Laboratory of Bacterial Pathogenesis
Emil C. Gotschlich
R. Gwin Follis-Chevron Professor
EMIL C. GOTSCHLICH
The studies of Emil C. Gotschlich and his colleagues have been concerned with surface and cellular components of pathogenic Neisseria, the meningococcus N. meningitidis, and the gonococcus N. gonorrhoeae.
Pathogenic Neisseriae are unusual in that they produce large amounts of polyphosphate (PP) which they secrete on their surfaces. We have purified the synthesizing enzyme PP kinase and based on amino acid sequence data cloned the gene and determined its sequence. The PP kinase gene appears to be transcriptionally coupled to a previously undescribed lipoprotein. The PP kinase gene has been inactivated by insertion of a kanamycin marker and meningococcal or gonococcal transformants no longer produce PP kinase or polyphosphate. The resulting in vitro and in vivo phenotypic changes are currently under study.
Pathogenic Neisseriae and Haemophilus influenzae are the only organism known where the lipopolysaccharide antigen LPS characteristic of gram-negative organisms is subject to rapid antigenic variation. Some information on the genetic mechanism for this variation in Haemophilus influenzae has been reported, but in the case of the Neisseriae the mechanism has not been elucidated. We have identified an operon consisting of five open reading frames which is involved in the biosynthesis of the variable portions of the gonococcal LPS molecule. Deletion of one or more of these genes alters the structure of the LPS antigen. Three of the genes contain poly-G tracts ranging in length from 10 to 17 nucleotides and represent potential sites for high frequency mutational events that account for the antigenic variation. We are determining the role of each of the genes in the biosynthesis of LPS by genetic and immunochemical approaches. The role of the poly-G tracts in antigenic variation will be elucidated.
There are a number of instances where pathogenic bacteria respond to lactate in the environment to modify their virulence attributes. Meningococci and gonococci are able to utilize either D- or L-lactate. The D-lactate dehydrogenase (LDH) has ben purified, cloned, and meningococcal mutants with this gene inactivated have been constructed. The transformants were found to no longer produce D-LDH and were unable to utilize oxygen in the presence of D-lactate, but continued to consume oxygen in the presence of L-lactate. The availability of D-LDH mutants is allowing us to concentrate on the biochemical and molecular characterization of L-LDH, which in the human host is likely to be the important enzyme. The biochemical characterization has proved to be a difficult problem because the L-LDH activity is very unstable precluding significant purification. Genetic techniques are being used to identify the gene and characterize it.
The immunophilins, the cyclosporin A and FK-506-binding proteins, have been identified as crucial in intracellular signaling in T lymphocytes in addition to their prolyl peptidyl isomerases or rotamase activity. While enzymatically active homologs of cyclophilins have been described in a number of bacterial species, we have found that so far meningococcus is the only bacterial species that contains a gene encoding a 12-kD protein which is strongly homologous to the similarly sized human FK-506-binding protein, and when expressed in Escherichia coli, like its human counterpart, has FK-506-inhibitable rotamase activity. Surprisingly, this gene is silent in meningococcus and its regulation is under study.
Erwin, A. L., and D. S. Stephens. 1995. Identification and characterization of auxotrophs of Neisseria meningitidis produced by Tn916 mutagenesis. FEMS (Federation of European Microbiology Societies) Microbiology Letters. 127:223-228.
Erwin, A. L., M. J. Gill, and E. C. Gotschlich. 1995. Use of antibiotics to select auxotrophic mutants of Neisseria meningitidis. Microbial Pathogenesis. In press.
Gotschlich, E. C. 1994. Genetic locus for biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide. Journal of Experimantal Medicine. 180:2181-2190.
Tinsley, C. R., and E. C. Gotschlich. 1995. Cloning and characterization of the meningococcal polyphosphate kinase gene: production of polyphosphate synthesis mutants. Infection and Immunity. 63:1624-1630.
Yang, Q.-L., C. R. Tinsley, and E. C. Gotschlich. 1995. Novel lipoprotein expressed by Neisseria meningitidis but not by Neisseria gonorrhoeae. Infection and Immunity. 63:1631-1636.
VINCENT A. FISCHETTI
Our major interests are focused both on the mechanisms by which streptococci and staphylococci cause disease and the development of methods to induce a protective mucosal immune response. One of the systems we use is the M protein from group A streptococci. M protein is the major known virulence factor of this organism by virtue of its ability to impede attack by human phagocytes. Physicochemical and sequence analysis has revealed that M protein is an a-helical coiled-coil ropelike structure extending nearly 60 nm from the cell surface. DNA sequence analysis of the COOH-terminal end of the M protein gene (the region involved in its attachment to the cell) is highly homologous to comparable regions of nearly all known surface proteins from gram-positive bacteria. This indicates that the mechanism of anchoring surface proteins in these bacteria may also be conserved.
Since gram-positive bacteria use their surface molecules to colonize or invade tissues, a knowledge of the anchoring process will enable us to devise strategies to prevent their attachment to the cell and thus block infection. Furthermore, because surface molecules are used by disease bacteria to initiate and establish infection, we have been concerned with characterizing such molecules from gram-positive pathogens.
Capitalizing on the conservation of the anchoring process for surface proteins, we discovered that active polypeptides or proteins genetically fused to the common anchor region of the M protein could be used to deliver the active molecule to the surface of gram-positive bacteria. To test this, the gene coding for an allergen was fused to the M protein anchor segment. After this gene complex was introduced into the chromosome of a gram-positive commensal bacterium normally located in the human oral cavity, the allergen was found to be expressed on the surface of the commensal. When these recombinant bacteria were placed into the nasopharynx of mice, they remained there for up to 12 weeks. In these colonized mice, significant allergen-specific serum IgG, salivary IgA, and T cell response were produced. It is anticipated that this approach may be used for a variety of antigens to protect against invasion by disease organism. To date, a 122-amino acid fragment of the V3 loop of gp120 from HIV-1, a 15-amino acid T cell epitope from gp120, and HPV E7protein have been successfully expressed on the surface of commensal bacteria. Immune response and protective assays are now being tested.
Identification of a conserved region within the M proteins of at least 30 different serotypes of group A streptococci enabled us to design experiments to determine if vaccines comprising these regions would protect against streptococcal pharyngitis. Using a mouse mucosal model of infection, we found that mice--immunized intranasally with either conserved region peptides coupled to cholera toxin B subunit or vaccinia virus cloned to contain the conserved region of the M molecule--were protected from challenge by live streptococci. This suggests that immunization with conserved regions of the M protein can protect at the mucosa and may be the first step in designing an antistreptococcal vaccine. Human studies will soon be under way to test the protective efficacy of a conserved region construct engineered to be delivered on the surface of a human oral commensal.
A listing of streptococcal strains from the university's Lancefield collection is available at http://www.rockefeller.edu/vaf/.
Bessen, D., and V. A. Fischetti. 1992. Nucleotide sequence of two adjacent M or M-like protein genes of group A streptococci: different RNA transcript levels and identification of a unique IgA-binding protein. Infection and Immunity. 60:124-135.
Fischetti, V. A. 1991. Streptococcal M protein. Scientific American. 264:58-65.
Fischetti, V. A., D. Medaglini, M. Oggioni, and G. Pozzi. 1993. Expression of foreign proteins on Gram-positive commensal bacteria for mucosal vaccine delivery. Current Opinion in Biotechnology. 4:603-610.
Fischetti, V. A., V. Pancholi, and O. Schneewind. 1990. Conservation of a hexapeptide sequence in the anchor region of the surface proteins of gram-positive cocci. Molecular Microbiology. 4:1603-1605.
Hruby, D. E., O. Schneewind, E. M. Wilson, and V. A. Fischetti. 1991. Assembly and analysis of a functional vaccinia virus "amplicon" containing the C-repeat region from the M protein of Streptococcus pyogenes. Proceedings of the National Academy of Sciences USA. 88:3190-3194.
Medaglini, D., G. Pozzi, T. P. King, V. A. Fischetti. 1995. Mucosal immune response to a recombinant fusion protein expressed on the surface of the oral commensal Streptococcus gordonii after oral colonization. Proceedings of the National Academy of Sciences USA. In press.
Pozzi, G., M. Contorni, M. R. Oggioni, R. Manganelli, M. Tommasino, F. Cavalieri, and V. A. Fischetti. 1992. The delivery and expression of a heterologous antigen on the surface of streptococci. Infection and Immunity. 60:1902-1907.
Pozzi, G., M. R. Oggioni, R. Manganelli, D. Medaglini, V. A. Fischetti, D. Fenoglio, M. T. Valle, A. Kunkl, and F. Manca. 1994. Human T helper cell recognition of an immunodominant epitope of HIV-1 gp120 expressed on the surface of Streptococcus gordonii. Vaccine. In press.
Rakonjac, J. V., J. C. Robbins, and V. A. Fischetti. 1995. DNA sequence of the serum opacity factor of class II group A streptococci: Identification of a fibronectin binding repeat domain. Infection and Immunity. 63:622-631.
Schneewind, O., P. Model, and V. A. Fischetti. 1992. Sorting of protein A to the staphylococcal cell wall. Cell. 70:1-20.
My main research focus is on characterization of the sar locus, a unique regulatory locus which we identified by insertional mutagenesis in a wild-type Staphylococcus aureus strain. Inactivation of this locus as a result of a single Tn917 insertion into the chromosome of an S. aureus strain has resulted in a pleiotropic effect on the expression of several extracellular and cell wall proteins many of which are involved in pathogenesis. We have subsequently cloned and sequenced an intact sar gene (designated sarA). Sequence analysis of the sarA gene reveals an ORF of 372 bp. However, it does not have significant homology to sequence elements of the helix-turn-helix motif or to the two component regulatory systems as described in procaryotes and hence may represent a novel regulatory system. Recent experimental evidence from our laboratory suggests that sar may be a regulator of agr, a well described regulator of exoprotein synthesis in S. aureus. Preliminary gel shift studies suggested that the sarA gene product may be a DNA binding protein. One of our future projects will be to define the molecular interactions between sar and agr loci.
In another line of research, we have shown that fibrinogen can act as a bridging molecule in the adherence of S. aureus to catheters and cultured endothelial cells. We have subsequently cloned the gene for the putative adhesin (designated fibrinogen binding protein or FBP) and were able to express this protein in E. coli. We are in the process of generating polyclonal and monoclonal antibodies to characterize the biological significance of this protein.
In our third investigative program, we are interested in phase switching of the agr phenotype in S. aureus. The agr locus is a global regulator involved in the control of extracellular and cell wall proteins. Given that some of these proteins regulated by agr are potential virulence determinants, it is conceivable that this phase switching may be an important event in controlling the expression of these proteins in S. aureus infections. Studies are under way to characterize the genes involved in phase switching.
Cheung, A. L., J. M. Koomey, C. A. Butler, S. J. Projan, and V. A. Fischetti. 1992. Regulation of exoprotein expression in Staphylococcus aureus by a locus (sar) distinct from agr. Proceedings of the National Academy of Sciences USA. 89:6462-6466.
Cheung, A. L., M. Krishnan, E. A. Jaffe, and V. A. Fischetti. 1991. Fibrinogen acts as a bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial cells. Journal of Clinical Investigation. 87:2236-2245.
Cheung, A. L., and S. J. Projan. 1994. Cloning and sequencing of sarA: a gene required for the expression of agr. Journal of Bacteriology. 176:580-585.
Cheung, A. L., M. Yeaman, and A. S. Bayer. 1994. The role of the sar locus in the induction of experimental endocarditis in rabbits. Infection and Immunity. 62:1719-1725.
Cheung, A. L., and P. Ying. 1994. Regulation of a and b hemolysins by the sar locus of S. aureus. Journal of Bacteriology. 176:580-585.
Microbial attachment to epithelial cell surfaces may result in several effects including colonization, internalization, and possible intracellular proliferation before the onset of a successful infection. The most common site of group A streptococcal infection is the tonsil/pharynx. We have chosen to focus on the early events of streptococcal infection of tonsillar/pharyngeal cells by defining the biochemical events necessary for the attachment of group A streptococci to their cellular target. We have identified a novel protein on the surface of group A streptococci that has structural and functional similarities with eukaryotic glyceraldehyde-3-phosphate dehydrogenase. This protein, named streptococcal surface dehydrogenase (SDH), has multiple binding activities with many mammalian and cytoskeletal proteins. Recently, we have shown that SDH is in fact an ADP-ribosylating enzyme, the activity of which is enhanced in the presence of nitric oxide radical (NO.), a novel messenger molecule. Our ongoing project has also now indicated that SDH plays a regulatory role in the phosphorylation events occurring at the pharyngeal cell membrane proteins and hence may have an important role in signaling events in pharyngeal cells. Studies focusing on the mechanism of this regulation and its effects on the intracellular events in pharyngeal cells as a result of their interaction with SDH are under investigation. Structural and functional analysis of epitopes bearing the above-mentioned functions of SDH will be carried out by cloning and sequencing of the sdh gene and using sdh mutants and/or the mutants lacking the region responsible for the above-mentioned functional epitopes. Studies are under way to characterize the role of this protein in streptococcal invasion and poststreptococcal sequelae such as rheumatic fever and postglomerulonephritis.
Pancholi, V., and V. A. Fischetti. 1992. A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate dehydrogenase with multiple binding activity. Journal of Experimental Medicine. 176:415-426.
Pancholi, V., and V. A. Fischetti. 1993. Glyceraldehyde-3-phosphate dehydrogenase on the surface of group A streptococci is also an ADP-ribosylating enzyme. Proceedings of the National Academy of Sciences USA. 90:8154-8158.