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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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Related in: MedlinePlus

PO activity in dsCTL4 mosquitoes. PO enzymatic activity (detected as absorbance at 492 nm, OD492, after conversion of l-3,4-dihydroxyphenylalanine) was measured in hemolymph extracted from dslacZ and dsCTL4 mosquitoes 6 h post-injection of a mixture of E. coli and S. aureus. Noninjected wild type (WT) mosquitoes (controls) showed basal PO activity. The graph shows PO activity measured at 10-min intervals over a 1-h period. Means are calculated from three independent biological experiments. Error bars represent mean ± S.E.
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Figure 4: PO activity in dsCTL4 mosquitoes. PO enzymatic activity (detected as absorbance at 492 nm, OD492, after conversion of l-3,4-dihydroxyphenylalanine) was measured in hemolymph extracted from dslacZ and dsCTL4 mosquitoes 6 h post-injection of a mixture of E. coli and S. aureus. Noninjected wild type (WT) mosquitoes (controls) showed basal PO activity. The graph shows PO activity measured at 10-min intervals over a 1-h period. Means are calculated from three independent biological experiments. Error bars represent mean ± S.E.

Mentions: We have shown previously that dsCTL4- and dsCTLMA2-treated mosquitoes melanize and kill the majority of P. berghei ookinetes that invade the mosquito midgut epithelium, suggesting that CTL4 and CTLMA2 function as parasite agonists (27). The mechanism by which these CTLs function is not yet elucidated, but they seem to block the mosquito melanization response to invading ookinetes. To ensure that the death observed in dsCTL4 and dsCTLMA2 mosquitoes following infection with E. coli is not because of an enhanced melanotic response that could be toxic to the mosquitoes themselves, we measured the PO activity in dsCTL4 mosquitoes, 6 h post-infection, with a mixture of E. coli and S. aureus, and we compared it with that in infected dslacZ controls. The results clearly revealed that the PO activities in infected dsCTL4 and dslacZ mosquitoes were induced relative to noninfected controls, but to similar levels (Fig. 4), suggesting that the attrition observed in infected dsCTL4 mosquitoes is indeed because of E. coli proliferation and not an enhanced PO activity.


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)

PO activity in dsCTL4 mosquitoes. PO enzymatic activity (detected as absorbance at 492 nm, OD492, after conversion of l-3,4-dihydroxyphenylalanine) was measured in hemolymph extracted from dslacZ and dsCTL4 mosquitoes 6 h post-injection of a mixture of E. coli and S. aureus. Noninjected wild type (WT) mosquitoes (controls) showed basal PO activity. The graph shows PO activity measured at 10-min intervals over a 1-h period. Means are calculated from three independent biological experiments. Error bars represent mean ± S.E.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: PO activity in dsCTL4 mosquitoes. PO enzymatic activity (detected as absorbance at 492 nm, OD492, after conversion of l-3,4-dihydroxyphenylalanine) was measured in hemolymph extracted from dslacZ and dsCTL4 mosquitoes 6 h post-injection of a mixture of E. coli and S. aureus. Noninjected wild type (WT) mosquitoes (controls) showed basal PO activity. The graph shows PO activity measured at 10-min intervals over a 1-h period. Means are calculated from three independent biological experiments. Error bars represent mean ± S.E.
Mentions: We have shown previously that dsCTL4- and dsCTLMA2-treated mosquitoes melanize and kill the majority of P. berghei ookinetes that invade the mosquito midgut epithelium, suggesting that CTL4 and CTLMA2 function as parasite agonists (27). The mechanism by which these CTLs function is not yet elucidated, but they seem to block the mosquito melanization response to invading ookinetes. To ensure that the death observed in dsCTL4 and dsCTLMA2 mosquitoes following infection with E. coli is not because of an enhanced melanotic response that could be toxic to the mosquitoes themselves, we measured the PO activity in dsCTL4 mosquitoes, 6 h post-infection, with a mixture of E. coli and S. aureus, and we compared it with that in infected dslacZ controls. The results clearly revealed that the PO activities in infected dsCTL4 and dslacZ mosquitoes were induced relative to noninfected controls, but to similar levels (Fig. 4), suggesting that the attrition observed in infected dsCTL4 mosquitoes is indeed because of E. coli proliferation and not an enhanced PO activity.

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