Student Theses and Dissertations


Assaf Raz

Date of Award


Document Type


RU Laboratory

Fischetti Laboratory


streptococcal disease, gram-positive bacteria, surface proteins, sortase localization, protein anchoring, sub-surface antigens, LPXTGase


Cell wall peptidoglycan-anchored surface proteins are essential virulence factors in many Gram-positive bacteria. Attachment of these proteins to the peptidoglycan is achieved through a transpeptidation reaction, whereby sortase cleaves a conserved Cterminal LPXTG motif and attaches the protein to the peptidoglycan precursor lipid II. This thesis deals with the spatial regulation of the sorting reaction, and the role sortase localization plays in the correct distribution of surface proteins. We have introduced a new immunofluorescence procedure to study the distribution of sub-surface antigens in Streptococcus pyogenes, which utilizes the phage lysin PlyC to permeabilize the cell wall of to antibodies. We found that sortase localizes within distinct membranal foci, the majority of which are associated with the division septum and colocalize with areas of active M-protein anchoring. Protein anchoring takes place at two distinct cellular locations, the division septum and the poles. Anchoring of M-protein at the septum is a rapid process that occurs in concert with septal peptidoglycan synthesis. The localized secretion of M-protein, as well as the prevalence of sortase, lipid II, and PBPs at this location, promote efficient protein anchoring. Sortase localization and M-protein anchoring occurs simultaneously at the septum and the mature equatorial rings, pointing to the possibility that cell division begins in the daughter cells before the conclusion of the previous division cycle. Anchoring of SfbI at the poles is a much slower and more diffuse process, and therefore requires less sortase. The protein sorting reaction is therefore a dynamic, and a highly regulated process. In the absence of sortase A, surface proteins accumulate at the membrane-wall interface. We found that stalled surface proteins can be released from the secretion channel by an LPXTG-specific enzyme other than sortase A, whose identity is still unknown. Nonetheless, accumulation of missorted surface proteins has deleterious effects on the cell, resulting in selective pressure to repress surface protein expression. Inhibition of sortase may therefore not only prevent surface protein anchoring but also exert direct pressure on the cell’s secretion and folding pathways. A better understanding of the mechanisms controlling the biogenesis of surface molecules, aided by the ability to study sub-surface antigens through immunofluorescence, may yield promising new candidates for the development of new anti-infecting agents.


A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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