The type VI secretion system in Pectobacterium spp: Role in virulence and regulation by Zur

Abstract

Pectobacterium spp. together with Dickeya spp. belong to a group of soft rot disease causing pathogens known as the soft rot Enterobacteriaceae (SRE). Pectobacterium carotovorum subsp. brasiliense is an aggressive pathogen of potato within South Africa causing aerial stem rot, tuber soft rot and blackleg both in the field and during post-harvest storage. In this study, emphasis has been placed on characterization of the type VI secretion system as a virulence determinant for anti-bacterial competition in Pcb 1692, since this system has been linked to effector proteins capable of secretion affecting prokaryotes and in some cases eukaryotes. Chapter 1, provides a review of literature on the role of the type VI secretion system within prokaryotes, including the T6SS’s impact on virulence, disease progression and overall host fitness. The review discusses the T6SS as a virulence determinant through interacting with the host as well as neighbouring competing bacteria. The regulation thereof is also discussed. The zinc uptake regulator (Zur) is also described and its influence on the disease development and zinc uptake discussed. In Chapter 2, 3 & 4, the role of the type VI secretion system as a virulence determinant in Pcb 1692 was investigated. Utilizing a previously generated mutant of the TssC gene (encoding ClpV, a core component of the type VI secretion system) this study generated data which indicated that the Pcb1692Δt6 mutant strain’s competitive fitness was hindered, losing the ability to outcompete neighbouring bacteria in potato tubers. Similarly, a reduction in virulence in planta as well as the inability to produce standard levels of EPS within the T6SS mutant was observed. These observations suggest that the type VI secretion system plays a key function in competitive fitness in planta, ensuring that nutrients available are utilized by Pcb 1692 as opposed to other competing endophytes resulting in efficient colonization of the host. Similarly, the role of the zinc uptake transcriptional regulator (zur) as a regulator of the type VI secretion system as well as a virulence determinant was investigated. After iron, zinc is the most important transition metal, acquired through the ZnuABC zinc uptake system which is negatively regulated by the Zur protein. Zur has too been described as a multifunctional regulatory protein reported to regulate virulence determinants in various pathogens with a functional zur gene essential for virulence. This study identified a homolog of zur within Pcb 1692 and proceeded to disrupt the gene encoding for zur. Through bioinformatics procedures the binding site of Zur was shown to be located within the VipA promoter, responsible for transcribing the core components of the type VI secretion system. Data indicated that the Pcb1692Δzur mutant strain, where the zinc uptake regulator was non-functional, was deficient in production of a number of virulence determinants, namely the production of EPS, biofilm formation and oxidative stress protection resulting in a reduction of virulence in planta. In contrast to this, the competitive fitness of the mutant strain in planta was intensified due to the absence of the regulator Zur, resulting in the increased expression of the type VI secretion system. These observations suggest that through unknown mechanism/s, Zur directly or indirectly regulates virulence determinants and is required by Pcb 1692 for host colonization and disease progression.