Participation of the p38 pathway in Drosophila host defense against pathogenic bacteria and fungi

Abstract

The signaling network of innate immunity in Drosophila is constructed by multiple evolutionarily conserved pathways, including the Toll- or Imd-regulated NF-κB and JNK pathways. The p38 MAPK pathway is evolutionarily conserved in stress responses, but its role in Drosophila host defense is not fully understood. Here we show that the p38 pathway also participates in Drosophila host defense. In comparison with wild-type flies, the sensitivity to microbial infection was slightly higher in the p38a mutant, significantly higher in the p38b mutant, but unchanged in the p38c mutant. The p38b;p38a double-mutant flies were hypersensitive to septic injury. The immunodeficiency of p38b;p38a mutant flies was also demonstrated by hindgut melanization and larvae stage lethality that were induced by microbes naturally presented in fly food. A canonical MAP3K-MKK cascade was found to mediate p38 activation in response to infection in flies. However, neither Toll nor Imd was required for microbe-induced p38 activation. We found that p38-activated heat-shock factor and suppressed JNK collectively contributed to host defense against infection. Together, our data demonstrate that the p38 pathway-mediated stress response contribute to Drosophila host defense against microbial infection.

Fig. 1.

The p38-deficient flies are more sensitive to microbial pathogens in food. (A) The survival rate to pupahood of the wild-type yellow white strain (y w) and p38ab;p38a double-mutant (p38a¹³; p38b^156A) (Fig. S1) larvae cultured in standard food with or without a mixture of antibiotics (500 μg/mL ampicillin, 50 μg/mL tetracycline, and 200 μg/mL rifamycin), or in sterilized food. (B) The survival rate to pupahood of the wild-type and p38b;p38a larvae cultured in sterilized food with or without adding 10⁵/mL P. aeruginosa (PAO1, wild-type strain; PAOR3, a more virulent mutant strain repressing quorum-sensing controlled genes). (C) Third instar larvae of y w as wild-type control. (D) Hindgut melanization in the p38b;p38a double mutant. (E) The melanization rate in the hindgut of the p38b;p38a larvae cultured in standard food with or without a mixture of antibiotics as in A, or in sterilized food. (F) The melanization rate in the hindgut of the p38b;p38a larvae cultured in sterilized food with or without adding 10⁵/mL P. aeruginosa as in B. (G and H) Melanization in the hindgut can be induced by dextran sodium sulfate (DSS) or SDS with the combination of PAO1R3.

Fig. 2.

The p38-deficient flies are more sensitive to septic injury. (A–C) The survival rates of infected adult flies of p38a (p38a¹³), p38b (p38b^156A), and p38c (p38c⁷) mutants, and wild-type flies (y w). (D–F) The survival rates of infected adult flies of p38b;p38a (p38b^156A; p38a¹³) mutants, p38b;p38a mutants with overexpression of UAS-p38b driven by da-Gal4, and wild-type flies (y w). The data were obtained by using groups of 30 female or male adults, aged 2 to 4 d, pricked with E. cloacae or L. monocytogenes or rolled in B. bassiana spores. Six replicates were used for the determination of the survival rates. Error bars represent SEM in each group. p38a compared with y w, P < 0.02; p38b and p38b;p38 compared with y w, P < 0.002 and 0.0001, respectively.

Fig. 3.

Delayed induction of Hsp genes and Hsf activity in p38 mutants. Relative mRNA levels of Hsp26 (A) and Hsp27 (B) in wild-type, p38a, p38b, and p38b;p38a mutants were shown with qRT-PCR normalized with the internal control rp49. Data from three independent experiments are expressed as mean ± SEM; p38b;p38a compared with y w, P < 0.005. The third instar larval polytene chromosomes from wild-type (C) and p38b;p38a double mutants (D) were stained for DNA (DAPI, blue) and Hsf (red) 1 h after septic infection. (E) Hsf trimerization was shown with Western blotting after native gel electrophoresis. (F–H) Hsf (hsf⁴) and Hsp27 (hsp27^KG03935) mutants are more susceptible to septic infection of E. cloacae (F) and L. monocytogenes (G), and natural infection of B. bassiana (H). The susceptibility of Hsf and Hsp27 mutants can be rescued by introduction of a Hsf transgene (hsf⁴;hsf^+/+) and overexpression of Hsp27 (Hsp27^KG03935;da > hsp27), respectively. Forty males, aged 2 to 4 d were tested for each group as one experiment. Data from three independent experiments are expressed as mean ± SEM; Hsf and Hsp27 mutants compared with y w, P < 0.0001 and 0.001, respectively.

Fig. 4.

p38 attenuates JNK expression and activity. (A) Wild-type, p38a, p38b, and p38b;p38a mutant flies were infected with E. cloacae for a time period, as indicated. JNK activity determined by immunoblotting with phospho-JNK antibodies. (B) The relative intensity of phospho-JNK (P-JNK) in wild-type, p38a, p38b, and p38b;p38a mutants was measured by Western blotting bands of three independent experiments, with pixel information obtained using Kodak Molecular Imaging Software Version 4.0. (C) The rate of hindgut melanization and (D) rate of survival to adult of the wild-type, p38a, p38b, and p38b;p38a mutants cultured in standard food containing 0.3% SDS with or without the JNK inhibitor SP600125 (n > 200). Data in B represent mean (± SEM) fold changes of P-JNK compared with y w injured with PBS alone; p38b versus y w, P < 0.01; p38b;p38a versus y w, P < 0.001.

Fig. 5.

A canonical MAP3K-MKK cascade is required for p38 activation by septic infection, but Toll and Imd are dispensable for this pathway. (A) Phospho-p38 in wild-type flies was detected at different time points after septic injury with E. cloacae and L. monocytogenes. (B) Phosphorylation of p38 is unchanged in Imd, Tl, and Imd;spz double mutants (loss-of-function) as well as in Tl^10b mutants (a gain-of-function mutation of Tl) following infection with both Gram-positive L. monocytogenes and Gram-negative E. cloacae. (C) Toll homologs or licorne (lic, a MKK3/6 homolog), TAK1, ASK1, Mekk1, and Slpr were knocked down with dsRNAs and the L. monocytogenes- or E. cloacae-induced phophorylation of p38 in S2 cells are shown. Knockdown of Tak1, ASK1, and Mekk1 had no significant effect on bacteria-induced p38 phosphorylation, and lic and Slpr are required for p38 activation. Phosphorylation was assayed by Western blotting using phospho-specific antibodies of p38. α-Tubulin is shown as a loading control. All Western blots have been repeated at least twice and all showed the same tendency.