The Insight of Salmonella Infection and its Impact on Behavioral Plasticity in Caenorhabditis Elegans

Abstract

Salmonella is one of the intracellular pathogens causing millions of people to succumb to death every year. The two serovars, Salmonella enterica serovar Typhimurium (S. Typhimurium) and Salmonella enterica serovar Typhi (S. Typhi), cause infection in animals and humans, respectively. A free-living soil nematode Caenorhabditis elegans (C. elegans) is used as an established model system for studying host-pathogen interaction. S. Typhimurium is known to cause persistent infection and dauer larva development, but the mechanism behind this phenomenon is not well explained. Dauer is an alternative developmental stage that gives survival benefits under unfavorable and even infection conditions. In pathogenic Escherichia coli K-12 (E. coli K-12), the role of various genes (yjgB, elaA, mutS, fepB, fepC, cyaA, yrfD, srlA, etc.) in inhibiting the C. elegans dauer state has been reported by gene deletion study. In this study, Salmonella genes (cyaA, fepB, and fepC) are targeted, showing maximum homology with that of E. coli K-12, and checked the effect of that gene on C. elegans dauer larvae development. It was observed that absence of the fepB gene from the S. Typhimurium genome, bacteria become less virulent with decreased motility and biofilm-producing ability compared to WT-STM counterparts. Also, the worm exhibited better clearance of the fepB mutant bacterial strain with no damage to the worm’s pharynx. The fepB mutant strain also activated worms' early immune responses and gave worms better survival ability. There are four conserved dauer signaling pathways, i.e., Insulin-like signaling pathway, TGF-β signaling pathway, Guanylyl cyclase pathway, and Steroid hormone pathway found in worms. Here, infection with mutant Salmonella strain altered TGF- β pathway, which led to activate Insulin- like signaling pathway and raised a significantly increased number of dauer larvae in the second generation of worm population compared to WT-STM infection. Dauer formation always requires nuclear translocation of DAF-16, a forkhead transcription factor of the Insulin-like signaling pathway. The activity of DAF-16 plays a major role in determining whether to continue the reproductive growth of animals or develop dauer larvae. Often sensing environmental stress, DAF-16 translocates from cytoplasm to nucleus to activate the dauer signaling cascade. Transgenic animals expressing DAF-16::GFP exhibited more DAF-16::GFP nuclear localization under ΔfepB strain infection conditions than WT-STM. Besides, DAF- 16::GFP localization remained up to F2 generation and strongly implied the negative regulation of fepB gene of S. Typhimurium in modulating dauer larvae development in worms. Next, we wanted to understand how bacteria act as food signals modulating the worm’s chemosensory system to mediate such behavioral plasticity in its second generation. C. elegans possess a well- developed chemosensory system with 302 neurons, including gustatory neurons that sense water-soluble environmental cues and olfactory neurons that sense volatile, attractive, or repellent compounds from the environment. Worms’ chemosensory neurons play an important role in navigating food search, avoiding harmful compounds, larval development, and even male mating. These neurons are widely located in the head or tail region of the worm body and grouped into sense organs, i.e., amphid, phasmid, inner labial, and outer labial organs. Sensing environmental stress often induces worms to enter the dauer state to maintain cellular homeostasis. Here we initially evaluated the olfactory preference of C. elegans toward the pathogenic WT-STM and its mutant strain along with regular E. coli OP50 food and found worms’ initial approach over 1-2 hours was dominated by WT-STM but not for fepB mutant Salmonella strain. Besides, exposing longer time duration under infection conditions showed a less aversive response of worms against the fepB mutant Salmonella strain. Still, it exhibited a better associative learning response against the fepB mutant strain than the WT-STM counterpart. With these altered behavioral responses, we next looked into mRNA expression of certain genes playing an important role in worms’ olfaction. We found upregulation of odr- 3 and ceh-36 genes expression along with the secondary signal transduction genes, i.e. tax- 2/tax-4 and daf-11 gene located upstream of tax-2/tax-4, a cyclic nucleotide-gated channel at 24 hours of ΔfepB post-infection. However, using mutant C. elegans strains showing a defect in worms’ olfactory neuron mediated chemosensation implied the involvement of the ceh-36 gene, which encodes the CEH-36 transcription factor required for terminal differentiation of AWC neuron, for sensing fepB mutant bacterial strain. Further, exposing AWC ablated C. elegans strain to the infection strains in a time-dependent manner strongly indicated AWC neuron's participation in sensing ΔfepB strain. Further, to understand the involvement of AWC neuron in worms’ behavioral plasticity, we exposed mutant worms and AWC ablated worms and observed AWC neuron playing an important role in modulating worms' behavioral plasticity against mutant Salmonella strain. Overall, our study deciphering the relationship between chemosensory neurons and bacteria emitted signals that alter worms’ behavioral plasticity and can help us understand the complex phenomenon of host-pathogen interaction benefiting pathogen in host dissemination.