The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta

Abstract

SummaryBacterial recognition in the lepidopteran insect, Manduca sexta, is mediated by pattern recognition proteins including Hemolin, Peptidoglycan recognition protein (PGRP) and Immulectin-2. These proteins bind to molecular patterns present on the surface of bacteria and trigger a protective response involving humoral and cellular reactions. Cellular mechanisms mediated by haemocytes include phagocytosis, encapsulation, and the formation of melanotic nodules. Here, we show that a non-pathogenic strain of Escherichia coli induces mRNA transcription and protein expression of Hemolin and PGRP but not Immulectin-2 in Manduca haemocytes. This upregulation can be effectively prevented (knocked-down) using RNA interference (RNAi) following injection of double-stranded (ds) RNA. Knock-down of Hemolin significantly decreased the ability of insects to clear E. coli from the haemolymph and caused a reduction in the number of free haemocytes. RNAi of Hemolin reduced the ability of haemocytes to engulf bacteria through phagocytosis and to form melanotic nodules in vivo. Importantly, washed haemocytes taken from RNAi-treated insects showed reduced ability to form microaggregates around bacteria in vitro. This shows that the immune function affected by RNAi knockdown of Hemolin is intrinsic to the haemocytes. In contrast, RNAi of PGRP had no effect on any of these cellular immune functions. These results demonstrate the vital role of Hemolin in Manduca cellular immune responses.

Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection

“…These assays were conducted as previously (Eleftherianos et al, 2007). Briefly, M. sexta larvae were injected with dsRNAs or DMPC water and 18 h later were bled; hemocytes were centrifuged, washed and their number was adjusted to 10 6 cells ml -1 .…”

“…Labeled E. coli and P. luminescens were injected 6 h after the RNAi treatment, as described above, and hemolymph was extracted 18 h later. Hemocyte monolayers, prepared as previously (Eleftherianos et al, 2007), were incubated overnight at 4°C in hybridoma supernatant containing the monoclonal antibody MS77 (Willott et al, 1994) followed by a second incubation with a TRITC-conjugated goat anti-mouse IgG antibody (Sigma) at 1:100 dilution in 0.3% bovine serum albumin (BSA) in PBS. Coverslips were finally mounted in 5 ml of Mowiol-4-88 solution.…”

“…Bacterial cells were stained as before (Eleftherianos et al, 2007). Labeled E. coli and P. luminescens were injected 6 h after the RNAi treatment, as described above, and hemolymph was extracted 18 h later.…”

“…Knockdown of immulectin-2 was shown to interfere with the humoral response by preventing normal activation of phenoloxidase (Eleftherianos et al, 2006b). Knock-down of hemolin was shown to be involved in mediating hemocyte aggregation, nodule formation and phagocytosis upon Escherichia coli infection (Eleftherianos et al, 2007).…”

Noduler, A Novel Immune Up-Regulated Protein Mediates Nodulation Response in Insects

“…Eighteen hours following E. coli challenge, the larvae were dissected under a saturated solution of PTU prepared in 1ϫ IBS and nodule formation was assessed as described by Eleftherianos et al (21). The nodules were counted manually using a stereomicroscope.…”