Caenorhabditis elegans as a potential animal model to study the pathogenesis of Shigella flexneri
Creators: name: Somasiri, Pamodha
Publisher: name: The Australian National University
Year: 2021

description: This study investigates Caenorhabditis elegans (CE) as an animal model to study the human bacterial pathogen Shigella flexneri (SF). First, we determined the changes in the CE transcriptome during a SF infection using RNA-seq carried out on L4-stage wildtype (N2) CE exposed to SF for 6hours. This revealed 1631 differentially expressed genes, that include up-regulated genes encoding proteins for reactive oxygen species (ROS) defense, immune response (IR) and autophagy. Based on these findings, knockout (KO) strains were used to determine the importance of these processes during a SF infection. The 2nd aim determined the importance of the CE transcription factor DAF-16, ROS and antimicrobial effectors (AE) during a SF infection. RNA-seq comparing SF-fed L4 CE daf-16 KO and N2 revealed the down-regulation of genes involved in ROS activity. This includes genes encoding mitochondrial superoxide dismutase SOD-3 and peroxisomal catalase CTL-2. Compared to N2, sod-3 and ctl-2 KO strains were highly susceptible to SF, with higher SF accumulations. SF-fed N2 and daf-16 KO worms also express higher levels of ROS than their E.coli OP50-fed counterparts at later stages of infection. Meanwhile, the SF-fed sod-3 KO showed rising ROS levels at the early stages of infection. No change in ROS production was observed in the ctl-2 KO, although it accumulated significantly high ROS levels compared to N2. Overall, this suggests ROS regulation during early stages of infection in CE is vital to control SF accumulation and mount an effective IR. The expression of many AE encoding genes was down-regulated in SF-fed daf-16 KO worms. This includes C-type lectin domain-containing protein (clec)-61 and lysozymes lys-7 and ilys-3. Compared with N2, non-expression of these AE cause CE to become highly susceptible to SF. clec-61 and ilys-3 KO strains showed significantly higher SF accumulation and reduced viability, while lys-7 KO showed no increased SF accumulation. Additionally, AE encoded by clec-60 and clec-70 are also vital in maintaining SF accumulation and viability of infected worms. The 3rd aim was to determine the importance of iron and autophagy during the infection. ACO-1, a cytosolic aconitase theorized to mediate iron between ferritin (FTN) and the labile iron pool in intestinal cells, was shown to be up-regulated in SF-infected N2 worms. Reduced expression of ACO-1 cause the worms to be less susceptible to SF, with reduced accumulation. RNA-seq comparing SF-infected aco-1 KO and N2 showed genes encoding the Fe2+ transporter smf-3 and the transcription factor pqm-1 were upregulated. During a pathogen infection, PQM-1 would activate DAF-16-Class-2 genes such as smf-3. Increased SMF-3 accelerates Fe2+ uptake from the intestinal tract (limiting iron for the pathogen to utilize), emphasized by Calcein-AM-based fluorescence microscopy experiments that were carried out using N2 and FTN related mutants. Interestingly, a ftn-1 KO strain showed no change in SF accumulation or susceptibility compared to N2. However, a strain over-expressing (OE) ftn-1 showed higher accumulation, with increased susceptibility. This suggests while FTN-1 plays almost no role in the infected CE; likely that FTN-2 is a key component in the CE response against SF. In the FTN-1 OE strain, FTN-1 would over-compete with FTN-2 for Fe2+, reducing FTN-2 iron storage that mitigates its presence and IR. When an autophagy reporter strain was exposed to SF, an increase in GFP-puncta, indicating autophagosomes, was observed early in the infection. Unsurprisingly, a cdc-48.2 KO strain (gene encodes protein for autophagy induction) significantly accumulated SF with reducing viability. We also tested the importance of a regulatory protein (MPK-1) from the ERK/MAPK pathway involved in CDC-48.2 activation. The extreme SF accumulation and reduced viability observed in the mpk-1 KO suggest that other proteins regulated by this pathway is vital for the survival of the infected worms.