Nontypable Haemophilus influenzae is a common cause of localized respiratory tract disease, especially otitis media, sinusitis, pneumonia, and exacerbations of chronic lung disease. Following each episode of acute otitis media, fluid remains in the middle ear for weeks to months and is associated with significant hearing deficit, a sequela that in turn can impair language acquisition, speech development, and school performance. The initial step in the pathogenesis of disease due to nontypable H. influenzae involves colonization of the upper respiratory epithelium. We have demonstrated that two related high-molecular-weight proteins called HMW1 and HMW2 promote attachment to human epithelium, an essential step in the process of colonization. Examination of collections of epidemiologically unrelated strains reveals that HMW1- and HMW2-like proteins are present in 75-80% of all nontypable H. influenzae strains and are the predominant adhesins in these strains. We have established that the HMW1 and HMW2 proteins are prototype members of the two-partner secretion family and require a cognate dimeric outer membrane protein called HMW1B or HMW2B for presentation on the bacterial surface. In the process of surface localization, HMW1 and HMW2 undergo a cleavage event that eliminates a large N-terminal pro-piece. In addition, we have demonstrated that HMW1 and HMW2 are glycoproteins and are modified by a glycosyltransferase called HMW1C or HMW2C, capable of transferring hexose units to asparagine residues. Based on studies of HMW1, over 30 asparagines are modified with mono- or di-hexoses, including glucose and galactose. In the present proposal we plan to elucidate the mechanism of HMW1 and HMW2 surface localization and maturation. In particular, we will define the determinants of interaction between HMW1 and HMW1B, the importance of HMW1B dimer formation for HMW1 secretion, and the mechanism of cleavage of the HMW1 pro-piece. In additional experiments, we will elucidate the molecular details of glycosylation, defining the relationship between specific asparagine sites of glycosylation and the adhesive function of HMW1 and the relationship between glycosylation and immunogenicity of HMW1. Finally, we will characterize the HMW1C-like family of glycosyltransferases. From a practical perspective, the results of these studies may be directly relevant to the development of novel antimicrobials and vaccines effective in the treatment and prevention of nontypable H. influenzae disease. More generally, they may provide fundamental insights into host-microbial relationships, bacterial protein secretion, and prokaryotic protein glycosylation.

Public Health Relevance

Nontypable Haemophilus influenzae is a common cause of localized respiratory tract disease, especially otitis media, sinusitis, pneumonia, and exacerbations of chronic lung disease. From a practical perspective, the results of the proposed studies may be directly relevant to the development of novel antimicrobials and vaccines effective in the treatment and prevention of nontypable H. influenzae disease. More generally, these studies will provide fundamental insights into host-microbial relationships, bacterial protein secretion, and prokaryotic protein glycosylation.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
7R01DC002873-19
Application #
8728393
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Watson, Bracie
Project Start
1995-06-01
Project End
2015-02-28
Budget Start
2013-07-01
Budget End
2014-02-28
Support Year
19
Fiscal Year
2013
Total Cost
$199,079
Indirect Cost
$20,201
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
McCann, Jessica R; St Geme 3rd, Joseph W (2014) The HMW1C-like glycosyltransferases--an enzyme family with a sweet tooth for simple sugars. PLoS Pathog 10:e1003977
Kawai, Fumihiro; Grass, Susan; Kim, Youngchang et al. (2011) Structural insights into the glycosyltransferase activity of the Actinobacillus pleuropneumoniae HMW1C-like protein. J Biol Chem 286:38546-57
Grass, Susan; Lichti, Cheryl F; Townsend, R Reid et al. (2010) The Haemophilus influenzae HMW1C protein is a glycosyltransferase that transfers hexose residues to asparagine sites in the HMW1 adhesin. PLoS Pathog 6:e1000919
St Geme 3rd, Joseph W; Yeo, Hye-Jeong (2009) A prototype two-partner secretion pathway: the Haemophilus influenzae HMW1 and HMW2 adhesin systems. Trends Microbiol 17:355-60
Cholon, Deborah M; Cutter, David; Richardson, Stephen K et al. (2008) Serial isolates of persistent Haemophilus influenzae in patients with chronic obstructive pulmonary disease express diminishing quantities of the HMW1 and HMW2 adhesins. Infect Immun 76:4463-8
Gross, Julia; Grass, Susan; Davis, Alan E et al. (2008) The Haemophilus influenzae HMW1 adhesin is a glycoprotein with an unusual N-linked carbohydrate modification. J Biol Chem 283:26010-5
Yeo, Hye-Jeong; Yokoyama, Takeshi; Walkiewicz, Katarzyna et al. (2007) The structure of the Haemophilus influenzae HMW1 pro-piece reveals a structural domain essential for bacterial two-partner secretion. J Biol Chem 282:31076-84
Li, Huilin; Grass, Susan; Wang, Tao et al. (2007) Structure of the Haemophilus influenzae HMW1B translocator protein: evidence for a twin pore. J Bacteriol 189:7497-502
Surana, Neeraj K; Buscher, Amy Z; Hardy, Gail G et al. (2006) Translocator proteins in the two-partner secretion family have multiple domains. J Biol Chem 281:18051-8
Sukupolvi-Petty, Soila; Grass, Susan; St Geme 3rd, Joseph W (2006) The Haemophilus influenzae Type b hcsA and hcsB gene products facilitate transport of capsular polysaccharide across the outer membrane and are essential for virulence. J Bacteriol 188:3870-7

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