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