Immune response and anti-microbial peptides expression in Malpighian tubules of Drosophila melanogaster is under developmental regulation.

Malpighian tubules (MT) of Drosophila melanogaster are osmoregulatory organs that maintain the ionic balance and remove toxic substances from the body. Additionally they act as autonomous immune sensing organs, which secrete antimicrobial peptides in response to invading microbial pathogens. We show that the antimicrobial peptides (AMP) diptericin, cecropinA, drosocin and attacinA are constitutively expressed and are regulated in developmental stage specific manner. Their developmental expression begins from 3(rd) instar larval stage and an immune challenge increases the expression several folds. Spatial variations in the level of expression along the MT tissue are observed. The mortality of 3(rd) instar larvae fed on bacterial food is much less than that of the earlier larval stages, coinciding with the onset of innate immunity response in MT. Ectopic expression of AMP imparts better resistance to infection while, loss of function of one of the AMP through directed RNAi reduces host survival after immune challenge. The AMP secreted from the MT exhibit bactericidal activity. Expression of the NF-κB transcription factor, Relish, also coincides with activation of immune responsive genes in MT, demonstrating that immune regulation in MT is under developmental control and is governed by the Imd pathway

Genetic mapping of the amide response element (s) of the hsrω locus of Drosophila melanogaster

Small chromosomal deletions [Df(3R)eR-1 and Df(3R)eP] with intact hsrω transcription units but with variable deletions of the upstream region were used to map the upstream regions that regulate heat shock and amide responsivity of the 93D puff (hsrω locus) in salivary glands of late third instar larvae of Drosophila melanogaster. The Df(3R)eP deletion, generated by a P-element mobilization screen, removed the 93B6-7 to 93D3-5 cytogenetic region. [3H]uridine-labeled transcription autoradiograms revealed that normal developmental and heat shock-induced expression of the 93D puff remained unaffected in both the deficiency chromosomes. However, the amide responsivity of the 93D site was lost on the Df(3R)eP homolog while the Df(3R)eR-1 homolog responded normally to amides. Southern hybridizations with a series of upstream probes mapped the distal breakpoint of the Df(3R)eP deletion between -22 kb and -23 kb of the hsrω transcription unit. Since the distal breakpoint of Df(3R)eR-1 is at about -45 kb upstream of the hsrω gene it is inferred that the amide response element(s) that modulate the specific transcriptional activation of the 93D puff following treatment of salivary glands with a variety of amides is/are located in the -22 kb to about -45 kb upstream interval. The Df(3R)eP and Df(3R)eR-1 deletions also abolished dosage compensation at the 93D locus as well as the effect of β-alanine levels on its heat shock inducibility

Data_Sheet_1_Hsc70-4 aggravates PolyQ-mediated neurodegeneration by modulating NF-κB mediated immune response in Drosophila.PDF

Huntington’s disease occurs when the stretch of CAG repeats in exon 1 of the huntingtin (htt) gene crosses the permissible limit, causing the mutated protein (mHtt) to form insoluble aggregates or inclusion bodies. These aggregates are non-typically associated with various essential proteins in the cells, thus disrupting cellular homeostasis. The cells try to bring back normalcy by synthesizing evolutionary conserved cellular chaperones, and Hsp70 is one of the families of heat shock proteins that has a significant part in this, which comprises of heat-inducible and cognate forms. Here, we demonstrate that the heat shock cognate (Hsc70) isoform, Hsc70-4/HSPA8, has a distinct role in polyglutamate (PolyQ)-mediated pathogenicity, and its expression is enhanced in the polyQ conditions in Drosophila. Downregulation of hsc70-4 rescues PolyQ pathogenicity with a notable improvement in the ommatidia arrangement and near-normal restoration of optic neurons leading to improvement in phototaxis response. Reduced hsc70-4 also attenuates the augmented immune response by decreasing the expression of NF-κB and the antimicrobial peptides, along with that JNK overactivation is also restored. These lead to the rescue of the photoreceptor cells, indicating a decrease in the caspase activity, thus reverting the PolyQ pathogenicity. At the molecular level, we show the interaction between Hsc70-4, Polyglutamine aggregates, and NF-κB, which may be responsible for the dysregulation of signaling molecules in polyQ conditions. Thus, the present data provides a functional link between Hsc70-4 and NF-κB under polyQ conditions.</p

RT-PCR for detecting AMP gene transcripts during development and after LPS treatment.

<p>Expression of, <i>Diptericin (dipt), drosocin (drc), cecropinA (cecA) and attacinA (attA),</i> in MT from 1^st, 2^nd, 3^rd, prepupae, pupae and adult under normal conditions (A). The intensity of bands were measured and plotted on a graph (A’). <i>AttacinA</i> and <i>Drosocin</i> is highest in adult, whereas <i>diptericin</i> and <i>cecropinA</i> is highest in pupae. MT from 3^rd instar, prepupae, pupae and adult were treated with LPS and RT-PCR was done (B). Enhanced expression of all the AMP were observed after LPS treatment (LPS) and compared with control (C) without LPS treatment. The intensity of bands were measured and plotted on a graph (B’) which show that after immune challenge there is an enhanced expression of all AMP at all stages of development. <i>Glyceraldehyde-3-phosphate dehydrogenase (GPDH)</i> is used as an internal control to ensure the integrity of RT-PCR.</p

A. Mortality rate of 3^rd instar larvae increases after depleting diptericin in MT.

<p>Percentage of adult flies eclosed from 3^rd instar larvae of <i>c42>UAS-diptericin_RNAi</i> fed on <i>E. coli</i> is decreased (dotted graph) in comparison to control <i>c42</i>><i>UAS-diptericin_RNAi</i>3^rd instar larvae (black solid graph). Asterick (*) represents level of significance at p<0.05. <b>B. Survival rate following bacterial infection decreases after depleting </b><b><i>diptericin</i></b><b> in MT of adult flies</b>. Percentage survival of <i>c42</i>><i>UAS-diptericin_RNAi</i> flies fed on <i>E. coli</i> (red graph) significantly reduces in comparison to <i>c42>UAS-diptericin_RNAi</i> control unfed flies (blue graph). Asterick (*) represents level of significance at p<0.05.</p

<i>E. coli</i> killing assay.

<p>The plates have <i>E. coli</i> bacterial lawn grown upon them. No plaque was formed when 1^st instar <b>(</b>A, 1^st LMT) and 2^nd instar (A, 2^nd LMT) exudate from MT were plated on bacterial lawn (encircled region). Plaque was observed when exudate from wandering 3^rd instar larval MT (A, LMT), pupal MT (B, PMT) and adult MT (A, FMT) were plated on bacterial lawn. <i>cecropin</i> was used as +ve control which resulted in the formation of plaque.</p

A. Expression of Relish in MT during development.

<p>Immunostaining using anti-Relish showed that in the 1^st (A) and 2^nd (B) instar larvae Relish expression is not observed but in the 3^rd instar (C) we do observe Relish staining. Counterstaining was done with DAPI (A’, B’ and C’) pseudo color red. Merged images (A”, B” and C”) show expression to be cytoplasmic as well as nuclear. <b>B. </b><b>Relish expression after immune challenge.</b> Number of nuclei showing Relish expression is enhanced after LPS treatment of 3^rd instar larvae (B) compared to control (A). Nuclei are counter stained with DAPI (A’ and B’), pseudo color red and merged images are A” and B”.</p

A. <i>E. coli-GFP</i> expression in gut of larvae.

<p>No fluorescence is observed in the gut of control larvae, not fed on GFP bacteria of 1^st instar (A) 2^nd instar (B) and 3^rd instar (D). A’, B’ and C’ are DIC images merged with fluorescence shows that the gut region is devoid of GFP-bacteria. Green fluorescent is observed when 1^st (D), 2^nd (E) and 3^rd (F) instar larvae are fed on GFP-bacteria. DIC images and merged fluorescence (D’, E’ and F’) confirms that green fluorescence is in the gut. <b>B</b>. <b>Mortality rate of different stage larvae when fed on bacterial food.</b> Percentage of adults emerged shows that 1^st instar (green graph) wild type larvae are most susceptible to <i>E. coli</i> and <i>M. smegmatis</i> than 2^nd instar (yellow graph) and 3^rd instar (maroon graph). <i>imd</i> mutants (control) are less viable than <i>diap2</i> mutants (control) and also the mortality rate for <i>imd</i> mutants are significantly less than <i>diap2</i> mutant when fed on <i>E. coli.</i> Asterick (*) represents significance at p<0.05.</p