Bacterial surface proteins and proteinaceous pili extensions play crucial roles in microbial attachment, a critical first step in pathogenesis. The structure and assembly of """"""""Chaperon-mediated"""""""" Gram-negative bacterial pili have been studied in great detail, but very little is known about the assembly and architecture of Gram-positive bacterial pili. In our previous work, we have investigated the structures of enzyme sortases that are responsible for surface protein anchoring and identified a putative Cys-Arg catalytic dyad that is needed for sortase driven transpeptidation. We have investigated the structures of sortase anchored Gram-positive bacterial surface proteins and their complexes with host extracellular matrix proteins, and identified a new paradigm for host pathogen interactions. In our recent work, we identified specific sortases that are involved in Gram-positive pili assembly and also recognized individual subunits of Gram-positive pili that are necessary for bacterial adhesion and pathogenesis. Sortases are unique transpeptidases that recognize C-terminal sorting signals of proteins destined for bacterial surface, either to be attached to the peptidoglycan layer or to be incorporated into the pili of Gram-positive bacteria. Sortases that dictate surface protein destinations exhibit high substrate specificity and very little is known about the structural correlates that are responsible. In this proposal we will address the hypothesis that the structural correlates that dictate sortase substrate specificity extend beyond the substrates C-terminal sorting signals. In addition we suggest, while having similar primary substrate binding pockets, the second substrate binding sites exhibit distinct spatial, polar and structural characteristics between the sortases involved in surface protein anchoring and pilus assembly. Significant progress has been achieved;the work proposed here will build on the preliminary results with the following specific aims: 1) We would characterize sortases of both kind and their active sites with the help of respective substrates, substrate analogues and inhibitors, 2) We would explore the structures of individual major and minor pilus subunits, and finally 3) We would attempt to provide a model for the architecture and assembly of Gram-positive bacterial pili.

Public Health Relevance

Our previous work revealed that sortases belong to a unique class of cysteine transpeptidases that catalyzes the reaction with the help of a novel Cys-Arg catalytic dyad. We have determined the crystal structures of surface proteins anchored by sortases. Our present goal is to understand the structural correlates that dictate sortase substrate specificity and the enzymes'role in Gram-positive pili assembly.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI073521-04
Application #
8079021
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Korpela, Jukka K
Project Start
2008-07-01
Project End
2013-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
4
Fiscal Year
2011
Total Cost
$364,842
Indirect Cost
Name
University of Alabama Birmingham
Department
Other Health Professions
Type
Schools of Optometry/Ophthalmol
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Vengadesan, Krishnan; Macon, Kevin; Sugumoto, Shinya et al. (2013) Purification, crystallization and preliminary X-ray diffraction analysis of the Staphylococcus epidermidis extracellular serine protease Esp. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:49-52
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
Krishnan, Vengadesan; Narayana, Sthanam V L (2011) Crystallography of gram-positive bacterial adhesins. Adv Exp Med Biol 715:175-95
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
Ganesh, Vannakambadi K; Barbu, E Magda; Deivanayagam, Champion C S et al. (2011) Structural and biochemical characterization of Staphylococcus aureus clumping factor B/ligand interactions. J Biol Chem 286:25963-72
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
Vengadesan, Krishnan; Narayana, Sthanam V L (2011) Structural biology of Gram-positive bacterial adhesins. Protein Sci 20:759-72
Mishra, Arunima; Devarajan, Bharanidharan; Reardon, Melissa E et al. (2011) Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development. Mol Microbiol 81:1205-20
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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