In the project proposal, the researcher proposed to pursue the following objectives:
Objective 1: To identify and characterize host and pathogen factors controlling autophagy-cytoskeleton interactions and inflammation.
- Over recent years, the lab and others have shown in vitro that M. marinum and S. flexneri lead to rearrangements of the actin and septin cytoskeletons, forming actin tails and septin cages respectively. In particular, we showed that septin cages target bacteria to autophagy both in vitro and in vivo (Mostowy et al, Cell Host Microbe, 2010; Mostowy et al, Plos Pathog, 2013; Sirianni et al, EMBO Rep 2016; Torraca et al, Front Cell Dev Biol 2016, Torraca et al, Trends Cell Biol 2018).
- The fellowship has significantly contributed to the discovery of a previously unknown mechanism by which septins control activation of the inflammasome, and prevent detrimental inflammation during infection (Mazon-Moya et al, Plos Pathog 2017).
Objective 2: To investigate the discovered molecules and mechanisms in vivo using a M. marinum or Shigella-zebrafish infection model.
- Recruitment of septins was observed in zebrafish around S. flexneri in a tailfin and intramuscular infection model. In similar conditions, recruitment of septins around mycobacteria was also observed, but is limited to <5% of bacteria. Recruitment of autophagy to mycobacteria was also observed (limited to ~20% of bacteria), but poor co-localization of Mycobacterium-containing autophagosomes and septins was observed using tools currently available.
- Rearrangement of the actin cytoskeleton was monitored during early and late stages of mycobacterial infection. Phalloidin staining of fixed samples was initially used but failed to capture actin tail formation. However, the fellow generated a transgenic zebrafish line labelling the actin cytoskeleton in macrophages. Recruitment of actin to phagocytic cups and around Mycobacterium-containing phagosomes were observed, but actin tail formation was not clearly observed in vivo.
- The lab also generated a transgenic line to follow Septin 6 in macrophages (mpeg1:mcherry-Sept6) and a knock-in line with precise introduction of monomeric GFP in fusion at the Septin15 locus (Sept15:mGFP-Sept15).
- The fellowship contributed to the development of 5 septin gene KO zebrafish lines (at least one for each septin subgroup) namely Septin 2, Septin 6, Septin 7a, Septin 9a and Septin 15. All lines are viable and do not have any obvious phenotype in the heterozygote carrier stage.
- Using our Shigella-zebrafish infection model, the fellow contributed to the discovery of a novel mechanism by which emergency granulopoiesis induced upon sublethal bacterial infections can boost innate immune defence (Willis et al. mBio, 2018).
- The fellow also worked in collaboration with the Sanger Institute (Cambridge, UK) in a RNA-seq based screen for candidate genes conferring susceptibility/resistance to different bacteria, including Shigella. The screen successfully identified several candidates for downstream analysis.
- The fellow has extended the zebrafish-S. flexneri infection model to S. sonnei, an emerging global threat displacing a niche historically occupied by S. flexneri as the leading etiological cause of bacillary dysentery. Strikingly, S. sonnei is significantly more virulent than S. flexneri in vivo. The fellow identified the precise virulence factor responsible for increased virulence, and the mechanism by which this mediates immune evasion (Torraca et al, mns in prep).
Results obtained from this project will be further explored in the next 2 years by the fellow and the supervisor in their new research environment at the LSHTM.
- To continue research on septins and cell-autonomous immunity, the mutants and the transgenic lines obtained during this fellowship will be used to follow septin rearrangement during infection and precisely dissect the contribution of septins to counteract invading pathogens.
- To complete their research on S. sonnei pathogenesis, the fellow and the supervisor will generate results using human neutrophils to validate their findings from zebrafish. They are additionally setting up collaboration to obtain a cohort of clinical data to corroborate the relevance of their findings in humans. The lab is also generating a dual-host pathogen transcriptome dataset profiling S. sonnei infected zebrafish larvae. It is expected this will significantly contribute to future research avenues studying S. sonnei.
