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.
Pathogenic Neisseriae are unusual in that they produce large amounts
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
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
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
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
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
is available at ../../vaf/vaf.home.html.
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.
Fischetti, V. A. 1991. Streptococcal M protein. Scientific American.
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.
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
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.
Cheung, A. L., and S. J. Projan. 1994. Cloning and sequencing of sarA: a
gene required for the expression of agr. Journal of Bacteriology.
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
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