Pili or fimbriae provide adhesive functions for the pathogenesis of bacterial infections and play important roles during the establishment of respiratory, urinary, and intestinal infectious diseases. The mechanism of pilus assembly in gram-negative bacteria has been well characterized; however, an assembly pathway for pili of gram-positive pathogens has not yet been revealed. The long-term goal of this proposal is to elucidate the mechanisms of pillus assembly in gram-positive microbes. Our specific hypothesis is that gram-positive bacterial pili are covalently linked by sortase to the bacterial cell wall. This hypothesis is based on the following observations: i) Gram-positive microbes employ the cell wall peptidoglycan as a surface organelle for the covalent attachment of proteins, a mechanism that requires sorting signals of surface protein precursors and sortase, which cleaves sorting signals at the LPXTG motif and links the C-terminus of surface proteins via an amide bond to the peptidoglycan cross-bridge; ii) Pilus assembly in Corynebacterium diphtheriae occurs by a mechanism of ordered cross-linking, whereby pilin-specific sortases cleave precursor proteins at sorting signals and involve the side chain amino groups of pilin motif sequences to generate links between pilin subunits; iii) Muramidase treatment releases pili into the medium; iv) Sortase and pilin genes with sorting signal and pilin motif are found in many gram-positive pathogens. Based on these observations, we propose the following specific aims: 1. Determination of the molecular architecture of corynebacterial pili. The proposed experiments will characterize the biochemical linkages between subunits of corynebacterial pili and the requirements of sortases and pilin motif sequences for assembly. 2. Biochemical analysis of pilus assembly in corynebacteria. 3. Determination of the universality of pilus architecture and assembly mechanisms in gram-positive bacteria. We will determine, by biochemical and microscopic methods, whether Streptococcus agalactiae employes the same mechanism for pilus assembly. We will also evaluate the role of pilus-specific genes in S. agalactiae pathogenesis using rodent models of infection based on the observation that corynebacterial pili plays an important role in bacterial adherence. Together these studies will reveal the assembly mechanism of pili in gram-positive bacteria and the contribution of these organelles during the pathogenesis of infection. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI061381-02
Application #
7156950
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Rubin, Fran A
Project Start
2005-12-15
Project End
2010-11-30
Budget Start
2006-12-01
Budget End
2007-11-30
Support Year
2
Fiscal Year
2007
Total Cost
$323,343
Indirect Cost
Name
University of Connecticut
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
022254226
City
Farmington
State
CT
Country
United States
Zip Code
06030
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Chang, Chungyu; Huang, I-Hsiu; Hendrickx, Antoni P A et al. (2013) Visualization of Gram-positive bacterial pili. Methods Mol Biol 966:77-95
Khare, Baldeep; Fu, Zheng-Qing; Huang, I-Hsiu et al. (2011) The crystal structure analysis of group B Streptococcus sortase C1: a model for the ""lid"" movement upon substrate binding. J Mol Biol 414:563-77
Vengadesan, Krishnan; Ma, Xin; Dwivedi, Prabhat et al. (2011) A model for group B Streptococcus pilus type 1: the structure of a 35-kDa C-terminal fragment of the major pilin GBS80. J Mol Biol 407:731-43
Chang, Chungyu; Mandlik, Anjali; Das, Asis et al. (2011) Cell surface display of minor pilin adhesins in the form of a simple heterodimeric assembly in Corynebacterium diphtheriae. Mol Microbiol 79:1236-47
Khare, B; Krishnan, V; Rajashankar, K R et al. (2011) Structural differences between the Streptococcus agalactiae housekeeping and pilus-specific sortases: SrtA and SrtC1. PLoS One 6:e22995
Rogers, Elizabeth A; Das, Asis; Ton-That, Hung (2011) Adhesion by pathogenic corynebacteria. Adv Exp Med Biol 715:91-103
Sillanpaa, Jouko; Nallapareddy, Sreedhar R; Singh, Kavindra V et al. (2010) Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection. Virulence 1:236-46
Khare, Baldeep; Samal, Alexandra; Vengadesan, Krishnan et al. (2010) Preliminary crystallographic study of the Streptococcus agalactiae sortases, sortase A and sortase C1. Acta Crystallogr Sect F Struct Biol Cryst Commun 66:1096-100
Vengadesan, Krishnan; Ma, Xin; Dwivedi, Prabhat et al. (2010) Purification, crystallization and halide phasing of a Streptococcus agalactiae backbone pilin GBS80 fragment. Acta Crystallogr Sect F Struct Biol Cryst Commun 66:1666-9

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