Oligopeptide Transporters of the SLC15 Family Are Dispensable for Peptidoglycan Sensing and Transport in Drosophila

Abstract

Peptidoglycan (PGN) detection by PGN recognition proteins (PGRP) is the main trigger of the antibacterial immune response in Drosophila. Depending on the type of immune cell, PGN can be sensed either at the cell membrane by PGRP-LC or inside the cell by PGRP-LE, which plays a role similar to that of Nod2 in mammals. Previous work, mainly in cell cultures, has shown that oligopeptide transporters of the SLC15 family are essential for the delivery of PGN for Nod2 detection inside of the cells, and that this function might be conserved in flies. By generating and analyzing the immune phenotypes of loss-of-function mutations in 3 SLC15 Drosophila family members, we tested their role in mediating PGRP-LE-dependent PGN activation. Our results show that Yin, CG2930, and CG9444 are required neither for PGRP-LE activation by PGN nor for PGN transport from the gut lumen to the insect blood. These data show that, while intracellular PGN detection is an essential step of the antibacterial response in both insects and mammals, the types of PGN transporters and sensors are different in these animals.

Keywords: Drosophila; NF-κB; Peptidoglycan; Peptidoglycan recognition proteins; SLC15 transporters.

Fig. 1

Drosophila SLC15 vertebrate orthologs. a Phylogenetic tree including the following protein sequences: Drosophila SLC15, human SLC15, human SLC16, human SLC17, and human SLC37. SLC15: oligopeptide transporter family, SLC16: monocarboxylate transporter family, SLC17: organic anion transporter, and SLC37: sugar-phosphate exchanger. This neighbor-joining tree was created using Clustal Omega. b Amino acid sequence alignment of SLC15 transporters. The following 4 sequences were aligned using Clustal Omega: human solute carrier family 15 member 1 (SLC15A1, Uniprot ID: P46059), Drosophila peptide transporter family 1 (Yin, Uniprot ID: P91679), Drosophila CG2930 (Uniprot ID: Q9W4P6), and Drosophila CG9444 (Uniprot ID: Q9VH93). Asterisks indicate positions that have a single, fully conserved residue. Colons and periods indicate conservation between groups of strongly and weakly similar properties, respectively (see Clustal Omega website for more details). According to Smith et al. [42], underlined amino acids have been identified in human SLC15A1 as essential to the transport function. In bold are the amino acids that play a role in substrate affinity. The amino acid sequence in italics corresponds to a PTR motif involved in proton binding.

Fig. 2

Drosophila SLC15 mRNA levels in immune tissues. a Relative gene expression of Yin, CG2930, and CG9444 mRNAs in axenic adult immune tissues compared to the whole body. b Relative gene expression of Yin, CG2930, and CG9444 mRNA in different gut domains from axenic adults compared to the midgut. Pv, proventriculus; Vtr, ventriculus; Cc, copper cells; Pmg, posterior midgut; Hg, hindgut. Histograms correspond to the mean ± SEM of 3 independent experiments. * p < 0.01, *** p < 0.0001; ns indicates p > 0.05 (unpaired 2-way ANOVA vs. a reference value set to 1 for each genotype).

Fig. 3

Drosophila SLC15 transporters are not required for NF-κB activation in the posterior midgut. a Attacin-D, Pirk, and PGRP-LB mRNA expression in the adult posterior midgut 4 h after Ecc feeding. The mRNA level in axenic control flies was set to 1, and values obtained with other genotypes are expressed as a fold of this value. Histograms correspond to the mean ± SEM of 3 independent experiments. * p < 0.05. ns indicates p > 0.05 (unpaired Kruskal-Wallis test vs. indicated controls). The following genotypes were used: control (w¹¹¹⁸), PGRP-LE mutant (PGRP-LE¹¹²), and SLC15 transporter mutant flies (Yin^KO, CG2930^KO, and CG9444^KO). b Bacterial loads of the adult gut 4 h after Ecc feeding. ** p < 0.01, **** p < 0.0001.

Fig. 4

Increased systemic activation of the immune response in Drosophila SLC15 mutants. Induction level of Diptericin mRNA in the fat body of adult flies 24 h after Ecc feeding (a) and 6 h after septic injury with Ecc (b). mRNA levels in axenic control flies were set to 1, and values obtained with other genotypes were expressed as a fold change relative to this value. Histograms correspond to the mean ± SEM of 3 independent experiments. * p < 0.05, ** p < 0.01. ns indicates p > 0.05 (unpaired Kruskal-Wallis test vs. indicated controls). The following genotypes were used: control (w¹¹¹⁸), PGRP-LE mutant (PGRP-LE¹¹²) and SLC15 transporter mutant flies (Yin^KO, CG2930^KO, and CG9444^KO).

Fig. 5

Lifespan reduction of Yin mutants after Ecc feeding is due to an overactivation of NF-κB signalling in the fat body. a Survival curves of Ecc orally infected flies revealing that YinKO and PGRP-LE112 mutant are more susceptible than CG2930KO, CG9444KO and control flies (w1118). bYinKO susceptibility to Ecc feeding is suppressed when NF-κB signalling is specifically blocked in the fat body, using CgGal4 driving UAS-dFaddIR. Survival curves are representative of at least three independent trials.