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Subdued, a TMEM16 family Ca²⁺-activated Cl⁻channel in Drosophila melanogaster with an unexpected role in host defense.

Wong XM, Younger S, Peters CJ, Jan YN, Jan LY - Elife (2013)

Bottom Line: Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question.Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells.This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, United States.

ABSTRACT
TMEM16A and TMEM16B are calcium-activated chloride channels (CaCCs) with important functions in mammalian physiology. Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question. Here we report that a TMEM16 family member from Drosophila melanogaster, Subdued (CG16718), is a CaCC. Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells. This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family. Additionally, we show that knockout flies lacking subdued gene activity more readily succumb to death caused by ingesting the pathogenic bacteria Serratia marcescens, suggesting that subdued has novel functions in Drosophila host defense. DOI: http://dx.doi.org/10.7554/eLife.00862.001.

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Feeding flies gentamicin greatly reduces Db11 counts from gut dissections.Nehme et al. observed that after 24 hr of Db11 feeding, a significant amount of bacteria had entered the hemocoel and adhered to dissected guts in hemocyte-impaired flies. To explore the possibility that KO flies accumulated bacteria in the hemocoel which then adhered to the guts, flies were fed with Db11/sucrose solution for 24 hr and then switched to vials containing 500 µg/ml gentamicin with Db11/sucrose solution. After another 48 hr, four fly guts were dissected and the bacteria recovered from the guts were plated and counted. The low Db11 CFU counts from flies fed with gentamicin show that hemocoel-resident Db11 that adhere to the gut do not contribute greatly to the population of bacteria recovered from gut dissections. The data also suggest that it is unlikely that KO flies have severe impairment of hemocyte and hemocoel defenses.DOI:http://dx.doi.org/10.7554/eLife.00862.009
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fig5s2: Feeding flies gentamicin greatly reduces Db11 counts from gut dissections.Nehme et al. observed that after 24 hr of Db11 feeding, a significant amount of bacteria had entered the hemocoel and adhered to dissected guts in hemocyte-impaired flies. To explore the possibility that KO flies accumulated bacteria in the hemocoel which then adhered to the guts, flies were fed with Db11/sucrose solution for 24 hr and then switched to vials containing 500 µg/ml gentamicin with Db11/sucrose solution. After another 48 hr, four fly guts were dissected and the bacteria recovered from the guts were plated and counted. The low Db11 CFU counts from flies fed with gentamicin show that hemocoel-resident Db11 that adhere to the gut do not contribute greatly to the population of bacteria recovered from gut dissections. The data also suggest that it is unlikely that KO flies have severe impairment of hemocyte and hemocoel defenses.DOI:http://dx.doi.org/10.7554/eLife.00862.009

Mentions: We hypothesized that since the knockout flies did not display prominent structural abnormalities in the alimentary canal, it was possible that the susceptibility to Db11 infection arose from defects in host defense. At 48 hr post-infection, whole flies were homogenized, and the homogenates were serially diluted and plated on LB agar plates with antibiotic selection. Colony forming units (CFU) were counted on each plate to estimate the number of Db11 bacteria present in the whole fly. Since only live flies were homogenized, this assay reports on the active host response the flies mount against Db11 infection. Significantly higher amounts of Db11 were isolated from knockout flies compared to wild type (Figure 5B). To control for the possibility that the higher titers of bacteria isolated from knockout flies could arise from increased feeding, we performed a feeding assay in which a food dye was introduced into the bacteria/sucrose solution (Ha et al., 2009). After 72 hr of feeding, flies were dissected to isolate the intact guts and crop, which were homogenized and analyzed for food dye content as a read-out for food consumption. Knockout fly guts did not contain more food dye and surprisingly, slightly but significantly less food dye was recovered from the guts of knockout compared to the wild-type flies (Figure 5C). Thus, we surmised that the knockout flies are unlikely to consume more food than wild-type flies. Differences in whole animal bacterial titers are thus likely to result from disparities in host defense. To see if bacteria also accumulated more in the guts as well as the whole animal, homogenates obtained from the feeding assay described above were serially diluted and plated on LB agar plates with antibiotic selection. Knockout fly guts had a significantly higher number of CFU of Db11 than wild-type flies (Figure 5D). Since it was reported that injection of latex beads into the hemocoel of Db11-fed flies to impair hemocytic phagocytosis causes significant Db11 proliferation in the hemocoel and adherence of Db11 to the gut wall (Nehme et al., 2007), defective hemocoel defenses could also give rise to an apparent increase in the CFU counts from gut dissections. However, homogenate Db11 counts were greatly reduced upon feeding the flies with gentamicin (Figure 5—figure supplement 2). This control reveals no significant contributions of hemocoel-resident Db11 to CFU counts from dissected guts. This result also rules out severe hemocoel defense impairment as an explanation for increased in vivo proliferation of Db11.


Subdued, a TMEM16 family Ca²⁺-activated Cl⁻channel in Drosophila melanogaster with an unexpected role in host defense.

Wong XM, Younger S, Peters CJ, Jan YN, Jan LY - Elife (2013)

Feeding flies gentamicin greatly reduces Db11 counts from gut dissections.Nehme et al. observed that after 24 hr of Db11 feeding, a significant amount of bacteria had entered the hemocoel and adhered to dissected guts in hemocyte-impaired flies. To explore the possibility that KO flies accumulated bacteria in the hemocoel which then adhered to the guts, flies were fed with Db11/sucrose solution for 24 hr and then switched to vials containing 500 µg/ml gentamicin with Db11/sucrose solution. After another 48 hr, four fly guts were dissected and the bacteria recovered from the guts were plated and counted. The low Db11 CFU counts from flies fed with gentamicin show that hemocoel-resident Db11 that adhere to the gut do not contribute greatly to the population of bacteria recovered from gut dissections. The data also suggest that it is unlikely that KO flies have severe impairment of hemocyte and hemocoel defenses.DOI:http://dx.doi.org/10.7554/eLife.00862.009
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814000&req=5

fig5s2: Feeding flies gentamicin greatly reduces Db11 counts from gut dissections.Nehme et al. observed that after 24 hr of Db11 feeding, a significant amount of bacteria had entered the hemocoel and adhered to dissected guts in hemocyte-impaired flies. To explore the possibility that KO flies accumulated bacteria in the hemocoel which then adhered to the guts, flies were fed with Db11/sucrose solution for 24 hr and then switched to vials containing 500 µg/ml gentamicin with Db11/sucrose solution. After another 48 hr, four fly guts were dissected and the bacteria recovered from the guts were plated and counted. The low Db11 CFU counts from flies fed with gentamicin show that hemocoel-resident Db11 that adhere to the gut do not contribute greatly to the population of bacteria recovered from gut dissections. The data also suggest that it is unlikely that KO flies have severe impairment of hemocyte and hemocoel defenses.DOI:http://dx.doi.org/10.7554/eLife.00862.009
Mentions: We hypothesized that since the knockout flies did not display prominent structural abnormalities in the alimentary canal, it was possible that the susceptibility to Db11 infection arose from defects in host defense. At 48 hr post-infection, whole flies were homogenized, and the homogenates were serially diluted and plated on LB agar plates with antibiotic selection. Colony forming units (CFU) were counted on each plate to estimate the number of Db11 bacteria present in the whole fly. Since only live flies were homogenized, this assay reports on the active host response the flies mount against Db11 infection. Significantly higher amounts of Db11 were isolated from knockout flies compared to wild type (Figure 5B). To control for the possibility that the higher titers of bacteria isolated from knockout flies could arise from increased feeding, we performed a feeding assay in which a food dye was introduced into the bacteria/sucrose solution (Ha et al., 2009). After 72 hr of feeding, flies were dissected to isolate the intact guts and crop, which were homogenized and analyzed for food dye content as a read-out for food consumption. Knockout fly guts did not contain more food dye and surprisingly, slightly but significantly less food dye was recovered from the guts of knockout compared to the wild-type flies (Figure 5C). Thus, we surmised that the knockout flies are unlikely to consume more food than wild-type flies. Differences in whole animal bacterial titers are thus likely to result from disparities in host defense. To see if bacteria also accumulated more in the guts as well as the whole animal, homogenates obtained from the feeding assay described above were serially diluted and plated on LB agar plates with antibiotic selection. Knockout fly guts had a significantly higher number of CFU of Db11 than wild-type flies (Figure 5D). Since it was reported that injection of latex beads into the hemocoel of Db11-fed flies to impair hemocytic phagocytosis causes significant Db11 proliferation in the hemocoel and adherence of Db11 to the gut wall (Nehme et al., 2007), defective hemocoel defenses could also give rise to an apparent increase in the CFU counts from gut dissections. However, homogenate Db11 counts were greatly reduced upon feeding the flies with gentamicin (Figure 5—figure supplement 2). This control reveals no significant contributions of hemocoel-resident Db11 to CFU counts from dissected guts. This result also rules out severe hemocoel defense impairment as an explanation for increased in vivo proliferation of Db11.

Bottom Line: Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question.Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells.This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, United States.

ABSTRACT
TMEM16A and TMEM16B are calcium-activated chloride channels (CaCCs) with important functions in mammalian physiology. Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question. Here we report that a TMEM16 family member from Drosophila melanogaster, Subdued (CG16718), is a CaCC. Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells. This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family. Additionally, we show that knockout flies lacking subdued gene activity more readily succumb to death caused by ingesting the pathogenic bacteria Serratia marcescens, suggesting that subdued has novel functions in Drosophila host defense. DOI: http://dx.doi.org/10.7554/eLife.00862.001.

Show MeSH
Related in: MedlinePlus