Identification and initial characterization of matrix metalloproteinases in the yellow fever mosquito, Aedes aegypti

Abstract

Aedes aegypti is a major vector for arboviruses such as dengue, chikungunya, and Zika viruses. During acquisition of a viremic bloodmeal, an arbovirus infects mosquito midgut cells before disseminating to secondary tissues, including the salivary glands. Once virus is released into the salivary ducts it can be transmitted to another vertebrate host. The midgut is surrounded by a basal lamina (BL) in the extracellular matrix, consisting of a proteinaceous mesh composed of collagen IV and laminin. BL pore size exclusion limit prevents virions from passing through. Thus, the BL likely requires remodelling via enzymatic activity to enable efficient virus dissemination. Matrix metalloproteinases (MMPs) are extracellular endopeptidases that are involved in remodelling of the extracellular matrix. Here, we describe and characterize the nine Ae. aegypti encoded MMPs, AeMMPs 1-9, which share common features with other invertebrate and vertebrate MMPs. Expression profiling in Ae. aegypti revealed that Aemmp4 and Aemmp6 were upregulated during metamorphosis, whereas expression of Aemmp1 and Aemmp2 increased during bloodmeal digestion. Aemmp1 expression was also upregulated in the presence of a bloodmeal containing chikungunya virus. Using polyclonal antibodies, AeMMP1 and AeMMP2 were specifically detected in tissues associated with the mosquito midgut.

Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion

“…The zymogen (50 kDa) and the active form of AeMMP1 (37 kDa) lacking its propeptide domain were highly expressed between 3 and 24 h pbm ( Fig 3A ) concurring with the time period during which collagen IV was less detectable ( Fig 2A ). Similar to what we had recently observed [ 27 ], the catalytically active form of AeMMP1 was barely detectable in midguts between 36 and 72 h pbm ( Fig 3A ). Typically, most of the bloodmeal has been digested by 48 h post-acquisition allowing the midgut tissue to contract and the BL to rebuild.…”

“…Recently, we showed that two of the nine MMPs of Ae . aegypti , AeMMP1 and AeMMP2, were active in midgut tissue and responded to the presence of a bloodmeal [ 27 ]. AeMMP2 was associated with tracheal cells surrounding the midgut, whereas AeMMP1 was highly expressed in epithelial cells, the site at which CHIKV infection occurs.…”

“…In vertebrates, MMPs and ADAMTSs are the most important enzymes responsible for extracellular matrix (ECM) degradation and remodeling [ 51 ]. Similar to MMP1 of vertebrates, Dm1-MMP1 which is orthologous to AeMMP1 [ 27 ], can degrade mammalian fibronectin and type IV collagen and the enzyme's degrading activity can be completely blocked by generic MMP inhibitors such as EDTA and BB-94 [ 26 , 52 ]. Importantly, Stevens and Page-McCaw [ 24 ] discovered that after tissue injury, secreted Dm1-MMP facilitated restoration of continuous tissue by remodeling the BL, promoting cell elongation and actin cytoskeletal reorganization, and activating extracellular signal-regulated kinase signaling.…”

“…In contrast, transient silencing of the MMP1 homologue in Anopheles gambiae caused a profound reduction in protein abundance resulting in an impaired phenotype [ 33 ]. Possibly, the putatively membrane-bound AeMMP1 [ 27 ] is a relatively stable protein that is maintained as a zymogen for extended periods of time despite protein production being temporarily cut off at the transcriptional and translational levels [ 53 ]. As a consequence, our observations so far do not allow us to conclude whether AeMMP1 is responsible for collagen IV degradation/diminishment during bloodmeal digestion or for collagen IV remodeling/restoration.…”

“…Recently, we described the MMPs in Ae . aegypti as a family of proteases that could be potentially involved in BL modification/remodeling during virus dissemination from the midgut [ 27 ]. In particular, two of the nine AeMMPs, AeMMP1 and AeMMP2, were found to be associated with midgut tissue and they also responded to the presence of a bloodmeal containing or not containing CHIKV.…”