Functional Analyses And Molecular Evolution On C-type Lysozyme In Bats

Mammalian chicken-type(c-type)lysozymes are critical for host immunity by degrading the peptidoglycan of the bacterial cell walls.Many studies have been carried out on the evolution of c-type lysozymes and found its new function for digestion,such as the duplicated stomach lysozymes in ruminants.Interestingly,researchers found that gene duplications and subsequent adaptive evolution of c-type lysozyme have also occurred in a clade of insect-eating bats,which could be related to chitin digestion.Moreover,four c-type lysozyme gene copies were uncovered in the bat genus Myotis.In the case of Myotis ricketti,we calculated the isoelectric point for each of the four lysozymes.The calculation results showed that the isoelectric points of the four lysozymes were quite different.In order to determine the functional differences of the four enzymes,we successfully expressed three of them in vitro(Mric Lyz-1,Mric Lyz-3 and Mric Lyz-4),and then carried out functional assays.The results showed that in terms of antimicrobial,Mric Lyz-1 may retain the original immune function of lysozyme.Compared with Mric Lyz-1,the optimal p H value of Mric Lyz-3 is more acid,which may be expressed in the stomach,while Mric Lyz-4 adapts to extremely acid environments and may play an immune role in the stomach.For digestion,compared with Mric Lyz-1,Mric Lyz-4 did not evolve a better enzymatic activity related to chitin digestion.As the second largest group of bats after the Vespertilionidae,bats from the family Phyllostomidae have a wide variety of feeding habits,including insectivorous,frugivorous,nectarivorous and sanguivorous species.However,there is no research has been carried out on the evolution of c-type lysozymes in the bat family Phyllostomidae.Here,we sequenced and analyzed the c-type lysozymes from four phyllostomid bats,including common vampire bat(Desmodus rotundus),white-winged vampire bat(Diaemus youngi),lesser long-nosed bat(Leptonycteris yerbabuenae)and big fruit-eating bat(Artibeus lituratus).The sequencing results indicated that only one lysozyme gene copy was identified in each of the all four species.According to the molecular evolutionary analyses,we found that only the lysozyme genes from vampire bats underwent positive selection among the single copy lysozyme genes.The analyses for amino acid substitutions of lysozymes also indicate that only the function of c-type lysozyme in vampire bats may be changed.We suggest that the adaptive evolution of lysozyme genes in vampire bats could be related to immunity and might be caused by their specialized blood-feeding related habits.