Structure and function of the accessory secretion system in gut symbionts

Griffiths, Ryan (2022) Structure and function of the accessory secretion system in gut symbionts. Doctoral thesis, University of East Anglia.

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Abstract

The accessory secretion (aSec) system is a protein export pathway uniquely present in gram-positive bacteria and dedicated to the secretion of large, glycosylated cell-wall anchored adhesins called serine rich repeat proteins (SRRPs). This system has been primarily characterised in pathogens in the context of biofilm formation and virulence. The recent discovery of aSec and SRRPs in Limosilactobacillus reuteri strains ATCC 53608 and 100-23 provided new insights into the structure, strain specific glycosylation and function of SRRPs in symbiotic gut bacteria but the aSec machinery has not been investigated. In L. reuteri, aSec consists of the translocation machinery (SecA2, SecY2, Asp4), chaperones (Asp1, Asp2, Asp3) and a variable number of glycosyltransferases (GTs) that O-glycosylate the secretory target SRRP. Here, a combination of biochemical, biophysical, and structural approaches was used to investigate the structure and function of aSec components in L. reuteri ATCC 53608 and 100-23 strains. To this aim, recombinant proteins of the L. reuteri aSec pathway were produced in E. coli and purified individually or in complex.

The crystal structure of LrGtfC100-23, one of the GTs involved in strain-specific glycosylation of SRRP, was determined by X-ray crystallography showing a classical GT-B fold. Site-directed mutagenesis of LrGtfC100-23 revealed the importance of Ser238 in conferring UDP-Glc specificity as shown using thermal shift assays while LrGtfC C240W53608 mutation introduced promiscuity with positive thermal shifts for both UDP-Glc and UDP-GlcNAc ligands.

Small-angle X-ray scattering (SAXS) and Alphafold2 approaches were used to construct structural models of individual aSec components and complexes. The LrSecA2-SecY2-Asp453608 complex consisted of a predicted dimerised motor-ATPase SecA2, membrane protein SecY2 and Asp4. LrAsp1-Asp2-Asp353608 formed a 1:1:1 complex, where LrAsp253608 exhibited acetylesterase activity. Native SRRPs purified from both L. reuteri ATCC 53608 and 100-23 strains showed high binding avidity to both LrAsp1-Asp2-Asp353608 and the secretion complex LrSecA2-SecY2-Asp453608 with cross-strain recognition.

Together these data suggest a highly regulated and conserved secretion process that progresses the SRRP secretion cargo through O-glycosylation, O-acetylation, and transport in a sequential manner. This work opens new avenues of research for further biophysical characterisation by cryo-EM and potential for glycoengineering applications.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	02 Aug 2023 09:27
Last Modified:	31 Dec 2023 01:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/92758
DOI:

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