Molecular cloning and functional characterization of tenebrio PGRP-LE, PGRP- LB and autophagy-related genes ATG3, 5 and 8 in response to listeria monocytogenes and pseudomonas geniculata HT1 infection

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Date

2015

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Chonnam National University

Abstract

The Current study investigated innate immune responses of the mealworm beetle, Tenebrio molitor, against microbial infections. We, therefore, dealt with two major themes, the immune functions of autophagy-reiated genes TmATG3, TmATG5 and TmATG8 on the one hand and, roles of TmPGRP-LE and TmPGRP-LB in immune responses to microbial infections on the other hand. In the first pail, the three autophagy-related genes, TmATG3, TmATG5^ and TmATG8 were identified and characterized for their immunological functions in the beetle against infections by an intracellular pathogen, Listeria monocytogenes. TmATG39 TmATG5 genes were identified from T. molitor EST and RNAseq databases. The cDNA of TmATG3 and TmATG5 comprise of ORF sizes of 963 and 792 bp encoding proteins with 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 transcripts are detected in all developmental stages analyzed, and primarily in fat body and hemocytes of larvae. TmATG3 and TmATG showed high amino acid sequence identity (58-95%) with corresponding homologues from various insects and were closer to their orthologs in T. castaneum. Loss of function of TmATG3 and TmATG5 by RNAi led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, L. monocytogenes. Six days post-Listeria challenge, the survivability in dsTwJTGJ- and dsTmATG5-treated larvae was significantly reduced to 3 and 4%, respectively, when compared with dsEGFP-injected control larvae. The cDNA cfiTmATG8 comprises of an ORF of 363 bp encoding a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 contains a highly conserved C-terminal glycine residue (G116) and shows high amino acid sequence identity (98%) to its T. castaneum homologue, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in mortality of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, Tw/lTUS-silenced larvae failed to turn-on autophagy in hemocytes after L. monocytogenes injection. Taken together, these data suggest that TmATG3, TmATG5 and TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor. In the second part, TmPGRP-LE and TmPGRP-LB were identified and characterised for their immunological functions in T. molitor. A PGRP-LE homologue, from T. molitor was identified and characterized for its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. TmPGRP-LE cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a protein of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis indicated a broad constitutive expression of the TmPGRP-LE transcripts at various stages of development spanning from larva to adult. RNAi-mediated knockdown of the TmPGRP-LE transcript, followed by a challenge with L. monocytogenes, resulted in a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control ofL. monocytogenes infection in T. molitor. PGRP-LB, one of several amidase-capable members of the PGRP family has been demonstrated to specifically recognize DAP-type PGN, thereby preventing over-activation of the IMD pathway upon infections Gram-negative bacteria. We have identified, cloned and partially characterized the immunological functions of a PGRP-LB homologue from T. molitor against a newly isolated Gram-negative bacterium Pseudomonas geniculata HT1 infection. TmPGRP-LB has an ORF of 597 bp encoding a protein with 198 amino acid residues and contains the conserved PGRP domain. TmPGRP-LB is closest to its orthologous TcPGRP-LBl and TcPGRP-LB2 isoforms in T. castaneum with which it shares the highest (73 %) percentage identity. TmPGRP-LB transcripts were detected in all developmental stages examined spanning from the late-instar larva to adult day 1 and 2. TmPGRP-LB transcripts were also detected in all tissues examined including the gut, hemocytes, fat body, Malphigian tubules, integuments, ovaries and testes. Infection of Tenebrio larvae with HT1 resulted in increased expression of TmPGRP-LB but not other IMD pathway-related genes TmPGRP-LC and TmPGRP-LE. TmPGRP-LB loss of function by RNAi resulted in increased susceptibility of larvae to infections by Gram-negative bacteria HT1 and Escherichia coli KI2 but not Gram-positive bacteria Staphylococcus aureus RN4220. Our results suggest that TmPGRP-LB plays a role in control of Gram-negative infections in T. molitor. Overall, we have demonstrated that both autophagy and peptidoglycan recognition proteins of the IMD pathway are deployed to counter bacterial infections in T. molitor

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Keywords

Molecular cloning, Listeria monocytogenes, Pseudomonas geniculata, HT1 infection, TmATG3,, Characterization of tenebrio

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