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Tracing T3SS effectors in vivo during Salmonella infection in the zebrafish model

Periodic Reporting for period 1 - Salmofish (Tracing T3SS effectors in vivo during Salmonella infection in the zebrafish model)

Reporting period: 2019-05-01 to 2021-04-30

Type III secretion systems (T3SS) are sophisticated “molecular needles” that many pathogenic Gram-negative bacteria use to establish infection. Salmonella enterica encodes two T3SS that translocate a number of proteins, called effectors, from bacteria to the cytosol of the infected eukaryotic cell. These proteins manipulate host signal transduction pathways and cellular processes to the pathogen’s advantage. However, how these effectors promote virulence in vivo remains poorly understood. The main objective of Salmofish was to shed light to the contribution of SlrP, and, potentially, other Salmonella T3SS effectors, to the dynamic interface between Salmonella and its hosts at the organism level. To do so, we have implemented the relevant vertebrate model zebrafish in my hosting laboratory, we have set up different conditions for infection and develop protocols to evaluate the degree of attenuation of Salmonella T3SS- strains in comparison with the wild type.

Additionally, we have described new host substrates for SlrP and other effectors, like SspH1, SspH2 and SseK. We have opened new lines of research in the hosting laboratory:
1. Functional analysis of Salmonella T6SS.
2. C. elegans as Salmonella infection model.

In addition to conduct a research project and produce relevant results regarding the impact of Salmonella T3SS effectors to the infection process, The MCSA action has allowed me to mentor 4 master students, 2 undergraduate students, 2 PhD students and participate in teaching activities of the Genetics department.
We have performed a deep study of SlrP and other Salmonella T3SS effectors of the same family of E3 ubiquitin ligases (SspH1 and SspH2). Briefly, we have identified the ubiquitinome” (set of ubiquitinated proteins in the cell) in the presence of these three effectors. Importantly, we have constructed a library of S. Typhimurium mutants covering all T3SS effectors describe so far.

In addition to envisaged work packages in Salmofish action, I have been involved in ongoing projects of the hosting laboratory to analyze the biological role of the T3SS effector SseK1 and the supervision of a PhD student who is defending his thesis within the coming months. Remarkably, I have started a new and independent line of research in the hosting department aiming to dissect the ecological and molecular mechanisms underlying the bacterial interference provided by S. Typhimurium T6SS against the indigenous microbiota in the zebrafish model and its contribution to the infection. The overall aim of this research is to better understand the contribution of these anti-bacterial nanomachines to the dynamic interface between Salmonella and the host. We have generated so far relevant preliminary data that have been presented in National congresses of Genetics and with good perspectives of publication.

During the course of the MSCA-IF-H2020-842629 we have sadly experienced the deep impact of the Coronavirus disease (COVID) pandemic. In particular, last March 2020 was marked by the onset of a strict confinement, not just in my country (Spain), but in the rest of the world, in order to prevent the quick dissemination of the SARS-Cov-2. As expected, this fact has had an important influence in the course of our planned work. However, I took advantage of the lock-down period to analyse previous generated data and I have been able to publish one original article and one review.
Briefly, this action encompassed 2 Major Objectives distributed in 2 main work packages. Almost all milestones have been accomplished throughout the course of the last 24 months. However, deliverables 1.4 and 2.2 were not fully addressed. Perhaps, this is the most important consequence of the above-mentioned confinement during 2020. Since we have gathered all experimental data, we are currently analysing the data about Salmonella manipulation of host cell ubiquitination pathway. We aim at being ready for publication within the next months. Besides, I have built an important network of national and international collaborations
During the course of the MSCA action, we have published 2 original papers, 1 revision and present 5 communications in national congresses (2 talks and 3 poster).
Finally, the MCSA actions has allowed me to lead a full research program, with substantial formative contents, in one of the leading groups worldwide in the study of protein effectors of Salmonella type III secretion systems and it has also put me in an optimal position to obtain other long-term competitive Spanish grants to ultimately run my own lab and research projects and become a reference research group in Salmonella-host interactions.
Implementation of oral-infection model for Salmonella infection