An essential complementary role of NF-kappaB pathway to microbicidal oxidants in Drosophila gut immunity

Abstract

In the Drosophila gut, reactive oxygen species (ROS)-dependent immunity is critical to host survival. This is in contrast to the NF-kappaB pathway whose physiological function in the microbe-laden epithelia has yet to be convincingly demonstrated despite playing a critical role during systemic infections. We used a novel in vivo approach to reveal the physiological role of gut NF-kappaB/antimicrobial peptide (AMP) system, which has been 'masked' in the presence of the dominant intestinal ROS-dependent immunity. When fed with ROS-resistant microbes, NF-kappaB pathway mutant flies, but not wild-type flies, become highly susceptible to gut infection. This high lethality can be significantly reduced by either re-introducing Relish expression to Relish mutants or by constitutively expressing a single AMP to the NF-kappaB pathway mutants in the intestine. These results imply that the local 'NF-kappaB/AMP' system acts as an essential 'fail-safe' system, complementary to the ROS-dependent gut immunity, during gut infection with ROS-resistant pathogens. This system provides the Drosophila gut immunity the versatility necessary to manage sporadic invasion of virulent pathogens that somehow counteract or evade the ROS-dependent immunity.

Figure 1

IMD/NF-κB-dependent innate immunity is indispensable for host protection from ROS-resistant pathogen. (A) KNU5377 yeast is a ROS-resistant yeast strain. The standard yeast strain (W303) and the KNU5377 strain were exposed to 10 mM H₂O₂ for different times (0, 30, 60, 90 and 120 min). The aliquots were spotted on YPD agar plates, and were incubated at 30°C in order to determine their survival rates. (B) Wild-type flies are equally resistant to both normal and ROS-resistant yeasts. The adult male flies were subjected to natural infection with W303 or KNU5377. (C) IMD/NF-κB pathway mutant flies are susceptible to KNU5377 but not to W303. The IMD/NF-κB pathway mutant flies (Dredd^B118, key¹ and Relish^E20) and the Toll/NF-κB pathway mutant flies (spz^rm7 and Dif¹) were subjected to natural infection with W303 or KNU5377. (D) Flies exhibiting impaired regulation of the Toll pathway showed wild-type level resistance against KNU5377. Loss-of-function flies for Toll pathway (J4 and Pelle-RNAi/+; Da-GAL4/+) or gain-of-function flies for Toll pathway (cact^A2) were subjected to natural infection with W303 or KNU5377. The Pelle-RNAi/+; Da-GAL4/+ flies used in this study showed severely reduced level of infection-induced Drosomycin gene expression following systemic infection (data not shown). The flies exhibiting impaired potential for both Toll and IMD pathways (Dredd; Pelle-RNAi/+; Da-GAL4/+) were also used in this experiment. These flies showed similar immune susceptibility to that of flies carrying IMD pathway mutation alone. In all cases, survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05).

Figure 2

The susceptibility of Relish^E20 flies to natural KNU5377 infection can be ameliorated via the re-introduction of Relish in the intestine but not in the fat body. For the rescue experiment, the Relish^E20 flies were crossed with flies carrying UAS-Relish. The cad-GAL4 and c564-GAL4 drivers were used for intestine-specific and fat body/hemocyte-specific Relish expression, respectively. The genotypes of the flies used in this study were as follows: control (cad-GAL4/+); Relish^E20 (cad-GAL4/+; Relish^E20); Relish^E20+Relish (intestine) (cad-GAL4/UAS-Relish; Relish^E20); Relish^E20+Relish (fat body/hemocytes) (c564-GAL4/UAS-Relish; Relish^E20). Natural gut infection (A) and septic infection (B) were performed with KNU5377 and Erwinia carotovora carotovora 15 (Ecc15), respectively. In all cases, survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05).

Figure 3

ROS-removing activity can act as a virulence factor to the host lacking IMD/NF-κB pathway potential. (A) KatN-overexpressing Salmonella can significantly decrease infection-induced host's ROS level. The total in vivo intestinal ROS levels were quantified (Ha et al, 2005a) with flies both before and after natural infection with control Salmonella (SL1344) or KatN-overexpressing Salmonella (SL1344-KatN). The ROS level in the uninfected control intestine was taken arbitrarily to be 100, and the results are presented as relative levels. Results are expressed as the mean and the standard deviations of three different experiments. (B) IMD/NF-κB pathway mutant flies are susceptible to bacteria overexpressing the Salmonella KatN catalase. Natural infection was performed with Salmonella enterica serotype Typhimurium (SL1344), SL1344 overexpressing Salmonella catalase, KatN (SL1344-KatN), SL1344 overexpressing mutant form of KatN (SL1344-KatN-mut), E. coli DH5α strain (E. coli) and DH5α strain overexpressing KatN (E. coli-KatN). (C) The susceptibility of Relish^E20 flies to natural SL1344-KatN infection can be greatly ameliorated via the re-introduction of Relish in the intestine. The genotypes of the flies used in this study are described in Figure 2. In all cases, survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05).

Figure 4

Intestinal AMP expression is indispensable for host protection from attack of ROS-resistant pathogens. (A) Natural yeast infection induces Cecropin (Cec) expression in the midgut via IMD/NF-κB pathway. Wild-type flies (WT) or IMD/NF-κB pathway mutant flies (Dredd^B118) were subjected to natural infection (0 and 12 h) with either the W303 or KNU5377 strains. Quantitative real-time PCR analysis of Cec gene transcription was performed using dissected midguts. Cec expression in the tissues of uninfected WT flies was taken arbitrarily as 1, and the results are shown as relative expressions. Results are expressed as the means±s.d. (P<0.05) of three different experiments. (B) Both KNU5377 and W303 strains are found to be equally susceptible to low concentrations of synthetic Cec A1 peptide. Yeast cells were incubated with serially diluted Cec A1 peptide at 28°C for 18 h. Antifungal activity was performed as described in Materials and methods. Results are expressed as the means±s.d. (P<0.05) of three different experiments. (C) Both SL1344 and SL1344-KatN strains are equally susceptible to synthetic Cec A1. To measure the antibacterial activity, inhibition zone assay were performed with serially diluted Cec A1, as described in Materials and methods. (D) The susceptibility of the Dredd^B118 flies to natural KNU5377 infection can be dramatically ameliorated by the ectopic expression of Cec A1 in the intestine. For the rescue experiment, Dredd^B118 flies were crossed with flies carrying UAS-Cec A1. The cad-GAL4 driver was used for intestine-specific Cec expression. The genotypes of the flies used in this study were as follows: control (cad-GAL4/+); Dredd^B118 (Dredd^B118; cad-GAL4/+); Dredd^B118+Cec (Dredd^B118; cad-GAL4/UAS-Cec). Natural infection was performed with KNU5377. Survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05). (E) The susceptibility of IMD/NF-κB pathway mutant flies to ROS-resistant Salmonella infection can be dramatically ameliorated by ectopic Cec A1 expression in the intestine. The genotypes of the flies used in this study are shown in panel (D). Natural infection was performed with SL1344-KatN. Survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05).

Figure 5

Intestinal NF-κB-dependent AMP expression is required for the inhibition of the proliferation of ROS-resistant virulent pathogens. (A) KNU5377 persists in the guts of the Dredd^B118 flies, but not in the guts of either the wild-type or Dredd^B118 flies expressing intestinal Cec A1. In order to construct a rescue line, Dredd^B118 flies were crossed with flies carrying UAS-Cec A1. The genotypes of the flies used in this study are listed in Figure 4D. Natural infection was performed with G418-resistant KNU5377. KNU5377 persistence was measured by plating appropriate dilutions of homogenates of five surface-sterilized intestines, which had been collected at different times after infection. Microbes were grown on YPD plates containing G418 (200 μg/ml). The number of CFUs per adult intestine obtained at each time point after infection represents the means±s.d. (P<0.05) of three different experiments. (B) The high persistence of ROS-resistant bacteria can be controlled by NF-κB/AMP pathway in the intestine of live Drosophila. E. coli overexpressing KatN, but not E. coli that does overexpress KatN, persists in the guts of Dredd^B118 flies. GFP-tagged E. coli DH5α strain (E. coli-GFP) and E. coli-GFP overexpressing KatN (E. coli-KatN-GFP) were used for the in vivo real-time analysis of bacterial persistence. The genotypes of the flies used in this study are shown in Figure 4D. Representative images of naturally infected flies (upper panels), dissected intestines (middle panels) and representative plates of E. coli recovered from the intestines (lower panels), at 72 h after infection, are shown.

Figure 6

The high persistence of ROS-resistant bacteria causes a severe abnormality in the intestine of NF-κB mutant Drosophila. (A, B) Wild-type and Relish mutant flies were naturally infected with E. coli or E. coli-KatN. Midguts were dissected out at 72 h post-infection and examined under light and fluorescence microscopy (A). Nuclear staining was performed with DAPI. Transverse sections of the midgut were stained with toluidine blue and analyzed under light microscopy (B). Magnification is × 400 in each panel. Insets show high magnification ( × 1000) of the boxed area.

Figure 7

Ingestion of ROS-resistant bacteria induces loss of typical intestinal cell shape and the apoptosis of Relish^E20 intestinal cells. Wild-type and Relish mutant flies were naturally infected with E. coli or E. coli-KatN. Midguts were dissected out at 72 h post-infection. Transverse sections of the midgut were used in this experiment. (A) Actin staining of the midgut cells. The F-actin was stained with Alexa 568 phalloidin (red) and nuclear staining was performed with DAPI (blue). Tissues were examined under fluorescence microscopy and merged images were presented. (B) Apoptosis of the midgut cells. DNA strand breaks of the apoptotic cells were detected by incorporation of the fluorescein-labeled nucleotide. Nuclear staining was performed with DAPI (blue). The samples were examined under fluorescence microscopy. Intestinal cells were randomly selected for apoptosis index analysis and the number of apoptotic cells was calculated. Apoptosis index was determined by dividing the number of cells by total number of cells and multiplying by 100. Values represent the means±s.d. (P<0.05) of three independent experiments.