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.
Keywords
Reverse Transcriptase Polymerase Chain Reaction Alpha Toxin Lysozyme Gene Bacterial Accumulation Complete KillingAbbreviations
- 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
Notes
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).
Supplementary material
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)
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)
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)
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)
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