Structural and Functional Analysis of PGRP-LC Indicates Exclusive Dap-Type PGN Binding in Bumblebees

Abstract

Peptidoglycan recognition proteins (PGRPs) play an important role in the defense against invading microbes via the recognition of the immunogenic substance peptidoglycan (PGN). Bees possess fewer PGRPs than Drosophila melanogaster and Anopheles gambiae but retain two important immune pathways, the Toll pathway and the Imd pathway, which can be triggered by the recognition of Dap-type PGN by PGRP-LCx with the assistance of PGRP-LCa in Drosophila. There are three isoforms of PGRP-LC including PGRP-LCx, PGRP-LCa and PGRP-LCy in Drosophila. Our previous study showed that a single PGRP-LC exists in bumblebees. In this present study, we prove that the bumblebee Bombus lantschouensis PGRP-LC (Bl-PGRP-LC) can respond to an infection with Gram-negative bacterium Escherichia coli through binding to the Dap-type PGNs directly, and that E. coli infection induces the quick and strong upregulation of PGRP-LC, abaecin and defensin. Moreover, the Bl-PGRP-LC exhibits a very strong affinity for the Dap-type PGN, much stronger than the affinity exhibited by the PGRP-LC from the more eusocial honeybee Apis mellifera (Am-PGRP-LC). In addition, mutagenesis experiments showed that the residue His³⁹⁰ is the anchor residue for the binding to the Dap-type PGN and forms a hydrogen bond with MurNAc rather than meso-Dap, which interacts with the anchor residue Arg⁴¹³ of PGRP-LCx in Drosophila. Therefore, bumblebee PGRP-LC possesses exclusive characteristics for the immune response among insect PGRPs.

Keywords: Bombus lantschouensis; Dap-type PGNs; PGRP-LC; bumblebee; innate immune.

Figure 1

Changes in the expression levels of the PGRP-LC, abaecin, defensin and hymenoptaecin genes in the bumblebee Bombus lantschouensis after challenge with the Gram-negative bacterium E. coli and the Gram-positive bacterium S. aureus. (A) Changes in the expression levels of the PGRP-LC, abaecin, defensin and hymenoptaecin genes induced by the infection with E. coli at 6 h, 12 h and 24 h in B. lantschouensis workers. (B) Changes in the expression levels of the PGRP-LC, abaecin, defensin and hymenoptaecin genes in B. lantschouensis workers triggered by the S. aureus infection at 6 h, 12 h and 24 h. The X-axis indicates the gene names, and the Y-axis indicates the 2^−△△Ct value. ** indicates p-value < 0.01, and * indicates p-value between 0.01 and 0.05. The dashed line indicates the differential expression level of the four genes in the control group (a 2^−ΔΔCt value of 1).

Figure 2

Analysis of the peptidoglycan (PGN)-binding ability of the wild-type bumblebee Bombus lantschouensis PGRP-LC (Bl-PGRP-LC) and honeybee Apis mellifera PGRP-LC (Am-PGRP-LC). Each sample includes four lanes. The first lane (M) is the marker; the second lane (+Lys) shows the corresponding protein samples after pull-down with the meso-diaminopimelic acid (Dap-type) PGN from B. subtilis; the third lane (+Dap) shows the corresponding protein samples after pull-down with the L-lysine type (Lys-type) PGN from M. luteus; and the fourth lane shows the normal corresponding protein as a control. (A) Analysis of the PGN-binding ability of the wild-type Bl-PGRP-LC. (B) The analysis of the PGN-binding ability of the wild-type Dm-PGRP-LC. (C) Analysis of the PGN-binding ability of the wild-type Am-PGRP-LC. (D) Analysis of the PGN-binding ability of the GST-tagged protein.

Figure 3

Analysis of the PGN-binding ability of the bumblebee Bombus lantschouensis PGRP-LC (Bl-PGRP-LC) mutants. Each sample includes four lanes. The first lane (M) is the marker; the second lane (+Lys) shows the corresponding protein samples after pull-down with the Dap-type PGN from B. subtilis; the third lane (+Dap) shows the corresponding protein samples after pull-down with the Lys-type PGN from M. luteus; and the fourth lane shows the Bl-PGRP-LC mutant protein. (A) Analysis of the PGN-binding ability of the twelve Bl-PGRP-LC mutants, namely, H283A and T283A, H305A, R309A, S312A, Y316A, R330A, H338A, F340A, N343A, S394A, R395A and L397A. (B) Analysis of the PGN-binding ability of the Bl-PGRP-LC mutant H390A.

Figure 4

Analysis of the bumblebee Bl-PGRP-LC structure and the sequence alignment of PGRP-LC from bees. (A) Comparison of the Bl-PGRP-LC/TCT complex and the Dm-PGRP-LCx/TCT complex, colored in gray and magenta, respectively. TCT is shown as a line and the contact residues are shown as sticks. The yellow dashed line indicates the bonds between the TCT and the contact residues in the Bl-PGRP-LC/TCT structure. (B) Amino acid sequence alignment of PGRP-LC from different bee species (Melipona quadrifasciata GenBank No. KOX71517.1, Apis dorsata GenBank No. XP_006618726.1, Apis florea GenBank No. XP_003693124.1, Osmia bicornis GenBank No. XP_029039739.1, Apis mellifera X3 GenBank No. XP_006565566.1, Megachile rotundata X4 GenBank No. XP_003702406.2, Megachile rotundata X3 GenBank No. XP_012138567.1, Dufourea novaeangliae GenBank No. KZC08445.1, Apis cerana GenBank No. XP_AGM19450.1, Apis mellifera X5 GenBank No. XP_006565568.1, Apis mellifera X4 GenBank No. XP_006565567.1, Apis mellifera X3 GenBank No. XP_392452.2 and Drosophila PGRP-LCx GenBank No. 2f2l_X). The residues of Bl-PGRP-LC contributing to the TCT binding are boxed with red rectangles.