A Model of Bacterial Intestinal Infections in Drosophila melanogaster

Serratia marcescens is an entomopathogenic bacterium that opportunistically infects a wide range of hosts, including humans. In a model of septic injury, if directly introduced into the body cavity of Drosophila, this pathogen is insensitive to the host's systemic immune response and kills flies in a day. We find that S. marcescens resistance to the Drosophila immune deficiency (imd)-mediated humoral response requires the bacterial lipopolysaccharide O-antigen. If ingested by Drosophila, bacteria cross the gut and penetrate the body cavity. During this passage, the bacteria can be observed within the cells of the intestinal epithelium. In such an oral infection model, the flies succumb to infection only after 6 days. We demonstrate that two complementary host defense mechanisms act together against such food-borne infection: an antimicrobial response in the intestine that is regulated by the imd pathway and phagocytosis by hemocytes of bacteria that have escaped into the hemolymph. Interestingly, bacteria present in the hemolymph elicit a systemic immune response only when phagocytosis is blocked. Our observations support a model wherein peptidoglycan fragments released during bacterial growth activate the imd pathway and do not back a proposed role for phagocytosis in the immune activation of the fat body. Thanks to the genetic tools available in both host and pathogen, the molecular dissection of the interactions between S. marcescens and Drosophila will provide a useful paradigm for deciphering intestinal pathogenesis

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

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

Munc13-4*rab27 complex tethers secretory lysosomes at the plasma membrane

Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTL) are critical for the immune response against virus infections or transformed cells. They kill target cells via polarized exocytosis of lytic proteins from secretory lysosomes (SL). Rab27a and munc13-4 interact directly and are required for target cell killing. How they cooperate in the intricate degranulation process is not known. We identified critical residues in munc13-4 for rab27 interaction and tested binding mutants in several complementation assays. In a rat mast cell line we replaced endogenous munc13-4 with ectopically expressed munc13-4 constructs. Unlike wild type munc13-4, binding mutants fail to rescue β-hexosaminidase secretion. In accord, expression of binding mutants in CTL of Familial Hemophagocytic Lymphohistiocytosis type 3 patients, does not rescue CD107 appearance on the plasma membrane. Total Internal Reflection Fluorescence (TIRF) imaging shows that munc13-4*rab27a restricts motility of SL in the subapical cytoplasm. We propose that rab27*munc13-4 tethers SL to the plasma membrane, a requirement for formation of a cognate SNARE complex for fusion

Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex

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The soluble PRRs GNBP1, PGRP-SA, and PGRP-SD are unlikely to function as opsonins. A-C.

<p>Flies were either preinjected with latex beads (LXB) or nontreated and then submitted to a septic injury with <i>M. luteus</i> (A), <i>E. faecalis</i> (B) and <i>S. aureus</i> (C). LXB injection has a strong effect on the survival of <i>PGRP-SA^seml</i> and <i>GNBP1^osi</i> as well as <i>PGRP-SD^Δ3</i> mutants after <i>M</i>. <i>luteus</i> infection (A). The results were less pronounced for <i>PGRP-SA^seml</i> and <i>Dif</i> when we used <i>E. faecalis</i> (B) and <i>S. aureus</i> (C) as pathogens. (<b>A.</b> wt <i>vs</i>. wt + LXB : p = 0.01; <i>seml vs. seml</i> + LXB : p = 0.0005; <i>PGRP-SD vs. PGRP-SD</i> + LXB : p = 0.0004; <i>osi vs. osi</i> + LXB : p = 0.0001. <b>B.</b> wt <i>vs</i>. wt + LXB : p = 0.0005; <i>key vs. key</i> + LXB : p<0.0001; <i>seml vs. seml</i> + LXB : p = 0.26; <i>PGRP-SD vs. PGRP-SD</i> + LXB : p<0.0001; <i>osi vs. osi</i> + LXB : p = 0.001; <i>Dif vs. Dif</i> + LXB : p = 0.13. <b>C.</b> wt <i>vs</i>. wt + LXB : p = 0.004; <i>key vs. key</i> + LXB : p = 0.006; <i>seml vs. seml</i> + LXB : p = 0.49; <i>PGRP-SD vs. PGRP-SD</i> + LXB : p<0.0001; <i>osi vs. osi</i> + LXB : p<0.0001.) The survival rate expressed in percentage is shown. <i>PGRP-SD^Δ3</i> (<i>PGRP-SD</i>); <i>GNBP1^osi</i> (<i>osi</i>). <b>D, E.</b> Quantification of in vivo phagocytosis of Alexa-fluor labeled <i>S. aureus</i>. 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).</p

Overexpression of <i>Defensin</i> or Toll pathway can enhance host resistance to some Gram-positive bacteria.

<p>Flies were either preinjected with latex beads (LXB) or nontreated and then submitted to an immune challenge with <i>M. luteus</i> (A), <i>E. faecalis</i> (B and D) and <i>S. aureus</i> (C and E). LXB-injected flies in which <i>Defensin</i> was constitutively overexpressed (<i>UAS</i>-<i>Defensin</i>) using <i>hsp</i>-<i>GAL4</i> driver (<i>hsp</i>) were resistant to a <i>M. luteus</i> challenge (A). A protective effect was not observed for <i>E. faecalis</i> or <i>S. aureus</i> infections (B-C). LXB-injected flies in which Toll (UAS-<i>Toll^10b</i>) was constitutively active were resistant to <i>E. faecalis</i>, but not to <i>S. aureus</i> (D-E). (<b>A.</b> wt <i>vs</i>. wt + LXB : p = 0.0014; <i>Dif vs. Dif</i> + LXB : p<0.0001; <i>seml vs. seml</i> + LXB : p = 0.002; <i>hsp</i>*<i>UAS</i>-<i>Defensin vs</i>. <i>hsp</i>*<i>UAS</i>-<i>Defensin</i> + LXB : p = 0.71; <b>wt + LXB </b><b><i>vs</i></b><b>. </b><b><i>hsp*UAS-Defensin</i></b><b> + LXB : p = 0.03</b>. <b>B.</b> wt <i>vs</i>. wt + LXB : p<0.0001; <i>Dif vs. Dif</i> + LXB : p<0.0001; <i>hsp*UAS-Defensin vs. hsp*UAS-Defensin</i> + LXB : p<0.0001; <b>wt + LXB </b><b><i>vs. hsp*UAS-Defensin</i></b><b> + LXB : p = 0.80</b>. <b>C.</b> wt <i>vs</i>. wt + LXB : p = 0.02; <i>seml vs. seml</i> + LXB : p = 0.09; <i>hsp*UAS-Defensin vs. hsp*UAS-Defensin</i> + LXB : p = 0.02; <b>wt + LXB </b><b><i>vs. hsp*UAS-Defensin</i></b><b> + LXB : p = 0.55</b>. <b>D.. </b><i>hsp*UAS- Toll^10b vs. hsp* UAS- Toll^10b</i> + LXB : p = 0.25; <b>wt + LXB </b><b><i>vs. hsp* UAS-Toll^10B</i></b><b> + LXB : p<0.0001</b>. <b>E.. </b><i>hsp*UAS- Toll^10b vs. hsp* UAS- Toll^10b</i> + LXB : p = 0.0015; <b>wt + LXB </b><b><i>vs. hsp* UAS-Toll^10B</i></b><b> + LXB : p = 0.19</b>). The survival rate expressed in percentage is shown.</p

Phagocytosis in adult flies restricted Gram-positive bacterial infection independent of antimicrobial peptides induction.

<p><b>A–C</b>. Flies were either preinjected with latex beads (LXB) or nontreated and then submitted to a septic injury with <i>M. luteus</i> (<b>A</b>), <i>E. faecalis</i> (<b>B</b>) and <i>S. aureus</i> (<b>C</b>). LXB pre-injected flies were significantly more susceptible to infection than noninjected wild type flies. (<b>A.</b> wt <i>vs</i>. wt + LXB : p<0.0001; <i>key vs. key</i> + LXB : p = 0.0003; <i>Dif vs. Dif</i> + LXB : p<0.0001. <b>B.</b> wt <i>vs</i>. wt + LXB : p = 0.02; <i>key vs. key</i> + LXB : p = 0.01; <i>Dif vs. Dif</i> + LXB : p = 0.08. <b>C.</b> wt <i>vs</i>. wt + LXB : p<0.0001; <i>key vs. key</i> + LXB : p = 0.0004; <i>seml vs. seml</i> + LXB : p = 0.02.) The survival rate expressed in percentage is shown. <i>wt</i>, wild-type controls. <i>Dif</i>, and <i>PGRP-SA^seml</i> (<i>seml</i>) are mutants of the <i>Toll</i> pathway, whereas <i>key</i> (<i>kenny</i>) is a mutant of the <i>imd</i> pathway. Susceptibility of LXB-injected flies to <i>M. luteus,</i> although sometimes less pronounced (<i>e.g., </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014743#pone-0014743-g002" target="_blank">Fig. 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014743#pone-0014743-g003" target="_blank">3</a>) was always statistically significant. <b>D-G.</b> LXB-preinjection did not impair <i>Drosomycin</i> or <i>Defensin</i> 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. <i>Drosomycin</i> mRNA levels were monitored 24 hr after a challenge with <i>M. luteus</i> at 25 °C (D) and 48 hr after a challenge with <i>E. faecalis</i> or <i>S. aureus</i> at 20 °C (E and F). <i>Defensin</i> RNA levels were monitored 6 hr after a challenge with <i>M. luteus</i> at 25 °C (G). For <i>E. faecalis</i> or <i>S. aureus</i> the experiments were performed at a lower temperature because these bacteria are highly virulent, killing the flies rapidly. Error bars represent standard deviation (SD). <b>H.</b> Gram-positive bacteria did not induce <i>Defensin</i> 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. <i>Defensin</i> RNA levels were monitored 6 hr after a clean injury (CI), a challenge with <i>M. luteus</i> or <i>E. coli</i> at 25 °C. Error bars represent SD.</p

The phagocytic receptor Eater plays an important role in the <i>Drosophila</i> host defense against <i>E. faecalis</i> and <i>S. aureus</i> but not <i>M. luteus</i>.

<p><b>A.</b> Flies were either preinjected with latex beads (LXB) or nontreated and then submitted to a septic injury with <i>M. luteus</i> (A), <i>E. faecalis</i> (B) and <i>S. aureus</i> (C). <i>Eater</i> mutant flies succumbed rapidly to a challenge with <i>S. aureus</i> and <i>E. faecalis</i> but not with <i>M. luteus</i>. (A. wt <i>vs</i>. wt + LXB : p = 0.0176; wt <i>vs. eater</i> : p = 0.0214. B. wt <i>vs. eater</i> : p = 0.0003. C. wt <i>vs. Dif</i> : p = 0.13; wt <i>vs. eater</i> : p<0.0001; wt vs. seml : p<0.0001). The survival rate expressed in percentage is shown. <b>B-E.</b> 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>B, C.</b> RNAi knock down of Eater in S2 cells affects phagocytosis and binding of FITC-<i>E. faecalis</i> and <i>S. aureus.. </i><b>D, E.</b> RNAi knock down of Eater in S2 and Kc167 cells does not affect phagocytosis (D) and binding (E) of <i>M. luteus.. </i><b>F.</b> 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 <i>eater</i>, or in untreated Kc167 cells. Control knockdown had no effect on <i>eater</i> expression. A nonspecific band at around 70 kDa (open arrow) served as an internal loading control.</p