Structure, function and genetic diversity of glucosyltransferase IV (GtrIV) of Shigella flexneri
Creators: name: Nair, Anesh
Publisher: name: The Australian National University
Year: 2012
Subjects: subject: QR82.E6 N35 2012
subject: Shigella flexneri Genetics
subject: Glycosyltransferases
subject: Virulence (Microbiology)

description: There are 15 known S. flexneri serotypes, which differ in their virulence, prevalence, distribution and the nature of their O-antigens. The three bacteriophage genes gtrA, gtrB and gtr(type) are responsible for O-antigen glucosylation in Shigella flexneri. Both gtrA and gtrB have been demonstrated to be highly conserved and interchangeable among serotypes while gtr(type) was found to be specific to each serotype, leading to the hypothesis that the Gtr(type) proteins are responsible for attaching glucosyl groups to the O-antigen in a site- and serotype- specific manner. Such interaction and attachment of the glucosyl groups to the O-antigen has been postulated to occur in the periplasm. In this study, the topology of GtrIV, a S. flexneri glucosyltransferase responsible for converting serotype Y to serotype 4a, was experimentally determined by creating different fusions between GtrIV and a dual-reporter protein, PhoA/LacZ. This study shows that GtrIV consists of 8 transmembrane helices, 2 large periplasmic loops, 2 small cytoplasmic N- and C- terminal ends and a re-entrant loop that occurs between transmembrane helices III and IV. This topology is similar to that of GtrIc. Based on the newly elucidated topology of GtrIV, the roles of the N-terminal and C-terminal periplasmic loops of GtrIV (loop No. 2 and loop No. 6) were investigated through loop deletions. By sequentially deleting loop segments in loop No. 6, the presence of a potential catalytic site located between resides D260 to W269 was hypothesised. The roles of conserved or specific functions of the two periplasmic loops of GtrIV was also investigated by creating loop swap chimeras between GtrIV and its closest structural homologue, GtrIc. The resulting hybrids lost their native function and were unable to substitute function to the other protein thus, signifying the importance of both loops in GtrIV function. A total of 20 negatively charged amino acids that occur throughout GtrIV were mutated to alanine to investigate their role in GtrIV function. Three of these acidic residues located in the periplasmic loop No. 6, D261, E262 and D267, when mutated in tandem, were found to abolish function of GtrIV. They are thought to be involved in the interaction with the donor and acceptor substrates that help confer serotype specificity in O-antigen glucosylation. 28 wildtype strains isolated from Japan, Bangladesh and Vietnam that were originally typed as serotype 4a, 4b or just 4 were subjected to a series of slide agglutination experiments to confirm their serotype. A number of strains had atypical agglutination patterns, highlighting the possibility that they are potentially new serotypes or subserotypes. The GtrIV sequences obtained from these strains revealed the presence of point mutations within several strains. Southern blots carried out using DIG-labelled gtrIV suggested that two different serotype 4a strains, with the possibility of one of them being originally a 4b strain, have been circulating within these populations. Taken together, this study has provided valuable insight into the possibly unique mode of action of GtrIV and has provided grounds for further investigation into the evolution of one of the most important human pathogens.