Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6 (3), e14743

Relative Roles of the Cellular and Humoral Responses in the Drosophila Host Defense Against Three Gram-Positive Bacterial Infections

Affiliations

Relative Roles of the Cellular and Humoral Responses in the Drosophila Host Defense Against Three Gram-Positive Bacterial Infections

Nadine T Nehme et al. PLoS One.

Abstract

Background: Two NF-kappaB signaling pathways, Toll and immune deficiency (imd), are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense.

Methodology/principal findings: In order to dissect the relative importance of humoral and cellular defenses after septic injury with three different gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus), we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival--independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response.

Conclusions/significance: Our results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Phagocytosis in adult flies restricted Gram-positive bacterial infection independent of antimicrobial peptides induction.
A–C. Flies were either preinjected with latex beads (LXB) or nontreated and then submitted to a septic injury with M. luteus (A), E. faecalis (B) and S. aureus (C). LXB pre-injected flies were significantly more susceptible to infection than noninjected wild type flies. (A. wt vs. wt + LXB : p<0.0001; key vs. key + LXB : p = 0.0003; Dif vs. Dif + LXB : p<0.0001. B. wt vs. wt + LXB : p = 0.02; key vs. key + LXB : p = 0.01; Dif vs. Dif + LXB : p = 0.08. C. wt vs. wt + LXB : p<0.0001; key vs. key + LXB : p = 0.0004; seml vs. seml + LXB : p = 0.02.) The survival rate expressed in percentage is shown. wt, wild-type controls. Dif, and PGRP-SAseml (seml) are mutants of the Toll pathway, whereas key (kenny) is a mutant of the imd pathway. Susceptibility of LXB-injected flies to M. luteus, although sometimes less pronounced (e.g., Fig. 2, 3) was always statistically significant. D-G. LXB-preinjection did not impair Drosomycin or Defensin induction. Expression of the AMP gene was determined by real-time PCR. Results are expressed as a percentage of the induction observed in wt control flies. Drosomycin mRNA levels were monitored 24 hr after a challenge with M. luteus at 25 °C (D) and 48 hr after a challenge with E. faecalis or S. aureus at 20 °C (E and F). Defensin RNA levels were monitored 6 hr after a challenge with M. luteus at 25 °C (G). For E. faecalis or S. aureus the experiments were performed at a lower temperature because these bacteria are highly virulent, killing the flies rapidly. Error bars represent standard deviation (SD). H. Gram-positive bacteria did not induce Defensin expression. Expression of the AMP gene was determined by real-time PCR. Results are expressed as a percentage of the induction observed in wt control flies. Defensin RNA levels were monitored 6 hr after a clean injury (CI), a challenge with M. luteus or E. coli at 25 °C. Error bars represent SD.
Figure 2
Figure 2. The soluble PRRs GNBP1, PGRP-SA, and PGRP-SD are unlikely to function as opsonins. A-C.
Flies were either preinjected with latex beads (LXB) or nontreated and then submitted to a septic injury with M. luteus (A), E. faecalis (B) and S. aureus (C). LXB injection has a strong effect on the survival of PGRP-SAseml and GNBP1osi as well as PGRP-SDΔ3 mutants after M. luteus infection (A). The results were less pronounced for PGRP-SAseml and Dif when we used E. faecalis (B) and S. aureus (C) as pathogens. (A. wt vs. wt + LXB : p = 0.01; seml vs. seml + LXB : p = 0.0005; PGRP-SD vs. PGRP-SD + LXB : p = 0.0004; osi vs. osi + LXB : p = 0.0001. B. wt vs. wt + LXB : p = 0.0005; key vs. key + LXB : p<0.0001; seml vs. seml + LXB : p = 0.26; PGRP-SD vs. PGRP-SD + LXB : p<0.0001; osi vs. osi + LXB : p = 0.001; Dif vs. Dif + LXB : p = 0.13. C. wt vs. wt + LXB : p = 0.004; key vs. key + LXB : p = 0.006; seml vs. seml + LXB : p = 0.49; PGRP-SD vs. PGRP-SD + LXB : p<0.0001; osi vs. osi + LXB : p<0.0001.) The survival rate expressed in percentage is shown. PGRP-SDΔ3 (PGRP-SD); GNBP1osi (osi). D, E. Quantification of in vivo phagocytosis of Alexa-fluor labeled S. aureus. Each dot corresponds to the amount of fluorescence signal in the abdomen of one individual fly (a phagocytic index was derived by multiplying the area with the mean intensity of the fluorescence signal measured). Pair wise P-values are indicated by black bars. A horizontal red bar indicates the average phagocytic index for each group. No significant differences were observed between mutants and their corresponding wild-type controls (Oregon-R, w iso and DD1).
Figure 3
Figure 3. The phagocytic receptor Eater plays an important role in the Drosophila host defense against E. faecalis and S. aureus but not M. luteus.
A. Flies were either preinjected with latex beads (LXB) or nontreated and then submitted to a septic injury with M. luteus (A), E. faecalis (B) and S. aureus (C). Eater mutant flies succumbed rapidly to a challenge with S. aureus and E. faecalis but not with M. luteus. (A. wt vs. wt + LXB : p = 0.0176; wt vs. eater : p = 0.0214. B. wt vs. eater : p = 0.0003. C. wt vs. Dif : p = 0.13; wt vs. eater : p<0.0001; wt vs. seml : p<0.0001). The survival rate expressed in percentage is shown. B-E. FACS analysis of phagocytosis and cell surface binding of heat-killed fluorescent bacteria to hemocyte-derived cell lines. To assess phagocytosis, extracellular fluorescence was quenched by trypan blue. The amount of phagocytosis (or cell surface binding) was quantified as percentage of cells phagocytosing (or binding) multiplied by mean fluorescence intensity. Error bars represent SD between four samples. * indicates : significantly different (p<0.01). B, C. RNAi knock down of Eater in S2 cells affects phagocytosis and binding of FITC-E. faecalis and S. aureus. D, E. RNAi knock down of Eater in S2 and Kc167 cells does not affect phagocytosis (D) and binding (E) of M. luteus. F. Eater protein is not detectable after RNAi knockdown in S2 cells and in Kc167 cells: Western Blot of cell extracts corresponding to 84 µg of protein separated on a 10% SDS-gel. A 128 kDa band corresponding to the Eater protein (black arrow) was present in S2 cells, whereas it was undetectable in S2 cells after RNAi knockdown of eater, or in untreated Kc167 cells. Control knockdown had no effect on eater expression. A nonspecific band at around 70 kDa (open arrow) served as an internal loading control.
Figure 4
Figure 4. Overexpression of Defensin or Toll pathway can enhance host resistance to some Gram-positive bacteria.
Flies were either preinjected with latex beads (LXB) or nontreated and then submitted to an immune challenge with M. luteus (A), E. faecalis (B and D) and S. aureus (C and E). LXB-injected flies in which Defensin was constitutively overexpressed (UAS-Defensin) using hsp-GAL4 driver (hsp) were resistant to a M. luteus challenge (A). A protective effect was not observed for E. faecalis or S. aureus infections (B-C). LXB-injected flies in which Toll (UAS-Toll10b) was constitutively active were resistant to E. faecalis, but not to S. aureus (D-E). (A. wt vs. wt + LXB : p = 0.0014; Dif vs. Dif + LXB : p<0.0001; seml vs. seml + LXB : p = 0.002; hsp*UAS-Defensin vs. hsp*UAS-Defensin + LXB : p = 0.71; wt + LXB vs . hsp*UAS-Defensin + LXB : p = 0.03. B. wt vs. wt + LXB : p<0.0001; Dif vs. Dif + LXB : p<0.0001; hsp*UAS-Defensin vs. hsp*UAS-Defensin + LXB : p<0.0001; wt + LXB vs. hsp*UAS-Defensin + LXB : p = 0.80. C. wt vs. wt + LXB : p = 0.02; seml vs. seml + LXB : p = 0.09; hsp*UAS-Defensin vs. hsp*UAS-Defensin + LXB : p = 0.02; wt + LXB vs. hsp*UAS-Defensin + LXB : p = 0.55. D. hsp*UAS- Toll10b vs. hsp* UAS- Toll10b + LXB : p = 0.25; wt + LXB vs. hsp* UAS-Toll10B + LXB : p<0.0001. E. hsp*UAS- Toll10b vs. hsp* UAS- Toll10b + LXB : p = 0.0015; wt + LXB vs. hsp* UAS-Toll10B + LXB : p = 0.19). The survival rate expressed in percentage is shown.

Similar articles

See all similar articles

Cited by 36 articles

See all "Cited by" articles

References

    1. Lemaitre B, Hoffmann J. The Host Defense of Drosophila melanogaster. Annu Rev Immunol. 2007;25:697–743. - PubMed
    1. Ferrandon D, Imler JL, Hetru C, Hoffmann JA. The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat Rev Immunol. 2007;7:862–874. - PubMed
    1. Royet J, Dziarski R. Peptidoglycan recognition proteins: pleiotropic sensors and effectors of antimicrobial defences. Nat Rev Microbiol. 2007;5:264–277. - PubMed
    1. Leone P, Bischoff V, Kellenberger C, Hetru C, Royet J, et al. Crystal structure of Drosophila PGRP-SD suggests binding to DAP-type but not lysine-type peptidoglycan. Mol Immunol. 2008;45:2521–2530. - PubMed
    1. Wang L, Gilbert RJ, Atilano ML, Filipe SR, Gay NJ, et al. Peptidoglycan recognition protein-SD provides versatility of receptor formation in Drosophila immunity. Proc Natl Acad Sci U S A. 2008;105:11881–11886. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback