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Physiological and Immunological Regulations in Caenorhabditis elegans Infected with Salmonella enterica serovar Typhi

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Indian Journal of Microbiology Aims and scope Submit manuscript

Abstract

Studies pertaining to Salmonella enterica serovar Typhimurium infection by utilizing model systems failed to mimic the essential aspects of immunity induced by Salmonella enterica serovar Typhi, as the determinants of innate immunity are distinct. The present study investigated the physiological and innate immune responses of S. Typhi infected Caenorhabditis elegans and also explored the Ty21a mediated immune enhancement in C. elegans. Ty21a is a known live vaccine for typhoidal infection in human beings. Physiological responses of C. elegans infected with S. Typhi assessed by survival and behavioral assays revealed that S. Typhi caused host mortality by persistent infection. However, Ty21a exposure to C. elegans was not harmful. Ty21a pre-exposed C. elegans, exhibited significant resistance against S. Typhi infection. Elevated accumulation of S. Typhi inside the infected host was observed when compared to Ty21a exposures. Transcript analysis of candidate innate immune gene (clec-60, clec-87, lys-7, ilys-3, scl-2, cpr-2, F08G5.6, atf-7, age-1, bec-1 and daf-16) regulations in the host during S. Typhi infection have been assessed through qPCR analysis to understand the activation of immune signaling pathways during S. Typhi infections. Gene silencing approaches confirmed that clec-60 and clec-87 has a major role in the defense system of C. elegans during S. Typhi infection. In conclusion, the study revealed that preconditioning of host with Ty21a protects against subsequent S. Typhi infection.

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Abbreviations

Ach:

Acetylcholine

ASH:

Amphied sensilla neuron of head

ATCC:

American Type Culture Collection

CFU:

Colony forming unit

CGC:

Caenorhabditis Genetic Centre

DEPC:

Diethylpyrocarbonate

IMTECH:

Institute of Microbial Technology

LB:

Luria–Bertani

LPS:

Lipopolysaccharide

MAPK:

Mitogen activated protein kinase

MTCC:

Microbial type culture collection

NGM:

Nematode growth medium

PAMP(s):

Pathogen associated molecular pattern(s)

PMK:

p38 MAP kinase

SCP/TAPS:

Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7

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Authors and Affiliations

  1. Department of Biotechnology, Alagappa University, Karaikudi, 630 003, India

    Bhagavathi Sundaram Sivamaruthi & Krishnaswamy Balamurugan

Authors
  1. Bhagavathi Sundaram Sivamaruthi
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  2. Krishnaswamy Balamurugan
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Correspondence to Krishnaswamy Balamurugan.

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12088_2013_424_MOESM1_ESM.tif

Fig. S1 Olfactory responses of C. elegans against S. Typhi and S. Typhi Ty21a. a NGM plate spotted with E. coli OP50 (Zone: A) and S. Typhi (Zone: B). b NGM plate spotted with E. coli OP50 (Zone: A) and S. Typhi Ty21a (Zone: B). c NGM plate spotted with S. Typhi (Zone: A) and S. Typhi Ty21a (Zone: B). The bacterial spots were indicated as a black circle. In all the combinations, worms were freely crawling over the bacterial spots which indicated that C. elegans does not exhibit the bacterial avoidance behavior against both S. Typhi and S. Typhi Ty21a. Supplementary material 1 (TIFF 592 kb)

12088_2013_424_MOESM2_ESM.tif

Fig. S2 Bacterial accumulations in C. elegans. The proliferation of Salmonella strains inside C. elegans was analyzed as described in “Materials and Methods” section. Bacterial load of S. Typhi and S. Typhi Ty21a over the course of infection. (TIFF 127 kb)

12088_2013_424_MOESM3_ESM.tif

Fig. S3 Diagrammatic representation of osmosensation assay. Salmonella enterica serovar Typhi infected or control worms were placed on NGM plate (a) and High osmolarity region of medium (b) was created around the worms. A Control animals recognized the high osmolarity zone and they have avoided the crossing of the high osmolarity area, whereas S. Typhi infected C. elegans B has lost their osmosensation and crossed the zone. (TIFF 443 kb)

12088_2013_424_MOESM4_ESM.tif

Fig. S4 Regulation of candidate immune genes. Expression patterns of clec-60, clec-87, age-1, bec-1, daf-16 during S. Typhi infection. Fold difference (2−ΔΔCt) of the expression of candidate genes were calculated after normalization with act-2 expression (housekeeping gene) and basal level expression of corresponding gene in control worms. The regulations of these genes indicated that infection also altered the aging related genes. (TIFF 337 kb)

12088_2013_424_MOESM5_ESM.tif

Fig. S5 Regulation of candidate immune genes. Expression patterns of lys-7, ilys-3, scl-2, cpr-2, F08G5.6, atf-7 during S. Typhi infection in naive host. Fold difference (2−ΔΔCt) of the expression of candidate genes were calculated after normalization with act-2 expression (housekeeping gene) and basal level expression of corresponding gene in control worms. (TIFF 387 kb)

12088_2013_424_MOESM6_ESM.tif

Fig. S6 Representative images of C. elegans depicting the phenotypic variation in clec-60 silenced C. elegans. Status of C. elegans after 36 h from the treatment with siRNA specific to clec-60 for 24 h and the tail swelling was highlighted by a circle (a, b) when compared to control (f) and the swelling phenotype of C. elegans after 72 h (c, d). The swelling at the posterior end is due to the vacuole formation (e, clearly indicated with arrows). The reason for the vacuole formation is not known. (TIFF 1969 kb)

12088_2013_424_MOESM7_ESM.tif

Fig. S7 Expression pattern of clec-60 and clec-87 in silenced and control C. elegans. Lane 1 shows the 100 bp ladder. No amplification was observed in clec-60 and clec-87 silenced worms for their respective gene. This results suggested that worms treated with RNAi were silenced for respective genes and also indicated the specificity of siRNA. (TIFF 184 kb)

12088_2013_424_MOESM8_ESM.tif

Fig. S8 Survival rate of clec-60 silenced C. elegans during S. Typhi infection. Rapid mortality was observed in S. Typhi infected clec-60 RNAi worms compared to the mortality of naïve worms exposed to S. Typhi. (TIFF 102 kb)

12088_2013_424_MOESM9_ESM.tif

Fig. S9 Survival rate of clec-87 silenced C. elegans during S. Typhi infection. Rapid mortality was observed in S. Typhi infected clec-87 RNAi worms compared to the mortality of naïve worms exposed to S. Typhi. (TIFF 103 kb)

Supplementary material 10 (DOCX 14 kb)

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Sivamaruthi, B.S., Balamurugan, K. Physiological and Immunological Regulations in Caenorhabditis elegans Infected with Salmonella enterica serovar Typhi. Indian J Microbiol 54, 52–58 (2014). https://doi.org/10.1007/s12088-013-0424-x

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  • DOI: https://doi.org/10.1007/s12088-013-0424-x

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