Preferential binding of DAP-PGs by major peptidoglycan recognition proteins found in cell-free hemolymph of Manduca sexta

Udeshika Kariyawasam, Mansi Gulati, Yang Wang, Haibo Bao, Tisheng Shan, Xiuru Li, Xiaolong Cao, Niranji Sumathipala, Yingxia Hu, Xiufeng Zhang, Geert Jan Boons, Haobo Jiang*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


Peptidoglycan recognition proteins (PGRPs) detect invading bacteria to trigger or modulate immune responses in insects. While these roles are established in Drosophila, functional studies are not yet achieved at the PGRP family level in other insects. To attain this goal, we selected Manduca sexta PGRP12 and five of the nine secreted PGRPs for recombinant expression and biochemical characterization. We cloned PGRP2−5, 12 and 13 cDNAs, produced the proteins in full (PGRP2–5, 13) or in part (PGRP3s, 12e, 13N, 13C) in Sf9 cells, and tested their bindings of two muramyl pentapeptides by surface plasmon resonance, two soluble peptidoglycans by competitive ELISA, and four insoluble peptidoglycans and eight whole bacteria by a pull-down assay. Preferential binding of meso-diaminopimelic acid-peptidoglycans (DAP-PGs) was observed in all the proteins containing a peptidoglycan binding domain and, since PGRP6, 7 and 9 proteins were hardly detected in cell-free hemolymph, the reportoire of PGRPs (including PGRP1 published previously) in M. sexta hemolymph is likely adapted to mainly detect Gram-negative bacteria and certain Gram-positive bacteria with DAP-PGs located on their surface. After incubation with plasma from naïve larvae, PGRP2, 3f, 4, 5, 13f and 13N considerably stimulated prophenoloxidase activation in the absence of a bacterial elicitor. PGRP3s and 12e had much smaller effects. Inclusion of the full-length PGRPs and their regions in the plasma also led to proHP8 activation, supporting their connections to the Toll pathway, since HP8 is a Spӓtzle-1 processing enzyme in M. sexta. Together, these findings raised concerns on the common belief that the Toll-pathway is specific for Gram-positive bacteria in insects.

Original languageEnglish
Article number103827
Pages (from-to)1-10
Number of pages10
JournalInsect Biochemistry and Molecular Biology
Publication statusPublished - Sept 2022
Externally publishedYes


  • Antimicrobial peptide
  • Hemolymph protein
  • Insect immunity
  • Melanization
  • Pattern recognition
  • Serine protease pathway


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