MMPs Regulate both Development and Immunity in the Tribolium Model Insect

Abstract

BackgroundMatrix metalloproteinases (MMPs) are evolutionarily conserved and multifunctional effector molecules in development and homeostasis. In spite of previous, intensive investigation in vitro and in cell culture, their pleiotrophic functions in vivo are still not well understood.Methodology/Principal FindingsWe show that the genetically amenable beetle Tribolium castaneum represents a feasible model organism to explore MMP functions in vivo. We silenced expression of three insect-type Tribolium MMP paralogs and their physiological inhibitors, TIMP and RECK, by dsRNA-mediated genetic interference (RNAi). Knock-down of MMP-1 arrested development during pupal morphogenesis giving phenotypes with altered antennae, compound eyes, wings, legs, and head. Parental RNAi-mediated knock-down of MMP-1 or MMP-2 resulted in larvae with non-lethal tracheal defects and with abnormal intestines, respectively, implicating additional roles of MMPs during beetle embryogenesis. This is different to findings from the fruit fly Drosophila melanogaster, in which MMPs have a negligible role in embryogenesis. Confirming pleiotrophic roles of MMPs our results also revealed that MMPs are required for proper insect innate immunity because systemic knock-down of Tribolium MMP-1 resulted in significantly higher susceptibility to the entomopathogenic fungus Beauveria bassiana. Moreover, mRNA levels of MMP-1, TIMP, and RECK, and also MMP enzymatic activity were significantly elevated in immune-competent hemocytes upon stimulation. To confirm collagenolytic activity of Tribolium MMP-1 we produced and purified recombinant enzyme and determined a similar collagen IV degrading activity as observed for the most related human MMP, MMP-19.Conclusions/SignificanceThis is the first study, to our knowledge, investigating the in vivo role of virtually all insect MMP paralogs along with their inhibitors TIMP and RECK in both insect development and immunity. Our results from the Tribolium model insect indicate that MMPs regulate tracheal and gut development during beetle embryogenesis, pupal morphogenesis, and innate immune defense reactions thereby revealing the evolutionarily conserved roles of MMPs.

Novel functions for Dorsocross in epithelial morphogenesis in the beetle Tribolium castaneum

“…For example, Drosophila Doc has a morphogenetic role in the wing imaginal disc, where it regulates extracellular matrix remodeling through Matrix metalloproteinase 2 (Mmp2, Sui et al, 2012). However, we found that parental RNAi against the Tribolium orthologue was lethal prior to the SW stage, in contrast to results from Knorr et al (2009) (Fig. S8).…”

Functional Characterization of Anopheles Matrix Metalloprotease 1 Reveals Its Agonistic Role during Sporogonic Development of Malaria Parasites

“…Moreover, the phenotype of AgTIMP silencing might also have implications for AgMMP2 (Fig. S3C) and AgMMP3 in parasite development, since it is an efficient inhibitor of all three metalloproteases of the mosquito, as supported by its homologues in Drosophila and Tribolium (7,56).…”

“…accumulates in large vesicles in the form of zymogen, which is also produced at high levels in Anopheles hemocyte-like Sua 4.0 cells and is constitutively secreted in the cell supernatant. Interestingly, the homologue of AgMMP1 in Tribolium is also shown to be expressed in hemocytes of the organism (7,39).…”

“…This assumption was based on previous reports showing that infection by several pathogens (parasites, viruses, or bacteria) is associated with increased MMP activity in the invaded tissue, facilitating pathogen migration and/or the establishment of systemic infections (7,62,63,72,73). Indeed, the number of oocysts developing in the midguts of AgMMP1 dsRNA-treated mosquitoes was severely reduced (in some cases 7-fold compared to the value for the control [see Table S1 in the supplemental material]).…”

“…In the moth Galleria mellonella, lipopolysaccharide (LPS) challenge induces G. mellonella MMP1 (GmMMP1) transcription in hemocytes and triggers MMP-dependent degradation of collagen IV (39), whereas in Tribolium, silencing of MMP1 results in higher susceptibility to the entomopathogenic fungus Beauveria bassiana (7). MMP activity is also induced by the insect baculovirus, Autographa californica, in the midgut of Trichoplusia ni, facilitating the escape and spreading of the virus through the tracheal system and promoting systemic infections (40).…”