Salmonella Infection and its Impact on Caenorhabditis Elegans Development

Abstract

Salmonella infection is known to cause various developmental defects in humans including still birth, abortion and delayed development. Also this pathogen causes 50% reduction in the lifespan of Caenorhabditis elegans (C. elegans). But the mechanism behind this reduction is not explained well. The current study aims to find out Salmonella infection mediated defects in C. elegans, and the etiology behind these defects by checking all life stages of worm sequentially for any defects caused by the microbe and also extending the infection to subsequent worm generations in order to observe the magnitude of infection. During the investigation, the study encompasses Salmonella infection mediated egg retention in the worm that leads to various developmental and morphological defects leading to disruption of temporal regulation of developmental timing in C. elegans. S. Typhimurium is not entering the worm either through the egg surface or able to pass on to the next generation. Still, it enters normally through the oral route as worms eat bacteria. The microbe’s virulence factors or effectors and its pathogenesis are transferred to the next generation directly or indirectly as an outcome of infection. Progeny of infected worms hasn’t shown alteration in the percentage of males and hermaphrodites, while the infected worm population exhibits reduced copulatory events. Thus, Salmonella infection modulates the reproductive behavior and hence reduced brood size in the C. elegans. Worm’s delayed egg-laying response to Salmonella infection causes several defects in eggs, including over-folding of developing embryo, increased egg size, and eggshell defect, possibly in the fifth layer causing it to lose the osmotic stress tolerance and observed penetration of hydrophobic dye inside the worm’s egg. The egg-laying/retention defects might be due to the S. Typhimurium virulence factors affecting the worm’s nervous system, mostly the neurotransmitter FMRF amide encoded by the flp-1 gene, which was found upregulated in the case of infected worms. Two different transgenic worms flp-1(ok2811) and flp-1(yn2) with single and two gene knockout respectively, show reduced egg laying, which supports the alteration found in flp-1 and daf-10 genes during infection-mediated egg-laying defects. The downregulated daf-10 (required for the production of chemosensory amphid and phasmid sensilla) and cat-4 (encoding dopamine and serotonin precursor) indicate their role along with flp-1 for egg retention in infected worms. Also, the infected eggs have shown delayed hatching. The reduced chitinase activity in infected worms indicated reduced hatching and caused a significant number of dead embryos and a reduced L1 population. Salmonella infection also affects cell division polarity as it alters par genes which have a role in embryonic development, showing expression with upregulated par-3 while downregulated par-5 and par-6 expression. With Salmonella infection, significant upregulation of the heterochronic gene, line-28a, in the L1 stage and downregulation in the L2 larval stage along with other L2 stage-specific collagen genes col-72 and col-87 were found, and a disrupted L2 stage in the worm was also observed. But, surprisingly L3, L4, and adult developmental stages of the worm reached earlier in the infection cases. The precocious development of L3, L4, and the adult developmental stage is associated with Salmonella infection-mediated alteration in the stage specific genes important for the development of the worm. The important stage-specific genes rnh-1.3, col-158 and col-176 (L3); col-17, col-38 and col-49 (L4); and col-19 and col-7 (adult stage) were altered. The expression of hbl-1, a hunchback ortholog in C. elegans, which controls temporal development, and heterochronic genes like lin-14 were downregulated in the L2 stage while lin-42 and daf-12 were upregulated at the L3 stage, which suggests the precocious L3 stage development. Further, the early commencement of L4 and adult stages due to infection were examined through microscopy and altered vulva as well as gonad morphology were detected at these particular stages of the worm. Salmonella infection altering vulval morphology might implicate reduced egg-laying in the worm. The downregulated gonad migration genes (mig-6, mig-17, and mig-23) in L2 and L3 and upregulated in L4 indicate a disparity in gonad development in these stages. The heterochronic genes specific for L4 (lin-41) and adult stage (lin-29) are in congruence with the precocious development of these stages. Also, the downregulated tubular lysosomal genes in L2 to adult stage worms indicate their precocious development. Salmonella infection causes fewer adults and more undeveloped larvae. When the bacterial factors causing the significant alteration in the worm development, were analysed hilA, the master regulator of SPI-1virulence genes loci, was hypothesised to produce the virulence factor or effectors that can induce the defects at various stages as the hilA, SPI-1 and SPI-1,2 mutant was compromised in showing the defects. The similar phenotypes were observed in multiple generations also, but the impact reduced beyond F5 and F6. Overall the study shows that the Salmonella infection causes a range of developmental anomalies and results in the shortening of worm life span through various regulatory genes alteration, and the effect can be seen in subsequent generations also.