Secretome analyses of selected Pectobacterium brasiliense 1692 secretion systems

Abstract

SUMMARY Soft Rot Pectobacteriaceae (SRP) is a family of Gram-negative bacteria (GNB) that causes damage to crop and ornamental plants. Pectobacterium brasiliense 1692 (Pbr1692) is among the most devastating Pectobacterium species. Therefore, it is important to understand the biology of this strain, including strategies used by SRP to adapt to changing environments, colonise susceptible hosts, and persist in the environment. Therefore, the contribution to the knowledge pool about Pbr1692 through this study entailed the determination of the secretome of two secretion systems, outer membrane vesicles (OMVs) and the type 6 secretion system (T6SS). These transport diverse proteins for multiple roles into the environment and target cells. In this study, their secretome profiles suggest that OMVs and the T6SS potentially contribute to the ecological success of Pbr1692.

Soft Rot Pectobacteriaceae (SRP) is a family of Gram-negative bacteria (GNB) that causes damage to crop and ornamental plants. Pectobacterium brasiliense 1692 (Pbr1692) is among the most devastating Pectobacterium species. Therefore, it is important to understand the biology of this strain, including strategies used by SRP to adapt to changing environments, colonise susceptible hosts, and persist in the environment. Therefore, the contribution to the knowledge pool about Pbr1692 through this study entailed the determination of the secretome of two secretion systems, outer membrane vesicles (OMVs) and the type 6 secretion system (T6SS). These transport diverse proteins for multiple roles into the environment and target cells. In this study, their secretome profiles suggest that OMVs and the T6SS potentially contribute to the ecological success of Pbr1692. Chapter One: Secretion systems form part of the mechanisms underlying bacteria-bacteria interactions, pathogenesis, and disease development in eukaryotic hosts. This chapter reviews the literature on Gram-negative bacteria secretion systems, the dialogues that possibly exist between them, and the implications of their interactions. Several cases of inter-secretion system functioning in bacterial rivalry, virulence, and survival, inter alia, are highlighted. The review also focuses on the importance of studying secretion systems as inter-dependent entities to unlock some fundamentals into the biology and functions of bacteria. Furthermore, it delves into the possible implications of the dialogues for bacteria in their natural niches. Chapter Two: In this chapter, vesicle-associated proteins were identified from isolated Pbr1692 OMVs. From these OMVs, 129 proteins in varied levels of abundance were functionally characterised using sequence analyses following identification by mass spectrometry. Functional annotation revealed that OMVs harbour and associate with cargo including proteins with phospholipase A1 or A2, polygalacturonase, peptidase, and cellulase activity that serve diverse roles beneficial to the phytopathogen, Pbr1692. Some of the OMV cargo proteins were predicted T6SS and T2SS substrates. Substrate signal peptide prediction analyses indicated possible signal sequence independent secretion by the OMVs. Using different phenotypic assays, we verified some of the in silico predicted functions. Protease activity of OMVs was shown using gelatine zymography. Competition assays showed that protein cargo of Pbr1692 OMV is involved in competitive fitness against Dickeya dadantii. Virulence on susceptible potato variety Solanum tuberosum cultivar (cv.) Mondial tubers was also observed. Furthermore, pathogen associated molecular pattern (PAMP) triggered immunity was observed by elicitation of a hypersensitive response (HR) in Nicotiana benthamiana leaves. Chapter Three: The focus of this chapter was to identify the role of the T6SS in Pbr1692 protein secretion and its role in the potential interplay by implementing a combination of computational and proteomic analyses. These revealed possible interdependence between other secretion systems and the T6SS. In silico prediction using the Bastion6 and BastionX computational tools revealed that there is potential substrate co-secretion between the T6SS and T1SS-T4SSs. Among the predicted shared substrates were the T2SS substrates, including Avirulence protein L (AvrL), which was also found as an OMV cargo in Chapter Two of this thesis. Therefore, the predictions suggested that the T6SS of Pbr1692 might assist in the secretion of T2SS substrates. To validate the in silico predictions, the Pbr1692 wild type (T6SS-active strain) and tssBC sheath mutant (T6SS-inactive strain) secretomes were compared to the predicted substrates. To this end, Pbr1692 strains were grown in virulence gene-inducing media to induce the T6SS and other virulence genes. Using mass spectrometry, 446 secreted proteins were identified from T6SS-inactive and T6SS-active cell-free supernatants. Comparing the T6SS-inactive and T6SS-active strain secretomes revealed 19 proteins to be secreted only by the wild type strain. Thirty proteins were differentially secreted by the T6SS-inactive and active strain. In total, 49 proteins were identified as T6SS associated proteins. Comparison of the in vitro T6SS substrates to predicted T1SS-T6SS substrates validated 25 candidates predicted via the computational pipeline. Overall, the results imply interplay between secretion systems and the T6SS via an unknown mechanism. Chapter Four: In Chapters Two and Three, Avirulence L (AvrL), an SRP T2SS substrate was identified as an OMV substrate and predicted as a T6SS substrate, respectively. In the current chapter, computational characterisation of AvrL was carried out to understand the protein that is predicted to have three secretion systems dedicated toward its secretion. This is a developmental chapter whose main goal is to build a solid basis for future work. Evolutionary analyses revealed that over 100 SRP encode avrL homologs. Further analyses predicted that AvrL contains a putative IpxD domain potentially involved in lipid A biosynthesis and a zinc-binding metallopeptidase domain involved in plant cell wall degradation. In addition, seven intrinsically disordered regions were identified in this protein which are predicted to form part of a host protein mimicry strategy. In terms of interacting partners, AvrL seems to be functionally linked to ten T2SS substrates, which further supports its association with the T2SS. A molecular interaction map of the AvrL with the model plant Arabidopsis thaliana protein-protein interaction network revealed 152 candidate host protein targets. A total of 150 Pbr1692 host potato (Solanum tuberosum) proteins were identified as A. thaliana interactor functional homologs. Nineteen differentially expressed genes, mostly transcription factors were identified to code for AvrL potential targets in Solanum tuberosum cv. Valor when infected by Pbr1692. The results of this chapter imply that AvrL potentially has spatial roles in Pbr1692 including host defence response manipulation for pathogen benefit. Altogether, the study concluded that GNB interactively use their secretion systems to achieve the secretion of a wide range of proteins that are crucial for the biology of Pbr1692. However, we recommend verification of these implications by future work.