Mechanisms of delivery and mode of action of type VI secretion system effectors

Ringel, Peter David. Mechanisms of delivery and mode of action of type VI secretion system effectors. 2017, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_12653

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In order to manipulate their environment, bacteria evolved a diverse set of secretion systems. Three of these were found to be able to inject their substrates directly into target cells, the type VI secretion system (T6SS) being the most recently discovered of these. The T6SS shares structural and functional homology with other contractile nanomachines such as the contractile phages. It is capable of delivering its substrates into both pro- and eukaryotes in a contact dependent manner and has become a major player in the field of microbial interactions. Recently, medium and high resolution structural data of T6SS subcomplexes and in situ structures provided detailed mechanistic insights into its functioning, further supported by live cell fluorescence microscopy of the assembly dynamics. Nonetheless, the role of some of the conserved core components is not yet fully understood even less so for the associated components. Moreover, despite its implication in numerous processes, the effector repertoire remains poorly characterized.
In this thesis, both the effector repertoire and the functional contribution of selected T6SS components were characterized in Acinetobacter baylyi ADP1. We developed a new scarless chromosomal mutagenesis method for A. baylyi ADP1 and fluorescently labeled structural components of the T6SS using this method. Furthermore, we constructed in frame deletions of selected T6SS components and evaluated their role by observing the T6SS dynamics, secretion capacity, target cell lysis and the ability to inhibit a competitor. The results of the fluorescence microscopy in combination with the sensitive lysis assay show that certain components, previously thought to be required for T6SS assembly, are in fact dispensable. Furthermore, we observed that most mutations which diminished the T6SS activity reduced the number of active T6SS structures but did not affect the sheath dynamics. This indicates, that these components are involved in a step preceding the contractile tail formation. Despite ongoing concerted efforts, we were so far unable to fluorescently label secreted components.
We identified and characterized five cargo effectors and their corresponding immunity proteins. One of the effectors was disrupted by an insertion element and could be restored. All five effectors exhibited antibacterial activity and did not cross-react with non-cognate immunity proteins. The morphological changes of prey cells targeted by the effectors were observed by fluorescence microscopy of competition mixtures and allowed us to confirm the predicted peptidoglycan amidase activity of Tae1 and the phospholipase activity of Tle1. Although the bioinformatic predictions together with the observed morphological changes and the lysis phenotype of prey cells targeted by the remaining effectors hinted at the subcellular location of their respective targets, the targets themselves remain to be identified. Furthermore, we constructed an effector deficient strain which retained wild-type T6SS activity and elicited the retaliatory attack of Pseudomonas aeruginosa, but failed to inhibit or lyse prey cells. Transcriptome data further indicated, that the damage inflicted by the effector deficient strain does not induce a stress response in the prey.
Recently the T6SS was shown to be involved in the horizontal gene transfer of naturally competent Vibrio cholerae. Since A. baylyi ADP1 is known to be naturally competent, we tested whether its T6SS also contributes to horizontal gene transfer. Not only could we demonstrate that the T6SS facilitates the acquisition of DNA from prey cells, but also that lytic effectors are superior to non-lytic effectors suggesting that a lytic effector set may increase the ability to acquire DNA from a diverse range of bacteria. These findings provide further evidence that the T6SS mediated horizontal gene transfer may be a general characteristic of naturally competent bacteria bearing a T6SS.
To better understand the role of the T6SS in shaping polymicrobial communities, we employed individual based modelling of interbacterial competition mixtures, the results of which we confirmed by performing the corresponding bacterial competition. We found that the contact dependent antagonistic interactions led to a segregation of the competitors minimizing their contact surface. Once segregated, the prey cells were able to survive or even outgrow the attack of a predator so long as the growth within the domain equaled or outweighed the killing on the surface of the domain. We further demonstrated that this critical domain size, beyond which the prey would survive, depends on the growth rate ratio of the competitors and the attack rate. Recently, others showed that this segregation of the competitors promotes the evolution of public goods.
Advisors:Basler, Marek
Committee Members:Jenal, Urs
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Infection Biology > Infection Biology (Basler)
UniBasel Contributors:Basler, Marek and Jenal, Urs
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12653
Thesis status:Complete
Number of Pages:1 Online-Ressource (II, 164 Seiten)
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Last Modified:08 Feb 2020 14:57
Deposited On:14 Jun 2018 09:25

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