Chair, Department of Pediatrics

Department / Division:
Pediatrics / Infectious Diseases

Address:
DUMC 3352
Durham, NC 27710

Appointment Telephone:
919-668-4000

Office Telephone:
919-681-4080

Fax Telephone:
919-681-2714

• MD, Harvard Medical School (Massachusetts), 1984

• Pediatrics, Children's Hospital of Philadelphia (Pennsylvania), 1984-1988

• Postdoctoral Fellow, Department of Microbiology and Immunology, Stanford University (California), 1988-1992
• Postdoctoral Fellow, Department of Pediatrics, Division of Infectious Diseases, Stanford University (California), 1991-1992

Clinical Interests:
Pediatric infectious diseases, antibiotic resistance, respiratory tract infections, central nervous system infections, tick-borne infections, vaccine development, microbial pathogenesis

Research Interests:
ST. GEME LAB

My laboratory is interested in host-pathogen interactions and is using genetic methods, protein chemistry, X-ray crystallography, high resolution microscopy, and microarray analysis to study the molecular and cellular determinants of disease due to Haemophilus influenzae, a model mucosal pathogen and a common cause of local respiratory tract disease and serious invasive infection.  H. influenzae expresses a number of adhesive proteins that mediate interaction with host epithelium, and we are studying the biogenesis, adhesive specificity, and regulation of these proteins.  H. influenzae is also capable of entering and surviving inside host cells, a strategy that may have evolved to provide the organism with a protected niche.  In ongoing work, we are examining the host cell receptors, cytoskeletal elements, and signaling molecules involved in cellular entry. Recent evidence indicates that H. influenzae forms biofilms, complex communities of organisms that likely facilitate persistence on the respiratory epithelial surface.  We are seeking to understand the bacterial and host factors that influence H. influenzae biofilm formation, and we are beginning to study the relationship between biofilms and evasion of innate immune mechanisms, such as cationic peptides, lactoferrin, and phagocytosis.  Using H. influenzae and human DNA microarrays, we hope to uncover novel pathways that are fundamental to the outcome of encounters between bacteria and the human host.

From a practical perspective, our work has relevance to development of novel antimicrobial compounds and to generation of a vaccine broadly effective against H. influenzae.

Projects

Haemophilus Influenzae Adherence

Most bacterial diseases begin with microbial colonization of a particular mucosal surface.  Bacterial attachment to host epithelial cells is a key event in this process and is mediated by specific interactions between microbial adhesins and complementary receptor structures on the epithelial cell surface.

Virtually all strains of H. influenzae express one or two high-molecular weight non-pilus adhesins, either Hia/Hsf or HMW1 and HMW2.  We are characterizing the structure, biogenesis, adhesive properties, and regulation of these adhesins.  We are also defining the host cell receptors with which these adhesins interact and the host cell pathways that are activated by H. influenzae adherence.


H. influenzae Hap-meditated Microcolony Formation

Microbial biofilms are structured microbial communities that are being recognized increasingly in human disease and appear to promote bacterial persistence on mucosal surfaces.  Several reports indicate that Haemophilus influenzae is capable of forming biofilms, in particular in patients with otitis media and probably in adults with chronic bronchitis.  We have identified an H. influenzae adhesive protein called Hap, which was first discovered based on the capacity to promote intimate interaction with cultured epithelial cells.  More recent evidence indicates that Hap mediates bacterial aggregation and microcolony formation.  Hap adhesive activity is augmented by a soluble host serine protease inhibitor called secretory leukocyte protease inhibitor (SLPI).  At the same time, Hap is a target for degradation by lactoferrin, a host protein present in a number of secretions and capable of cleaving arginine-rich sequences.  We hypothesize that Hap-mediated microcolony formation is a precursor to biofilm formation, and we are currently exploring this possibility.

Protein Secretion
               
The interaction between pathogenic bacteria and host cells is largely determined by microbial proteins presented on the bacterial surface or released extracellularly. In gram-negative bacteria, the process of protein secretion requires translocation across the inner membrane, the periplasm, and the outer membrane.  In general, protein secretion occurs via one of six different secretion pathways, distinguished at least in part by the mechanism of translocation across the outer membrane and referred to as types I-VI.  We have focused on characterizing the type V pathway, which is utilized by proteins belonging to the autotransporter family and the two-partner secretion family.

Proteins in the autotransporter family contain 3 functional domains, including an N-terminal signal peptide, an internal passenger domain, and a C-terminal outer membrane translocator domain.  The translocator domain forms a pore and facilitates surface localization of the passenger domain.  Once on the surface of the organism, the passenger domain usually undergoes cleavage, often with subsequent extracellular release.  The H. influenzae Hap protein is a conventional autotransporter and undergoes autoproteolytic cleavage, mediated by a serine protease motif defined by residues H98, D140, and S243.  The Hia and Hsf adhesins are examples of a subset of autotransporters referred to as trimeric autotransporters.  These proteins are characterized by a trimeric architecture and a short C-terminal translocator domain and remain uncleaved at the C-terminus and fully cell-associated.

The two-partner secretion pathway consists of an exoprotein and a pore-forming outer membrane translocator.  Upon secretion, the exoprotein either remains non-covalently associated with the bacterial surface or is secreted extracellularly.  The HMW1 and HMW2 adhesins are secreted by the two-partner secetion pathway and are glycosylated, representative of a subset of proteins in the two-partner secretion pathway family.

We are interested in defining the structural determinants of secretion by conventional autotransporters, trimeric autotransporters, and two-partner secretion proteins, using Hap, Hia/Hsf, and HMW1/HMW2 as prototypes.

Gene Regulation

During the course of natural infection, pathogenic bacteria encounter varied and changing environments.  To survive these diverse conditions, many pathogens have developed mechanisms that facilitate efficient adaptation. Phase variation represents one such mechanism and is characterized by the reversible loss or gain of a defined structure.  In most cases, the involved structure is expressed on the surface of the organism and varies between two states, namely off and on.  Sometimes three states exist, including off (-), weakly on (+), and strongly on (+++).

We have found that the H. influenzae HMW1 and HMW2 adhesins are subject to phase variation, mediated by variation in the number of tandem 7-base pair repeats upstream of the hmw1A and hmw2A structural genes.  It is notable that HMW1 and HMW2 are important colonization factors.  In addition, they are strongly immunogenic and are major targets of the antibody response during natural infection.  Based on examination of paired nasopharyngeal and middle ear isolates from children with otitis media and serial sputum isolates from patients with chronic bronchitis, we speculate that spontaneous variation in the number of tandem repeats enables the organism to vary between states associated with efficient adherence versus effective immune evasion.  Interestingly, in contrast to other known examples of phase variation, the phase-variable expression of HMW1 and HMW2 involves multiple states that range from weakly on to very strongly on, with a series of gradations in between.  We are exploring the mechanism by which repeat number influences gene expression.  In addition, we are interested in defining the selective advantage of graded phase variation, rather than off/on phase variation.

Laboratory Staff:

Sue Grass, Associate in Research
Amanda Sheets, Graduate Student
Nicole Spahich, Graduate Student
Eric Porsch, Graduate Student

Representative Publications:
St. Geme JWIII, Yeo H-Y. A prototype two-partner secretion pathway: the Haemophilus influenzae HMW1 and HMW2 system. Trends Microbiol. In press. (2009)

Radin JN, Grass S, Meng G, Cotter SE, Waksman G, St. Geme JWIII. Structural basis for the differential binding affinities of the HsfBD1 and HsfBD2 domains in the Haemophilus influenzae Hsf adhesin. J Bacteriol. In press. (2009)

Kehl-Fie TE, Porsch EA, Miller SE, St. Geme JWIII. Expression of Kingella kingae type IV pili is regulated by sigma54, PilS, and PilR. J Bacteriol. In press. (2009)

Sheets AJ, Grass S, Miller S, St. Geme JWIII.  Identification of a novel trimeric autotransporter adhesin in the cryptic genospecies of Haemophilus. J Bacteriol. 2008;190:43134320. (2008)

Meng G, St. Geme JWIII, Waksman G. Crystal structures of Hia fragments reveal a repetitive architecture in the Haemophilus influenzae Hia trimeric autotransporter. J Mol Biol. 2008; 384:824-836. (2008)

Kehl-Fie TE, Miller SE, St. Geme JWIII. Kingella kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells. J Bacteriol. 2008;190:7157-7163. (2008)

Gross J, Grass S, Davis AE, Gilmore-P, Townsend RR, St. Geme JWIII. The Haemophilus influenzae HMW1 adhesin is a glycoprotein with an unusual N-linked carbohydrate modification. J Biol Chem. 2008;283:26010-26015. (2008)

Cholon D, Cutter D, Richardson SK, Sethi S, Murphy TF, Look DC, St. Geme JWIII. Serial isolates of Haemophilus influenzae from patients with chronic obstructive pulmonary disease express diminishing quantities of the HMW1 and HWM2 adhesins. Infect Immun. 2008;76:4463-4468. (2008)

Yeo H-J, Yokoyama T, Walkiewicz K, Kim Y, Grass S, St. Geme JWIII. The structure of the Haemophilus influenzae HMW1 pro-piece reveals a structural domain that is essential for bacterial two-partner secretion. Journal of Biological Chemistry. 2007;42:31076-31084.
(2007)

Li H, Grass S, Wang T, Susan Grass2, Liu T, St. Geme JWIII. Structure of the Haemophilus influenzae HMW1B translocator protein: Evidence for a twin-pore. Journal of Bacteriology. 2007;189:7497-7502. (2007)

Kehl-Fie T, St. Geme JWIII. Identification of a novel cytotoxin in Kingella kingae. Journal of Bacteriology. 2007;189:430-436. (2007)

Webster P, Wu S, Gomez G, Apicella M, Plaut AG, St. Geme JWIII. Distribution of bacterial proteins in biofilms formed by non-typeable Haemophilus influenzae.  Journal of Histochemistry and Cytochemistry. 2006;54:829-842. (2006)

Surana NK, Buscher AZ, Hardy GG, Kehl-Fie T, Grass S, St. Geme JWIII. Translocator proteins in the two-partner secretion family have multiple domains. Journal of Biological Chemistry. 2006;281:18051-18058. (2006)

Sukupolvi-Petty S, Grass S, St. Geme JWIII.  The Haemophilus influenzae type b hcsA and hcsB gene products facilitate transport of capsular polysaccharide across the outer membrane and are essential for virulence. Journal of Bacteriology. 2006;188:3870-3877. (2006)

Meng G, Surana NK, St. Geme JWIII, Waksman G. Structure of the outer membrane translocator domain of the Haemophilus influenzae Hia trimeric autotransporter. EMBO Journal. 2006;25:2297-2304. (2006)

Cotter SE, Surana NK, Grass S, St. Geme JWIII. Trimeric autotransporters require trimerization of the passenger domain for stability and adhesive activity. Journal of Bacteriology.  2006;188:5400-5407. (2006)

Buscher AZ, Grass S, Heuser J, Roth R, St. Geme JWIII. Surface anchoring of a bacterial adhesin secreted by the two-partner secretion system. Molecular Microbiology. 2006;61:470-483. (2006)

Cotter SE, Yeo H-J, Juehne T, St. Geme JWIII. Architecture and adhesive activity of the Haemophilus influenzae Hsf adhesin. Journal of Bacteriology. 2005;187:4656-4664. (2005)

Cotter SE, Surana NK, St. Geme JWIII.  Trimeric autotransporters:  A distinct subfamily of autotransporter proteins.  Trends in Microbiology.  2005;13:199-205. (2005)

Yeo H-J, Cotter SE, Laarmann S, Juehne T, St. Geme JWIII, Waksman G.  Structural basis for host recognition by the Haemophilus influenzae Hia autotransporter.  EMBO J 2004;23:1245-1256. (2004)

Surana NK, Grass S, Hardy GG, Li H, Thanassi DG, St. Geme JWIII. Evidence for conserved architecture and physical properties of Omp85-like proteins throughout evolution. Proc Natl Acad Sci USA. 2004;101:14497-14502. (2004)

Surana NK, Cutter D, Barenkamp SJ, St. Geme JWIII. The Haemophilus influenzae Hia autotransporter contains an unusually short, trimeric translocator domain. J Biol Chem 2004;279:14679-14685. (2004)

Buscher AZ, Burmeister K, Barenkamp SJ, St. Geme JWIII.  Evolutionary and functional relationships among the nontypeable Haemophilus influenzae HMW family of adhesins.  J Bacteriol 2004;186:4209-4217. (2004)

Hendrixson DR, Qiu J, Shewry SC, Fink DL, Petty S, Baker EN, Plaut AG, St. Geme JWIII. : Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites. Mol Microbiol 2003;47:607-617. (2003)

Grass S, Buscher AZ, Swords WE, Apicella MA, Barenkamp SJ, Ozchlewski N, St. Geme JWIII. The Haemophilus influenzae HMW1 adhesin is glycosylated in a process that requires the HMW1C protein and an enzyme important for lipooligosaccharide biosynthesis. Mol Microbiol. 2003;48:737-751. (2003)

Fink DL, St. Geme JWIII.  Chromosomal expression of the Haemophilus influenzae Hap autotransporter allows fine-tuned regulation of adhesive potential via inhibition of intermolecular autoproteolysis. J Bacteriol 2003;185:1608-1615. (2003)

Fink DL, Buscher AZ, Green BA, Fernsten P, St. Geme JWIII: The Haemophilus influenzae Hap autotransporter mediates microcolony formation and adherence to epithelial cells and extracellular matrix via binding regions in the C-terminal end of the passenger domain. Cell Microbiol 2003;5:175-186. (2003)

St. Geme JWIII: Molecular and cellular determinants of nontypable Haemophilus influenzae adherence and invasion. Cell Microbiol 2002; 4:191-200. (2002)

Laarmann S, Cutter D, Juehne T, Barenkamp SJ, St. Geme JWIII: The Haemophilus influenzae Hia autotransporter harbors two adhesive pockets that reside in the passenger domain and recognize the same host cell receptor. Mol Microbiol 2002;46:731-743. (2002)

Cutter D, Mason KW, Howell A, Fink DL, Green BA, St. Geme JWIII. Immunization with the Haemophilus influenzae Hap adhesin protects against nasopharyngeal colonization in experimental mice. J Infect Dis 2002;186:1115-1121. (2002)

Fink DL, Cope LD, Hansen EJ, St. Geme JWIII: The Haemophilus influenzae Hap autotransporter is a chymotrypsin clan serine protease and undergoes autoproteolysis via an intermolecular mechanism. J Biol Chem 2001;276:39492-39500. (2001)

Dawid S, Grass S, St. Geme JWIII. Mapping of binding domains of nontypeable Haemophilus influenzae HMW1 and HMW2 adhesins.  Infect Immun 2001; 69:307-314. (2001)

St. Geme JWIII, Cutter D. The Haemophilus influenzae Hia adhesin in an autotransporter protein that remains uncleaved at the C-terminus and fully cell associated.  J Bacteriol 2000; 182:6005-6013. (2000)

Krasan GP, Sauer F, Cutter D, Farley MM, Gilsdorf JR, Hultgren SJ, St. Geme JWIII. Evidence for donor strand complementation in the biogenesis of Haemophilus influenzae hemagglutinating pili. Mol Microbiol 2000;35:1335-1347. (2000)

Grass S, St. Geme JWIII: Maturation and secretion of the nontypable Haemophilus influenzae HMW1 adhesin: Roles of the N-terminal and C-terminal domains. Mol Microbiol 2000;36:55-67. (2000)

Dawid S, Barenkamp SJ, St. Geme JWIII: Variation in expression of the Haemophilus influenzae HMW adhesins: A prokaryotic system reminiscent of eukaryotes. Proc Natl Acad Sci USA 1999;96:1077-1082. (1999)

St. Geme JWIII, Grass S. Secretion of the Haemophilus influenzae HMW1 and HMW2 adhesins involves a  periplasmic intermediate and requires the HMWB and HMWC proteins. Mol Microbiol 1998;27:617-630. (1998)

Qiu J, Hendrixson DR, Baker EN, Murphy TF, St. Geme JWIII, Plaut AG. Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae. Proc Natl Acad Sci, USA 1998;95:12641-12646. (1998)

Hendrixson DR, St. Geme JWIII: The Haemophilus influenzae Hap serine protease promotes adherence and microcolony formation, potentiated by a soluble host protein. Mol Cell 1998;2:841-850. (1998)

Hendrixson D, de la Morena ML, Stathopoulos C, St. Geme JWIII. Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein. Mol Microbiol 1997;26:505-518. (1997)

St. Geme JWIII, Pinkner JS, Krasan GP, Heuser J, Bullitt E, Smith AL, Hultgren SJ. Haemophilus influenzae pili are composite structures assembled via the HifB chaperone. Proc Natl Acad Sci USA 1996;93:11913-11918. (1996)