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Two C-type lectins cooperate to defend Anopheles gambiae against Gram-negative bacteria.

Schnitger AK, Yassine H, Kafatos FC, Osta MA - J. Biol. Chem. (2009)

Bottom Line: Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood.Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria.Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.

ABSTRACT
C-type lectins (CTLs) are a family of proteins that share a common structural motif, the carbohydrate recognition domain, and may act as receptors in pathogen recognition. Indeed, some vertebrate CTLs, particularly the collectins, are unequivocally implicated in the innate immune response to certain microbes. Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood. Here we report that two CTLs, CTL4 and CTLMA2, which were shown previously to inhibit Plasmodium berghei ookinete melanization in the malaria vector Anopheles gambiae, are transcriptionally induced by bacterial challenge. Using in vivo reverse genetic analysis, we show that both CTLs are required for the clearance of Escherichia coli, but not Staphylococcus aureus, from adult female mosquitoes. Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria. Furthermore, molecular characterization reveals that both CTLs are secreted into the mosquito hemolymph mainly in the form of a disulfide-linked heterodimer. This association explains the similar roles of these CTLs in bacterial defense as well as in the melanization response to P. berghei ookinetes. Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

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dsCTL4 mosquitoes are susceptible to Gram-negative infections. Four days post-injection of dslacZ (control) or dsCTL4, A. gambiae adult female mosquitoes were challenged with the Gram-negative bacteria E. cloacae (A600 nm = 0.004) and Pseudomonadaceae (A600 nm = 0.001) (A) and Gram-positive bacteria E. faecalis (A600 nm = 0.4) and M. luteus (A600 nm = 0.4) (B). Dead mosquitoes were counted daily over a period of 10 days (all except Pseudomonadaceae) or 30 h (Pseudomonadaceae) post-bacterial challenge. Graphs represent the cumulative survival as calculated by the Kaplan-Meier method over the indicated time points for one representative experiment of three biological replicates. The thin dotted lines represent the 95% confidence intervals. Statistical significance was calculated by the log rank test. Survival curves were considered to be significantly different if p < 0.05.
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Figure 7: dsCTL4 mosquitoes are susceptible to Gram-negative infections. Four days post-injection of dslacZ (control) or dsCTL4, A. gambiae adult female mosquitoes were challenged with the Gram-negative bacteria E. cloacae (A600 nm = 0.004) and Pseudomonadaceae (A600 nm = 0.001) (A) and Gram-positive bacteria E. faecalis (A600 nm = 0.4) and M. luteus (A600 nm = 0.4) (B). Dead mosquitoes were counted daily over a period of 10 days (all except Pseudomonadaceae) or 30 h (Pseudomonadaceae) post-bacterial challenge. Graphs represent the cumulative survival as calculated by the Kaplan-Meier method over the indicated time points for one representative experiment of three biological replicates. The thin dotted lines represent the 95% confidence intervals. Statistical significance was calculated by the log rank test. Survival curves were considered to be significantly different if p < 0.05.

Mentions: We tested whether the CTL heterodimer is involved in defense against Gram-negative bacteria other than E. coli. Because the depletion of either CTL abolished heterodimer formation in the mosquito hemolymph, we used dsCTL4 mosquitoes to address this question. Survival assays were performed as described previously for E. coli- and S. aureus-challenged dsRNA-treated mosquitoes. Interestingly, dsCTL4 mosquitoes were also susceptible to two other Gram-negatives, E. cloacae and Pseudomonadaceae H2.26 (Fig. 7A), an isolate from field-caught A. gambiae sensu lato mosquitoes (32). Because the Pseudomonadaceae isolate was highly pathogenic, the survival of mosquitoes infected with this bacterium was scored over 35 h rather than 10 days. In contrast, dsCTL4 mosquitoes survived infections with other Gram-positive bacteria, including M. luteus and E. faecalis, as efficiently as did dslacZ controls (Fig. 7B). These results strongly suggest that the CTL4-CTLMA2 heterodimer is involved in defense against a broad range of Gram-negative bacteria.


Two C-type lectins cooperate to defend Anopheles gambiae against Gram-negative bacteria.

Schnitger AK, Yassine H, Kafatos FC, Osta MA - J. Biol. Chem. (2009)

dsCTL4 mosquitoes are susceptible to Gram-negative infections. Four days post-injection of dslacZ (control) or dsCTL4, A. gambiae adult female mosquitoes were challenged with the Gram-negative bacteria E. cloacae (A600 nm = 0.004) and Pseudomonadaceae (A600 nm = 0.001) (A) and Gram-positive bacteria E. faecalis (A600 nm = 0.4) and M. luteus (A600 nm = 0.4) (B). Dead mosquitoes were counted daily over a period of 10 days (all except Pseudomonadaceae) or 30 h (Pseudomonadaceae) post-bacterial challenge. Graphs represent the cumulative survival as calculated by the Kaplan-Meier method over the indicated time points for one representative experiment of three biological replicates. The thin dotted lines represent the 95% confidence intervals. Statistical significance was calculated by the log rank test. Survival curves were considered to be significantly different if p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719400&req=5

Figure 7: dsCTL4 mosquitoes are susceptible to Gram-negative infections. Four days post-injection of dslacZ (control) or dsCTL4, A. gambiae adult female mosquitoes were challenged with the Gram-negative bacteria E. cloacae (A600 nm = 0.004) and Pseudomonadaceae (A600 nm = 0.001) (A) and Gram-positive bacteria E. faecalis (A600 nm = 0.4) and M. luteus (A600 nm = 0.4) (B). Dead mosquitoes were counted daily over a period of 10 days (all except Pseudomonadaceae) or 30 h (Pseudomonadaceae) post-bacterial challenge. Graphs represent the cumulative survival as calculated by the Kaplan-Meier method over the indicated time points for one representative experiment of three biological replicates. The thin dotted lines represent the 95% confidence intervals. Statistical significance was calculated by the log rank test. Survival curves were considered to be significantly different if p < 0.05.
Mentions: We tested whether the CTL heterodimer is involved in defense against Gram-negative bacteria other than E. coli. Because the depletion of either CTL abolished heterodimer formation in the mosquito hemolymph, we used dsCTL4 mosquitoes to address this question. Survival assays were performed as described previously for E. coli- and S. aureus-challenged dsRNA-treated mosquitoes. Interestingly, dsCTL4 mosquitoes were also susceptible to two other Gram-negatives, E. cloacae and Pseudomonadaceae H2.26 (Fig. 7A), an isolate from field-caught A. gambiae sensu lato mosquitoes (32). Because the Pseudomonadaceae isolate was highly pathogenic, the survival of mosquitoes infected with this bacterium was scored over 35 h rather than 10 days. In contrast, dsCTL4 mosquitoes survived infections with other Gram-positive bacteria, including M. luteus and E. faecalis, as efficiently as did dslacZ controls (Fig. 7B). These results strongly suggest that the CTL4-CTLMA2 heterodimer is involved in defense against a broad range of Gram-negative bacteria.

Bottom Line: Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood.Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria.Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom.

ABSTRACT
C-type lectins (CTLs) are a family of proteins that share a common structural motif, the carbohydrate recognition domain, and may act as receptors in pathogen recognition. Indeed, some vertebrate CTLs, particularly the collectins, are unequivocally implicated in the innate immune response to certain microbes. Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood. Here we report that two CTLs, CTL4 and CTLMA2, which were shown previously to inhibit Plasmodium berghei ookinete melanization in the malaria vector Anopheles gambiae, are transcriptionally induced by bacterial challenge. Using in vivo reverse genetic analysis, we show that both CTLs are required for the clearance of Escherichia coli, but not Staphylococcus aureus, from adult female mosquitoes. Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria. Furthermore, molecular characterization reveals that both CTLs are secreted into the mosquito hemolymph mainly in the form of a disulfide-linked heterodimer. This association explains the similar roles of these CTLs in bacterial defense as well as in the melanization response to P. berghei ookinetes. Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

Show MeSH
Related in: MedlinePlus