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Changes in Caenorhabditis elegans life span and selective innate immune genes during Staphylococcus aureus infection

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Abstract

Caenorhabditis elegans has been increasingly used to study the innate immunity and for the screening of microbe/host-specific pathogenic factors. Staphylococcus aureus-mediated infections with live C. elegans were performed on solid (full-lawn) and liquid assays. S. aureus required 90 ± 10 h for the complete killing of C. elegans, but the infection was started only after 32 h of exposure with 20% inoculum of S. aureus. The short time exposure studies revealed that, in 20% of inoculum, continuous exposure to the pathogen was required for the killing of nematode. In 100% of inoculum, only 8 h of exposure was sufficient to kill the C. elegans. To evaluate kinetically at the innate immune level, the regulation of representative candidate antimicrobial genes was investigated. Both semi-quantitative reverse transcriptase polymerase chain reaction (PCR) and real-time PCR analyses indicated the regulation of candidate immune regulatory genes of lysozyme (lys-7), cysteine protease (cpr-2), and C-type lectin (clec-60 and clec-87) family members during the course of S. aureus infections, indicating the possible contribution of the above players during the host immune response against S. aureus exposures.

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Abbreviations

TSS:

Toxic shock syndrome

PAMPs:

Pathogen-associated molecular patterns

PRRs:

Pattern-recognition receptors

HSMPs:

Host-specific molecular patterns

ATCC:

American Type Culture Collection

CGC:

Caenorhabditis Genetic Centre

NGM:

Nematode growth medium

CFU:

Colony-forming unit(s)

PCR:

Polymerase chain reaction

LT50 :

Median lethal time

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Acknowledgment

We thank Caenorhabditis Genetics Center for providing C. elegans N2 WT strain and E. coli OP50. KB thankfully acknowledges the financial support received through DBT-RGYI (BT/PR10270/GBD/27/83/2007 dated 18 Dec 2007) and UGC [F. No. 33-237/2007 (SR)], GOI, India. Authors gratefully acknowledge the Alagappa University BIF (BT/BI/25/001/2006).

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Authors and Affiliations

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

    Gnanasekaran JebaMercy, Shunmugiah Karutha Pandian & Krishnaswamy Balamurugan

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  1. Gnanasekaran JebaMercy
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  2. Shunmugiah Karutha Pandian
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  3. Krishnaswamy Balamurugan
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Corresponding author

Correspondence to Krishnaswamy Balamurugan.

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ESM 1

The level of mRNA expression of an immune effector gene lys-7 during the continuous infection of S. aureus. A semi-quantitative RT-PCR was performed using gene specific primers as described in Materials and methods. The pattern of expression was analyzed during the linear range of amplifications (22–28 cycle). A representative 12% PAGE showing the PCR products. (Lane: 1–0 h sample, 2–12 h OP50-exposed C. elegans, 3–12 h M9-buffer-exposed C. elegans, 4–12 h S. aureus-exposed C. elegans, 5–100 bp ladder; 6–24 h OP50-exposed C. elegans, 7–24 h M9-buffer-exposed C. elegans, 8–24 h S. aureus-exposed C. elegans, 9–36 h S. aureus-exposed C. elegans, 10–100 bp ladder; 11–48 h S. aureus-exposed C. elegans, 12–60 h S. aureus-exposed C. elegans, 13–72 h S. aureus-exposed C. elegans, 14–72 h OP50-exposed C. elegans) (JPEG 4 kb)

High-resolution image (TIFF 1,290 kb)

ESM 2

The level of mRNA expression of an immune effector gene, cpr-2. A semi-quantitative RT-PCR was performed using gene-specific primers as described in Materials and methods. The pattern of expression was analyzed during the linear range of amplifications (22–28 cycle). A representative 12% PAGE showing the PCR products. (Lane: 1–0 h sample, 2–12 h S. aureus-exposed C. elegans, 3–24 h S. aureus-exposed C. elegans, 4–36 h S. aureus-exposed C. elegans, 5-100 bp ladder; 6–48 h S. aureus-exposed C. elegans, 7–60 h S. aureus-exposed C. elegans, 8–72 h S. aureus-exposed C. elegans) (JPEG 14 kb)

High-resolution image (TIFF 2,323 kb)

ESM 3

The level of expression of a pathogen recognizing receptor gene, clec-60 of C. elegans during the continuous infection of S. aureus. A semi-quantitative RT-PCR was performed using gene-specific primers as described in Materials and methods. The pattern of expression was analyzed during the linear range of amplifications (22–28 cycle). A representative 12% PAGE showing the PCR products. (Lane: 1–100 bp ladder, 2–0 h sample, 3–12 h OP50-exposed C. elegans, 4–12 h M9-buffer-exposed C. elegans, 5–12 h S. aureus-exposed C. elegans, 6–24 h OP50-exposed C. elegans, 7–24 h M9-buffer-exposed C. elegans, 8–24 h S. aureus-exposed C. elegans, 9–36 h S. aureus-exposed C. elegans; 10-100 bp ladder, 11–48 h S. aureus-exposed C. elegans, 12–60 h S. aureus-exposed C. elegans, 13–72 h S. aureus-exposed C. elegans, 14–72 h OP50-exposed C. elegans) (JPEG 5 kb)

High-resolution image (TIFF 1,750 kb)

ESM 4

The level of expression of a pathogen recognizing receptor gene, clec-87 of C. elegans during the continuous infection of S. aureus.(Lane 1–0 h sample, 2–12 h OP50-exposed C. elegans, 3–12 h S. aureus-exposed C. elegans, 4–24 h S. aureus-exposed C. elegans, 5–36 h S. aureus-exposed C. elegans, 6-100 bp ladder; 7–48 h S. aureus-exposed C. elegans, 8–60 h S. aureus-exposed C. elegans, 9–72 h S. aureus-exposed C. elegans, 10–72 h OP50-exposed C. elegans) (JPEG 6 kb)

High-resolution image (TIFF 2,890 kb)

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JebaMercy, G., Pandian, S.K. & Balamurugan, K. Changes in Caenorhabditis elegans life span and selective innate immune genes during Staphylococcus aureus infection. Folia Microbiol 56, 373–380 (2011). https://doi.org/10.1007/s12223-011-0060-y

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