Microbiology & Immunology | Faculty | David J. Kolodrubetz, Ph.D.
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Microbiology & Immunology Faculty

  Research | Publications | Lab Members


David J. Kolodrubetz, Ph.D.
Professor

Room X.051V
Tel: (210) 567-3967
Fax: (210) 567-6612
Email: Kolodrubetz@uthscsa.edu
 

Research

 

Periodontitis is an inflammatory disease of tissues in the subgingival crevice that can lead to soft tissue damage and dramatic bone loss. Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans and Campylobacter rectus are gram negative bacteria involved in the etiology of particular periodontal diseases. A number of putative virulence factors have been identified in these organisms including a leukotoxin from A. actinomycetemcomitans and a proteinaceous layer, called the S-layer, external to the outer membrane of C. rectus. The two major goals in our lab are to determine the roles of these proteins, and other virulence factors, in pathogenesis and to unravel the mechanisms by which their synthesis is regulated by signals unique to the oral microenvironment. These two fundamental questions are being addressed using genetic, molecular, genomic and biochemical approaches.

 

Our work on the A. actinomycetemcomitans (Aa) leukotoxin has focused on the regulation of its transcription. In particular, leukotoxin (lktA) RNA synthesis is repressed in the presence of oxygen. Thus, the anaerobic environment of the subgingiva will have an effect on leukotoxin production. To define the region of the leukotoxin promoter that is involved in the regulation of lkt RNA by oxygen, we have assayed the effects of promoter region mutations. The results have delimited a 30 bp region that is a negative regulatory site which responds to oxygen. The DNA sequence in this element does not appear to be related to the sequences of aerobic/anaerobic responsive sequences found in other bacteria. Thus, aerobic/anaerobic regulation of leukotoxin RNA synthesis in Aa appears to be using an undefined mechanism. In addition, we have created mutants in the genes encoding Fnr and ArcAB, which are homologues of proteins involved in transcriptional regulation by oxygen in other bacteria, and demonstrated that the Aa leukotoxin is not regulated by Fnr or ArcAB. A systematic and comprehensive molecular genetic approach is currently being employed to identify the transcription factors involved in this potentially novel regulatory pathway.

 

Since other oxygen-responsive genes are also likely to have critical roles in the pathogenic potential of Aa in its microenvironment, we have used gene-expression microarrays to identify the full complement of Aa genes that are differentially synthesized in aerobic versus anaerobic growth. The transcription of approximately forty genes is regulated by oxygen in Aa. Not surprisingly, the majority of these genes encode metabolic proteins which are important for the growth of the bacterium in the aerobic or anaerobic environment. However, there are a handful of oxygen-regulated genes whose products have unknown functions. These are potential new virulence factors for Aa and their roles in pathogenesis are being investigated by the construction and analysis of appropriate mutants. Interestingly, we have also used microarrays to identify a set of oxygen-responsive genes that may be regulated by the same non-Fnr/non-ArcA pathway(s) as is leukotoxin. Studying the regulation of these genes and leukotoxin will allow us to begin to develop the first molecular models for the mechanisms of non-Fnr/non-ArcAB aerobic/anaerobic regulation in a periodontal pathogen.

 

The lab's studies on the C. rectus S-layer have emphasized the function and secretion of the 150 kd protein that makes up the S-layer. Isogenic S-layer-positive and S-layer-negative strains of C. rectus have been constructed and were used to show that the presence of the S-layer alters the nature of the binding of C. rectus to host cells. We are also evaluating the possible role of the S-layer in the formation of biofilms with other periodontal bacteria. Such experiments will critically evaluate the role of the C. rectus S-layer in pathogenesis and help determine the mechanisms of virulence. In addition, we have used genetic approaches to define the pathway used to secrete the S-layer protein to the cell surface. Our results show that the S-layer protein is transported to the cell surface by a type I secretion system. Interestingly, another gene, called crsC, is involved in the release of some S-layer protein from the cell surface. We are investigating the mechanism by which CrsC causes S-layer protein release because the S-layer protein released from the C. rectus cell surface appears to have a new function, possibly as a cytotoxin.

Leukotoxin (LKT) RNA levels increase when Aa is grown anaerobically


 

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Publications


 

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Lab Members

  Lab Rooms: 5.023V

  • Sandra Bryant, Research Assistant
  • Alex Burgum, Research Associate
  • Linda Phillips, Research Associate



    • Graduate Students:
     Catherine Childress
    Leigh Ann Feuerbacher

 

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