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

Influence of the structure of cornstarch dispersions on kinetics of aroma release

International audienceThis study deals with the impact of food structure and texture on aroma release. This was done with cornstarch dispersions with constant concentrations in starch but differing in their structures. The structure parameters of the cornstarch dispersions were varied by changing the shearing conditions during the pasting process. Linalool and isoamyl acetate were chosen as reference aroma compounds. Linalool is known to form complexes with amylose while isoamyl acetate does not. The release of aroma compounds from starch dispersions under stirring was studied at 2 temperatures by discrete sampling of the headspace. Aroma release curves were modeled and the kinetic and thermodynamic parameters were extracted. The release of linalool seemed to be governed mainly by the interactions (complexation) with starch. In contrast, the interactions between starch and isoamyl acetate were feebler, and the release of this aroma compound was governed by the structure of the starch dispersion. The observed interactions were better established at 20 °C than at 32 °C

The Drosophila Toll Pathway Controls but Does Not Clear Candida glabrata Infections

Item does not contain fulltextThe pathogenicity of Candida glabrata to patients remains poorly understood for lack of convenient animal models to screen large numbers of mutants for altered virulence. In this study, we explore the minihost model Drosophila melanogaster from the dual perspective of host and pathogen. As in vertebrates, wild-type flies contain C. glabrata systemic infections yet are unable to kill the injected yeasts. As for other fungal infections in Drosophila, the Toll pathway restrains C. glabrata proliferation. Persistent C. glabrata yeasts in wild-type flies do not appear to be able to take shelter in hemocytes from the action of the Toll pathway, the effectors of which remain to be identified. Toll pathway mutant flies succumb to injected C. glabrata. In this immunosuppressed background, cellular defenses provide a residual level of protection. Although both the Gram-negative binding protein 3 pattern recognition receptor and the Persephone protease-dependent detection pathway are required for Toll pathway activation by C. glabrata, only GNBP3, and not psh mutants, are susceptible to the infection. Both Candida albicans and C. glabrata are restrained by the Toll pathway, yet the comparative study of phenoloxidase activation reveals a differential activity of the Toll pathway against these two fungal pathogens. Finally, we establish that the high-osmolarity glycerol pathway and yapsins are required for virulence of C. glabrata in this model. Unexpectedly, yapsins do not appear to be required to counteract the cellular immune response but are needed for the colonization of the wild-type host

Effect of high hydrostatic pressure on the structure of the soluble protein fraction in Porphyridium cruentum extracts

International audienceHigh hydrostatic pressure (HHP) treatments are trending as “green” stabilization and extraction process. The extraction of B-phycoerythrin from microalgae is getting more and more interest due to its numerous potentialities in foods, cosmetics and medicine. Thus, the effects of high pressure on the structural characteristics of B-phycoerythrin extracted from Porphyridium cruentum are explored in this paper.Spectrophotometric methods allowed to measure B-phycoerythrin content (UV–visible) and gave an indication on the protein structure (fluorescence). Micro-DSC analysis and electrophoresis complemented this structural investigation for all the protein fractions of P. cruentum extracts.Applying high hydrostatic pressure treatments up to 300 MPa during 5 min had no significant effect on B-phycoerythrin content and structure in P. cruentum extracts. Nevertheless, conformational changes of the protein are suggested by fluorescence yield decrease at 400 MPa, and protein aggregation of B-phycoerythrin, observed by Micro-DSC and electrophoresis, occurred at 500 MPa.Industrial relevanceThe HHP process is an emerging technology for the microbiological stability of various food matrices, including the proteins of microalgae as natural colorant. The target pressure to stabilize is around 400 MPa. High hydrostatic pressure can be used on P. cruentum extracts up to 300 MPa without any change in protein structure, as the threshold of protein aggregation is observed at 400 MPa. The observed changes of the proteins structure after applying HHP above 400 MPa can have a strong impact at macroscopic scale on the food matrices: increase of turbidity, change of texture, stability of emulsion

Effect of high hydrostatic pressure on extraction of B-phycoerythrin from Porphyridium cruentum: Use of confocal microscopy and image processing

The aim of the study was to extract B-phycoerythrin from Porphyridium cruentum while preserving its structure. The high hydrostatic pressure treatments were chosen as extraction technology. Different methods have been used to observe the effects of the treatment: spectrophotometry and confocal laser scanning microscopy followed by image processing analysis. Image processing led to the generation of masks used for the identification of three clusters: intra, extra and intercellular. All methods showed that high hydrostatic pressure treatments between 50 and 500 MPa failed to extract B-phycoerythrin from Porphyridium cruentum cells. The fluorescence emission was negatively impacted by high hydrostatic pressure treatment from 400 MPa for the extracellular and intercellular cluster and from 500 MPa for the intercellular cluster. These results suggest that high pressure treatments could induce the denaturation of B-phycoerythrin in all clusters but with different intensities depending on the cluster

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