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. 2010 May 21;285(21):15731-8.
doi: 10.1074/jbc.M109.082115. Epub 2010 Mar 26.

Cooperative regulation of the induction of the novel antibacterial Listericin by peptidoglycan recognition protein LE and the JAK-STAT pathway

Akira Goto  1 , Tamaki Yano, Jun Terashima, Shinzo Iwashita, Yoshiteru Oshima, Shoichiro Kurata
Affiliations

Cooperative regulation of the induction of the novel antibacterial Listericin by peptidoglycan recognition protein LE and the JAK-STAT pathway

Akira Goto et al. J Biol Chem. .

Abstract

Intracellular bacteria cause serious infectious diseases such as tuberculosis, shigellosis, and listeriosis. The Drosophila peptidoglycan recognition protein (PGRP)-LE functions as an important host pattern recognition receptor against intracellular bacteria such as Listeria monocytogenes. One PGRP-LE-mediated intracellular response against L. monocytogenes infection is the induction of autophagy, a conserved intracellular degradation system. Here, to further elucidate PGRP-LE-mediated intracellular innate immune responses, we performed a strategic microarray analysis and identified the Listericin gene, whose expression is induced in response to L. monocytogenes infection in a PGRP-LE-dependent manner. RNA interference and overexpression experiments demonstrated that Listericin gene induction is cooperatively regulated by PGRP-LE and the JAK-STAT (Janus kinase-signal transducers and activators of transcription) pathway. An in vitro cell culture assay showed that Listericin is secreted as processed forms and suppresses the growth of L. monocytogenes and Gram-negative bacteria. A colony formation unit assay clearly demonstrated that induction of the Listericin gene suppresses not only the growth of L. monocytogenes but also the growth of Gram-negative bacteria in vivo. Based on these findings, we propose that the Listericin gene encodes a novel antibacterial peptide-like protein whose induction is cooperatively regulated by PGRP-LE and the JAK-STAT pathway.

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Figures

FIGURE 1.
FIGURE 1.
Identification of the Listericin gene by strategic microarray. A, schematic presentation of four microarray samples is shown. S2 and YFP-LE cells were infected by either Δhly mutant lacking listeriolysin O or wild-type (WT) L. monocytogenes (L.m.). B, shown is a signal average of Listericin gene expression from microarray analysis. C, real-time PCR analysis of Listericin gene expression in S2 cells and YFP-LE cells with different multiplicities of infection of L. monocytogenes (WT or Δhly) is shown. Ribosomal protein Rp49 was used as an internal control. Bars indicate S.D.
FIGURE 2.
FIGURE 2.
Control of PGRP-LE-dependent Listericin gene induction in response to immune stimulation by the JAK-STAT pathway. A, dsGFP (used as a negative double-stranded RNA control), dskey (for the imd pathway), dsDif and dsdorsal (for the Toll pathway), and dsStat92E (for the JAK-STAT pathway) were transfected into either S2 cells or Ind-LE cells (metallothionein promoter-dependent inducible-PGRP-LE), and the cells were stimulated by heat-killed E. coli (E.c (HK)), Δhly, and wild type (WT) L. monocytogenes (L.m). Listericin gene expression was then measured at 8 h after immune stimulation. Representative data of two independent experiments are shown. B, semiquantitative RT-PCR analysis to check RNAi effects on each gene is shown. Numbers in parentheses indicate PCR cycle number. Ribosomal protein Rp49 was used as an internal control.
FIGURE 3.
FIGURE 3.
Synergistic effect of activation of JAK-STAT pathway and expression of PGRP-LE on the expression of the Listericin gene. The ligand of the JAK-STAT pathway (Upd), the receptor for the imd pathway (PGRP-LC), and activated Toll receptor of the Toll pathway (TollΔLRR) were each expressed in S2 or Ind-LE cells, and Listericin, Diptericin, Drosomycin, and Vir-1 expression was measured by real-time PCR. The lower panel shows the forced expression of each gene analyzed by RT-PCR. Numbers in parentheses indicate PCR cycle number. Rp49 was used as the internal control. The mean of three independent experiments is shown. Bars indicate S.D.
FIGURE 4.
FIGURE 4.
Secretion of Listericin into the cell culture medium and suppression of L. monocytogenes and E. coli growth. A, cell lysate (Cell) and conditioned medium (Med) of 10% fetal bovine serum containing Schneider or serum-free SFX insect medium from either mock (pAC5.1 vector only) or Listericin-V5-HIS transfected cells were analyzed by immunoblotting using anti-V5 antibody. Asterisks indicate the positions of detected signals. The same amount of proteins used for the immunoblot was also visualized by CBB staining. B–E, conditioned medium from the Listericin gene-overexpressed S2 cell line was mixed with the appropriate numbers of E. coli K12 in 10% fetal bovine serum (FBS) medium (B), E. coli K12 in serum-free medium (C), L. monocytogenes (D), and S. saprophyticus (E), and the bacterial growth was analyzed by CFU assay. Stable GFP-expressing S2 cells were used as a negative control. The graph shows representative results from three independent experiments. WT, wild type.
FIGURE 5.
FIGURE 5.
Effect of overexpression of the Listericin gene on host survival against L. monocytogenes infection. Survival rates of Cg-Gal4-driven GFP as a control and Listericin gene-overexpressed flies were tested after injecting saline (used as a bacteria-free control) or L. monocytogenes (L.m (WT)) at 29 °C in a PGRP-LE112 mutant (A) and wild-type (B) background. The 69-nl injection of A600 = 0.00001 and A600 = 0.001 L. monocytogenes suspension approximately corresponds to ∼10 and ∼1000 bacteria/fly, respectively. *, p < 0.05 (Wilcoxon-Mann-Whitney test).
FIGURE 6.
FIGURE 6.
Effect of overexpression of the Listericin gene on growth of infected bacteria in vivo. At 24 h after injection of L. monocytogenes (Lm (WT)), L. monocytogenes (Δhly), E. coli, Ecc 15, S. aureus, and S. saprophyticus into Collagen (Cg)-Gal4-driven GFP as a control, Listericin gene-overexpressed flies were sterilized by ethanol on the surface, homogenized, and diluted serially in the appropriate medium. Bacterial growth in each fraction was quantified by determining CFU by plate assay. Data represent the mean of at least three independent experiments with more than 30 flies of each genotype examined. Bars indicate S.D. *, p < 0.05.
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