Molecular Mechanisms of Bacterial Pathogenesis
The Friedman Lab Group
PO Box #245221
Tucson, AZ, 85724-5221
Departmental phone number: 520-626-6409
Lab phone number: 520-626-4385
FAX: 520-626-2100
Mycobacterium Tuberculosis Research
Tuberculosis is probably the most important infectious disease which afflicts mankind. A recent World Health Organization report estimates that 1.86 billion people are infected with tuberculosis (32% of the world's population ) and that in 1997, eight million new cases occurred. Over three million people die yearly from tuberculosis, the largest single infectious cause of mortality worldwide. Tuberculosis is still a persistent health problem in the U.S.A. due in part to the human immunodeficiency virus (AIDS) epidemic. AIDS patients are highly susceptible to infection with M. tuberculosis and other atypical mycobacteria that seldom infect individuals with intact immune systems. This increased incidence of tuberculosis is also associated with the increased numbers of homeless and of alcohol- and drug-abusing populations, as well as with the emergence of multidrug resistant strains of M. tuberculosis. Such strains constitute a very serious problem because they cannot be treated with any of the most commonly used anti-tuberculosis antibiotics, resulting in high mortality and rates of transmission.
I began studies on Mycobacterium tuberculosis during a one year sabbatical I took in 1994-1995 in London, England, in the laboratory of Professor Douglas B. Young, St. Mary's Medical School at Imperial College. This research was supported by the award of a Fogarty Senior International Fellowship from NIH. Our research on M. tuberculosis centers on cloning and identifying genes of the microbe which are involved in its ability to enter and to survive within macrophages. In screening for M. tuberculosis genes that play a role in the intracellular survival, an M. tuberculosis gene (eis) was identified that enhanced survival of Mycobacterium smegmatis in both human macrophages and in the human macrophage-like cell line U-937 when introduced on the multi-copy plasmid pOLYG. When a single chromosomal copy of eis was also introduced into M. smegmatis, using an integrative vector, the 42-kDa Eis protein was expressed, and the construct still exhibited increased intracellular survival in U-937 cells.
The role of the eis gene in M. tuberculosis intracellular survival and multiplication was investigated by inactivation of the gene using allelic exchange. The eis gene is present and the Eis protein is produced in both M. tuberculosis strain H37Ra and H37Rv. A mutated eis allele (eis::hyg) was delivered at the eis locus, using the suicide vector pMJ10, in both strains. Southern and Western blot analysis demonstrated that the eis gene was disrupted and that no Eis protein was produced. Further studies are in progress to study the mutant strain for survival within macrophages and in animal studies.
In Western blot studies using purified Eis, 40% of sera from pulmonary TB patients gave positive reactions. In summary, these results suggest that the eis gene may play a critical role in M. tuberculosis replication within human macrophages and that the Eis protein is produced and induces an immune response during active human tuberculosis infection. This work is presently supported by a grant from NIH.
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Bordetella Pertussis
Additional research in my laboratory involves studies on the human pathogens Bordetella pertussis, the causative agent of whooping cough. Our work on B. pertussis presently centers on studies of two transcriptional regulators we have identified in the microbe, Btr and Baf. Btr appears to be involved in metabolic regulation, while Baf is criticalin the expression of several important virulence factors of the pathogen (pertussis toxin and adenylate cyclase toxin)
Btr (Bordetella transcriptional regulator) is a member of the FNR family of transcriptional regulators found in E. coli and in a broad range of microbial pathogens. FNR regulates gene expression in response to changing oxygen tensions in E. coli by activating expression of genes required for anaerobic metabolism and repressing genes for aerobic growth. We have shown that Btr is indeed an FNR homologue by demonstrating its ability to functionally replace FNR in E. coli. It is intriguing to find such a regulator in the strictly aerobic organism B. pertussis.
Btr appears to be important in the virulence of B. pertussis. A btr mutant is less able to enter and survive within macrophages, and in a mouse infection model the btr mutant was unable to replicate and did not persist in the lungs as did the wild-type control. These results clearly demonstrate that the btr mutant is virulence-deficient. By sensing the redox state of the environment, Btr may allow survival by activation of a set of unrecognized virulence genes or Bordetella biosynthetic or catabolic genes, which are not classically thought of as virulence factors. Activation of these genes may be required for B. pertussis survival and persistence within the human host. Action of recognized virulence genes has been well studied, but the metabolism of pathogens in the host has not, although it may be equally important in the disease process.
We have also developed a Btr titration assay, which enables us to identify B. pertussis genes which are Btr-regulated. One of the Btr-regulated genes so far identified is a LysR homologue, called BrgR, another transcriptional regulator of the pathogen. These lysR regulatory genes are generally transcribed divergently from the genes they regulate. The DNA sequence surrounding brgR was determined and three open reading frames (brgABC) were found orientated opposite of brgR transcription. These genes have similarity to metabolic genes found in other organisms. For example, brgB encodes a protein with homology to urea amidolyase from yeast which metabolizes allantoin to ammonia and carbon dioxide. It is possible that this putative enzyme in B. pertussis may be involved in the acquisition of nitrogen for bacterial growth. Studies are in progress to determine the role of BrgR for vivo survival of B. pertussis and to determine the environmental signals which regulate both btr and brgR expression.
The virulence genes of B. pertussis have been demonstrated to be regulated by the two-component regulatory system BvgAS. Both bvg and fha (filamentous hemagglutinin) are expressed, in E. coli in the presence of BvgAS. Transcriptional fusions of fhaB-lacZ and bvg-lacZ are activated in the presence of bvgAS in trans. Transcriptional fusions of ptx (pertussis toxin) and cya (adenylate cyclase) promoters to lacZ are not expressed in E. coli, with bvgAS in trans. These results have prompted the hypothesis that other factors (accessory regulatory factors), in addition to BvgAS, must be required for activation and expression of ptx and cya. Baf was identified by my laboratory and appears to be the previously postulated BvgAS accessory factor required, along with BvgAS, for the production of both pertussis toxin and adenylate cyclase toxin.
Further studies were done to construct a baf mutant. From these initial studies, no baf mutants were recovered, suggesting that baf may be essential. Other studies were successful in the construction of a baf-kan mutation in the B. pertussis chromosome but only when the wild type baf gene was present in trans on a plasmid.
These results suggest that baf is an essential gene. We are presently studying what global role baf may play for B. pertussis survival. Our research on these identified B. pertussis transcriptional regulators will add to our understanding of the control of Bordetella pathogenesis and gene regulation for this important human pathogen.
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Recent Publications
- Bannan, J.D, M.J. Moran, J.I. MacInnes, G.A. Soltes, and R.L. Friedman. 1993. Cloning and characterization of btr, a Bordetella pertussis gene, encoding an FNR-like transcriptional regulator. J. Bacteriol. 175:7228-7235.
- DeShazer, D., G. E. Wood, and R.L. Friedman. 1995. Identification of a Bordetella pertussis regulatory factor required for transcription of the pertussis toxin operon in Escherichia coli. J. Bacteriology 177:3801-3807.
- Wood, G.E., N. Khelef, N. Guiso and R.L. Friedman.1998. Identification of Btr-regulated genes using a titration assay. Search for a role for this transcriptional regulator in the growth and virulence of Bordetella pertussis. Gene. 209: 51-58.
- Wei, J., J.L. Dahl, J.W. Moulder, E. A. Roberts, P. O’Gaora, D.B. Young, and R.L.Friedman. 2000. Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages. J. Bacteriology 182: 377-384.
- Wood, G. E., and R. L. Friedman. 2000. The Bvg accessory factor (Baf) enhances pertussis toxin expression in E. coli and is essential for Bordetella pertussis viability. FEMS Microbiology Letters 193: 25-30.
- Dahl, J. L., J. Wei, J. W. Moulder, S. Laal, and R. L. Friedman. 2001. Subcellular localization of the intracellular survival-enhancing Eis protein of Mycobacterium tuberculosis. Infect. Immun. 69:4295-4302.
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Lab Members
Current Lab Members
- Rick Friedman, Ph.D.
Professor, lab director
Past members
- Jim Moulder, Ph.D.
Professor Emeritus
- Janet Hatt, Ph.D.
Postdoctoral researcher
- Christopher Alteri, B.S.
PhD, 2005
- Linoj Samuel, B.S.
PhD, 2005
- Esteban Roberts, B.S.
PhD, 2004
- Amy Windley, B.S.
Senior Research Specialist
- Jun Wei
PhD, Dec 2001
- John Dahl, PhD
Post-doctoral Fellow
1997-2000
- Antonio Izzo, M.A.
Research Specialist
1996-1998
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- Gwen Wood
PhD, May 1997
- David DeShazer
PhD, Aug 1994
- Jason Bannan
PhD, June 1992
- Lisa Steed
PhD, Sept 1991
- Mark S. Leusch
PhD, Dec 1990
- Michael J. Moran
PhD, Aug 1990
- Steven Kuhl
Post-doctoral fellow
1988-1990
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Collaborations
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Owned/shared lab equipment
Friedman Lab General Equipment list
- AlphaInnotech Photoimager w. UV box for gel documentation
- table top centrifuge
- 4 microfuges
- computer equipment
- PowerMacintosh 7200 - connected to ethernet
- PowerMacintosh 7600 - connected to ethernet
- IBM 486 PC - primarily for use with photoimager
- Mac LaserWriter 12/640PS Microtek E-Max Microtiter plate reader
- 2 Sorvall centrifuges
- 4 horizontal gel electrophoresis chambers
- electroporation equipment
- -70 deg C freezer
- -20 deg freezer
- 2 refridgerator/freezers
- 2 sterile hoods
- 2'X 4' shaker
- 2 tabletop shakers
- Gilsen Photospectrophotometer
- Vertical gel electrophoresis
- sonicator
- 5 water baths
Departmental shared equipment
- ultracentrifuge
- walk-in cold room storage
- dark room
- scintillation counter
- autoclave room
- dish washer
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Lab Links
Institutional Pages:
Search Tools:
Sequence Interpretation:
- Genquest - FASTA, BLAST
- BCM-SearchLauncher - sequence-search using FASTA, BLAST, BEAUTY, BLOCKS (and derivatives)
- BLOCKS
- BLAST Blastserver: Sanger,
- France
- BCM-SearchLauncher - multiple sequence alignment using MSA, Clustal, PIMA
- ISREC-Page - examine sequences, searching for specific features using SAPS, TMpred, BOXSHADE, Prositescan, PatternFind, Coils
- ProDom - protein domain database
- MOTIF - search a protein for common motifs
- ProteinMotifs - search AA or DNA sequences for your motif
- PredictProtein - server at the EMBL Heidelberg
- TESS - find transcription factorsites
- ExPasy - Enzyme Database
- ReBase - Restriction Enzyme Database
Tools:
UofA Biosciences:
Collaborations:
Journals:
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Biotech companies/services
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