Studies on the serotype modifying O-acetyltransferase of S. flexneri : understanding its structural and functional characteristics and its role in bacterial virulence
Creators: name: Thanweer, Farzaana
Publisher: name: The Australian National University
Year: 2011
Subjects: subject: QR82.E6 T53 2011
subject: Shigella flexneri
subject: Acetyltransferases
subject: Shigellosis Immunological aspects

description: Thirteen serotypes of S. flexneri have been recognised, all of which are capable of causing bacillary dysentery or shigellosis. Furthermore, the emergence of newer serotypes has posed additional challenges to the development of S. flexneri vaccines. One of the factors responsible for the generation of serotype diversity is an LPS O-antigen modifying, integral membrane protein known as O-acetyltransferase or Oac. Oac functions by adding an acetyl group to a specific O-antigen sugar, thus changing the antigenic signature of the parent S. flexneri strain. Being a membrane protein, Oac consists of hydrophobic and hydrophilic components and this study investigates the roles of some of these components. Three strategies have been employed to study this - the construction of Oac deletion mutants, the construction of Oac chimeric proteins and the construction of point mutations in Oac. From these studies an understanding of the mechanistics of Oac-mediated serotype modification has been gained. In the first part of the study, the roles of three hydrophilic loops of Oac - loop 3, loop 4 and loop 11 were investigated. These loops were chosen as targets for deletion on account of the high diversity in their amino acid sequence (when compared to homologous acetyltransferases of other organisms) to investigate whether the diversity offered Oac a characteristic unique to it. The deletion of loops 3 and 11 interfered with the appropriate levels of assembly of Oac in the membrane thus suggesting that these loops played a structural role in the overall functioning of the protein. The deletion of loop 4 manifested as a loss in Oac function without affecting the levels of protein assembled in the membrane suggesting a possible catalytic role for this loop. The second strategy for studying Oac involved the construction of Oac chimeras with two homologous proteins - Acyltransferase 3 of Pseudomonas fluorescenes and OafA of Salmonella enterica serovar Typhimurium. The exchange of conserved amino acid segments between the proteins was performed to investigate whether a 'domain' structure existed among the homologues. The absence of functionality for any of the chimeric proteins however would possibly indicate the absence of domains in these proteins. Oac amino acids which showed a high level of conservation with those of other homologous acetyltransferases were also subjected to mutagenesis in order to study their individual effects on Oac. Several conserved serine-glycine and phenylalanine-proline motifs found in the transmembrane segments were found to be critical to Oac. Critical arginine residues were also identified in the hydrophilic cytoplasmic loop 3. Based on their effects on Oac, possible roles for the critical amino acids in the overall mechanism of O-acetylation have been proposed. The physiological effect of Oac on the virulence of S. flexneri was also explored in the last part of this study. The oac gene in a virulent S. flexneri serotype 3b strain was made non{u00AD}functional by gene disruption. A comparison of the virulence capabilities of the parent serotype 3b strain and the newly constructed (oac negative) isogenic strain thus proved that Oac provided S. flexneri with survival advantages in the host system, possibly by helping the bacteria resist the host's immune clearance mechanisms better.