Characterisation of the type VI secretion system in Pantoea ananatis

Abstract

Pantoea ananatis is an important plant pathogen that causes disease symptoms in different plants worldwide. However, most of the virulence determinants of this pathogen have not been identified and functionally characterized. A previous study identified the type VI secretion system (T6SS) as a putative virulence determinant of P. ananatis strain LMG 20103, based on in silico analysis. This secretion system has been shown to play different roles in bacteria, including virulence, fitness and interbacterial competition. Therefore, the overall objective of this study was to determine the biological role (s) of the T6SS of P. ananatis. The first chapter of this thesis is a review of the literature, dealing with the different secretion systems used by Gram-negative bacteria to secrete effectors (toxins and proteins) from the cytoplasm to the exterior of the cell. Six different secretion systems have been identified in Gram-negative bacteria, i.e. T1SS to T6SS. These secretion systems have been functionally characterized and shown to play different roles related to virulence, fitness and inter-bacterial interactions. The T6SS represents the most recently described secretion system found in Gram-negative bacteria. Gene clusters encoding the T6SS are widespread in several pathogenic and non-pathogenic bacteria, with up to six genetically distinct T6SS gene clusters found in some bacterial species. This secretion system has been associated with different processes such as virulence, fitness, biofilm formation, niche colonization, and inter-bacterial competition. The T6SS can target cytotoxic effectors into either eukaryotes, prokaryotes or both. Genes encoding bactericidal and bacteriostatic effectors have been identified in the T6SS gene clusters of different bacteria. However, only a few of these effectors such as Hcp, VgrG, VasX, Tse, Tae, Tge, Tle, Ssp and Rhs toxins have been functionally characterized. In Chapter 2, a comparative analysis of the different T6SS gene clusters found in P. ananatis was undertaken. The T6SS-1 and T6SS-3 gene clusters of LMG 20103 were found to be conserved and syntenic in eight strains of P. ananatis of which genome sequences have been determined. Using PCR and probes, we also identified homologs of genes found in the T6SS-1 and T6SS-3 gene clusters in all 36 additional strains of P. ananatis of which the genome sequences have not been determined. The third cluster, T6SS-2 was found to be restricted to only three out of eight sequenced strains of P. ananatis, which included LMG 20103 (a pathogen of Eucalyptus spp.), PA-4 (a rice pathogen) and AJ13355 (a non-pathogenic strain isolated from the soil). Furthermore, T6SS-3 gene homologs were also identified in 12 out of 36 (33%) environmental strains of P. ananatis analyzed in this study. In Chapter 3, we functionally characterized the different T6SS gene clusters found in strains LMG 20103 and LMG 2665T. Our results indicated that the T6SS-1 of LMG 2665T plays a role in onion pathogenicity and growth inhibition of other bacteria. We also showed that the homologous T6SS-1 of strain LMG 20103 played a role in bacterial competition but was not required for pathogenicity in onion plants. Based on our assay conditions, no discernable phenotype was observed following deletion of the T6SS-2 and T6SS-3 gene clusters found in the genome sequences of either strains of P. ananatis. In Chapter 4, we carried out a genetic analysis of the tssA and tssD genes found in the T6SS-1 of strain LMG 2665T. This was done in part to validate results from Chapter 3, because the whole cluster deletion mutants were not complemented and secondly, to determine if these genes were required for T6S. Deletion of these genes abrogated pathogenicity in onion plants compared to the wild-type. In addition, the ΔtssA and ΔtssD mutants of strain LMG 2665T were unable to inhibit growth of other Gramnegative bacteria following co-culture on LB agar. In trans-expression of the fulllength tssA and tssD genes on a plasmid restored pathogenicity and inter-bacterial competition of the complemented T6SS mutants to near wild-type levels. These results, for the first time, demonstrated that the tssA and tssD genes of strain LMG 2665T are required for pathogenicity and inter-bacterial competition. We hypothesize that these genes encode proteins that are essential for the biosynthesis of a functional T6SS.