Defining the mechanism of Yersinia entomophaga MH96 exoprotein release : a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy

Abstract

There is an increasing need in New Zealand for the development of new solutions and improvement of currently available biological control agents of pasture pests. In the last two decades, the entomopathogen Yersinia entomophaga was isolated, characterised, and defined as an economically viable agent for use in biopesticides against pasture pests, such as the New Zealand grass grub Costelytra giveni, the black beetle Hetreronychus arator, and other insects from the orders Coleoptera and Lepidoptera. Y. entomophaga produces an ABC toxin complex, called the Yen-TC, which is its main orally active virulence factor. Although previous studies have revealed how the toxin is affecting the host, the mechanism for Yen-TC production and cellular release have yet to be fully elucidated. Furthermore, the roles of other virulence factors of Y. entomophaga and their effects and mechanisms of cellular release are yet to be determined. In silico analysis of the draft genome sequence revealed several gene clusters encoding for virulence factors such as Rhs with predicted function in the type 6 secretion system, YenT as a possible heat-stable enterotoxin, and PirAB, a haemocoelic-active toxin. This study aimed to identify regulators of exoproteome release in Y. entomophaga MH96. The Yersinia entomophaga region of exoproteome release (YeRER) was identified by an exoproteome screening assay developed in this study. The YeRER is comprised of the transcriptional regulator RoeA, a regulatory ncRNA, and the Yersinia lysis cassette (YLC). Transcriptomics, mutagenesis, and trans complementation experiments were used to elucidates the importance of YeRER in Y. entomophaga protein release and changes in cell morphology. The global regulator, RoeA, strictly down-regulates the YLC expression which is involved in protein release and vesicle formation. Expression levels of the encoded secretion systems in MH96, T1SS, T3SS, T3SS2, and T6SS are not under the control of YeRER or quorum sensing (QS), which are involved in protein secretion. The T2SS expression is increased in roeA and QS mutants, which in turn showed reduced global exoproteome concentration. It is unlikely that the MH96 secretion systems are involved in MH96 exoproteome production in vitro. While underlying mechanisms have yet to be investigated, this study strongly suggests that exoproteins such as the Yen-TC are secreted by membrane vesicles which are induced by activation of a holin-endolysin complex.