Host-Pathogen Interactions: Pseudomonas and Cystic Fibrosis
Principal Investigator: Fred Ausubel
Pseudomonas aeruginosa, a ubiquitous Gram-negative bacterium
isolated from soil, water, and plants (1), is an
pathogen that infects cystic fibrosis patients, patients with thermal
burns and patients who are immunodeficient or otherwise compromised. The
Ausubel laboratory has developed a novel multi-host pathogenesis system
that uses non-vertebrate hosts as an adjunct to mammalian models to identify
and study P. aeruginosa virulence-related genes as well as host genes
that are involved in the defense response to pathogen attack (2-7).
Remarkably, among 20 P. aeruginosa strain PA14 genes that are required
for pathogenesis in at least one of three different invertebrate hosts
(a plant, a nematode and an insect), 17 were also required for full
in a mouse burn model. This project concerns the use of genomic approaches
to extend this multi-host pathogenesis model to the pathogenic processes
that occur during the pulmonary infection of mice that contain a knock-out
mutation in the CFTR gene, the gene which is mutated in human cystic fibrosis
The first genomic approach, involves the analysis of the PA14 genome for
candidate virulence genes absent in less-pathogenic strains of
P. aeruginosa, as well as the generation of a non-redundant library of
P. aeruginosa mutants that contain a transposon insertion in each of the
estimated 4,800 non-essential genes. This library will greatly facilitate the
identification of P. aeruginosa virulence factors that play a role in the
colonization of the lungs of CF patients. The second genomic approach involves
the use of DNA microarray analysis to identify both P. aeruginosa and
mouse genes that are specifically activated or repressed during pulmonary
infection of CF mice. We predict that these latter experiments will lead to
the identification of mouse genes that are involved in the innate immune
response to P. aeruginosa colonization.
- Generate enhancements/refinements to the P. aeruginosa
PA14 non-redundant mutation library and related resources including
the PA14 genome sequence.
- Annotate the PA14 genome and transfer the annotated
genome data to PseudoCAP (Pseudomonas Community Annotation
- Identify and mutate 300-400 non-essential genes
estimated to be missing from the PA14 library.
- Distribute the non-redundant PA14 mutant library to
the public and transfer the responsibility for distributing
the library to the Pseudomonas FLEX Gene Repository at Harvard
- Design and manufacture a multi-purpose spotted
P. aeruginosa microarray for transcriptional profiling, transposon
site hybridization (TraSH), and phylogenetic studies, and make it
available to the public.
- Identify P. aeruginosa pathogenicity-related genes,
construct non-polar deletions of these genes, test them in mouse
infection models, and make the resulting collection of mutants
available to the public.
- Identify putative PA14 pathogenicity-related genes
by transcriptional profiling and Trash analysis.
- Prioritize putative PA14 pathogenicity-related
mutants for further characterization.
- Test putative PA14 pathogenicity-related genes
for virulence-related phenotypes.
- Correlate phenotypes of PA14 mutants with a
transposon insertion in a particular ORF.
- Construct non-polar in-frame deletions
corresponding to selected mutants identified in
- Test mutants from Aim 3e in murine CF
oropharyngeal and GI colonization models.
- Expand the public PA14 database so that it coordinates
three genomic resources: the annotated PA14 sequence, the PA14
mutant library, and the spotted oligonucleotide array. Transfer the
responsibility of maintaining the database to PseudoCAP.
Palleronic, J. N. 1984. Family I. Pseudomonadaceae. In Bergey's Manuel of systemative bacteriology. Bergey, ed, p. 141-172.
- Rahme, L. G., E. J. Stevens, S. F. Wolfort, J. Shao, R. G. Tompkins, and F. M. Ausubel. 1995. Common virulence factors for bacterial pathogenicity in plants and animals [see comments]. Science 268:1899-902.
- Rahme, L. G., M. W. Tan, L. Le, S. M. Wong, R. G. Tompkins, S. B. Calderwood, and F. M. Ausubel. 1997. Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc Natl Acad Sci U S A 94:13245-50.
- Mahajan-Miklos, S., M. W. Tan, L. G. Rahme, and F. M. Ausubel. 1999. Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model. Cell 96:47-56.
- Tan, M. W., S. Mahajan-Miklos, and F. M. Ausubel. 1999. Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. Proc Natl Acad Sci U S A 96:715-20.
- Tan, M. W., L. G. Rahme, J. A. Sternberg, R. G. Tompkins, and F. M. Ausubel. 1999. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc Natl Acad Sci U S A 96:2408-13.
- Jander, g., L. G. Rahme, and F. M. Ausubel. 2000 (submitted). Positive Correlation between Virulence in Pseudomonas aeruginosa mutants in mice and insects.
To learn more about the Host-Pathogen Interactions: Pseudomonas and Cystic
Fibrosis project, visit the Ausubel Lab Web Site.
For more information on specific sub-sections of this project, visit the
- PA14 Genomic
- CF Animal Models
For available resources pertaining to this project, visit the Bacterial
Pathogen Resources Page.