inoculation. The inoculum bar represents the number of CFU of P. aeruginosa injected into each animal. Results are from the BALB/c mouse model of septicemia induced by intraperitoneal injection (104 CFU/mouse; 3 days) (A) and the Sprague-Dawley rat model of chronic lung infection induced via intratracheal instillation of bacterial cells enmeshed in agar beads (5 × 105 CFU/rat; 5 days) (B). Cells were grown overnight at 37°C in rich (adenine-supplemented) laboratory medium. Strains 100 and 101 (Lac) and strain 102 (Lac+) were preselected purA-lac fusion strains. PAO909 is a P. aeruginosa auxotroph for adenine. Data are presented as averages of two to five independent assays ± standard deviations.

Characterization of ivi genes. Plasmid preparations from in vivo-selected PAO909 clones were electroporated into E. coli DH5pir to allow plasmid rescue. Next, rescued plasmids which had unique restriction patterns and which gave a Lac phenotype in vitro were selected for further analysis by DNA sequencing. ivi junctions were sequenced using primers homologous to the 5' region of the purA gene. Similarity searches with the P. aeruginosa genome were performed at the National Center for Biotechnology Information using the uncompleted P. aeruginosa sequence genome database (http://www.pseudomonas.com). Bioinformatics analysis was done using GeneMark and software in the University of Wisconsin Genetics Computer Group package (version 10.0). We identified three ivi genes with homology to known sequences: pvdD, ftsY, and hag2. The remaining six ivi genes were open reading frames (ORFs) found to have no DNA or protein similarity (Table 2).

View this table: [in this window] [in a new window]   TABLE 2.   P. aeruginosa in vivo-induced genes isolated in both animal modelsa

Strain 131-17, identified by IVET (henceforth IVET 131-17), contains an unidentified ORF of 15,450 nucleotides (named pvdI) coding for a 5,150-amino-acid synthetase having 43% identity with PvdD of P. aeruginosa. We refer to this synthetase gene located upstream and in the same orientation as pvdD. The PvdD pyoverdine synthetase is involved in the synthesis of the fluorescent siderophore pyoverdine that is essential for iron uptake (6, 7). The independent isolation of IVET 131-17 from both animal models reflects the relative importance of iron acquisition in the establishment and/or maintenance of P. aeruginosa mucosal and systemic infections. Large-scale isolation of candidate virulence genes of P. aeruginosa strain PAK identified the pyochelin receptor (fptA), known to be inducible under iron-deprived conditions, providing further evidence that animal host tissues are deficient in free iron due to the presence of high-affinity iron binding proteins like transferrin (12, 13).

IVET 134-21 carries an ORF that encodes a protein sharing 65% identity with E. coli FtsY, a docking protein that interacts with the prokaryotic signal recognition particle-like complex involved in protein targeting and membrane biogenesis (10). Several ivi genes are involved in bacterial membrane modifications, presumably in response to overcoming environmental stresses imposed on the pathogen during infection (4).

IVET 131-19 carries a predicted peptide which has 43% identity with hemagglutinin Hag2 of Eikenella corrodens, an oral bacterium found in dental plaque (9). Similarly, the adherence of P. aeruginosa to the mucosa of the oropharynx is believed to be the initial step in colonization of the lower respiratory tract (14). The ivi of IVET 131-19 in both infection models suggests a mucosal and systemic requirement for P. aeruginosa adhesins, as is the case for other mucosal and systemic infection models (4). Cross talk of virulence factors between different in vivo pathogenesis models has been described previously using plants as hosts to identify P. aeruginosa virulence factors (8). The remaining six ivi genes code for proteins having no significant homology to reported proteins found in databases.

Induction of fusions is required for in vivo survival. All eight ivi clones showed no or weak -galactosidase activity when in vitro promoter activity was tested as described by Slauch et al. (11) (data not shown). Results shown in Fig. 1 indicate that the mutant P. aeruginosa purA strain PAO909 could not be recovered from mouse liver and rat lung tissues, confirming the efficacy of the selection in both animal models. Moreover, the eight ivi fusions showed a 10³- to 10⁵-fold growth advantage in both infection models. Thus, induction of all eight ivi fusions is required for survival in both animal models under conditions of the IVET selection.

These eight ivi genes were shown to be required for survival under the conditions of IVET selection in both animal models, suggesting that at least some host signals present during mouse systemic infection are also present in the rat respiratory mucosa. The propensity to isolate ivi genes coding for proteins related to the expression of surface proteins such as FtsY, PvdI, and Hag2 may suggest that they play a role in virulence by some unknown mechanisms. IVET selects bacterial ivi genes that presumably contribute to the in vivo fitness of the pathogen host tissues. Many of the ivi genes that have been recovered from several pathogens infecting a wide variety of animal models are unknown (4). The high possibility of recovering ivi genes of unknown function may reflect our limited knowledge of the bacterial functions required to survive during infection. Many of these presumably reflect the unique lifestyle of each individual pathogen during growth in the host and may not be shared by other pathogens. Thus, further studies on both known and unknown P. aeruginosa ivi gene products will contribute to a better understanding of the pathobiology of P. aeruginosa as an opportunistic pathogen.

	ACKNOWLEDGMENTS

We express our gratitude to Bruce Holloway, Monash University, for strain PAO1; John J. Mekalanos, Harvard University, for plasmid pIVET1 and E. coli strains DH5pir and SM10pir; Paul Phibbs, Pseudomonas Genetic Stock Center, for strain PAO909; and J. Renaud and G. Cardinal, University of Laval, for excellent assistance in DNA sequencing.

This work was supported by the Medical Research Council of Canada. Work in R.C.L. lab is also funded by the Canadian Cystic Fibrosis Foundation and the Canadian Bacterial Diseases Network via the Canadian Centers of Excellence (R.C.L.) and by NIH grant AI36373, ACS Junior Faculty Research Award 554, and a Beckman Young Investigator Award (M.J.M.). R. C. Levesque is a Scholar of Exceptional Merit from Le Fonds de la Recherche en Santé du Québec, and M. Handfield obtained a studentship from the Canadian Cystic Fibrosis Foundation.

The first two authors (M.H. and D.E.L.) contributed equally to this work and are listed alphabetically.

	FOOTNOTES

^* Corresponding author. Mailing address: Microbiologie Moléculaire et Génie des Protéines, Pavillon Charles-Eugène Marchand, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4. Phone: (418) 656-3070. Fax: (418) 656-7176. E-mail: rclevesq{at}rsvs.ulaval.ca.

Editor:   J. T. Barbieri

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Infection and Immunity, April 2000, p. 2359-2362, Vol. 68, No. 4
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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