TG-dependent incorporation of monodansylcadaverine (MDC) or biotin pentylamine into drosocrystallin recombinants.

<p>(A) Wild-type drosocrystallin (WT), the KR mutant (KR), or bovine serum albumin (BSA) was incubated with chitin, and each fraction (Fr) was analyzed by SDS-PAGE in 10% slab gels (left panel). The intensity of each fraction relative to that of the input was summed as the bound fraction. Bars indicate the mean and standard deviations of experiments performed in triplicate (right panel). BSA was used as a negative control. Open bars, unbound fraction; closed bars, bound fraction. (B) Wild-type drosocrystallin (WT) or the KR mutant (KR) was incubated with MDC in the presence of TG, and analyzed by SDS-PAGE in 10% slab gels. The proteins were stained with Coomassie brilliant blue (CBB), and the MDC-incorporated protein was detected by the emission intensity of the dansyl group. Data are representative of three independent experiments (left panel). The relative emission intensity of each fraction compared to that of CBB-stained protein was calculated using ImageJ software (right panel). (C) Wild-type drosocrystallin (WT) or the KR mutant (KR) was incubated with or without biotin pentylamine (BPA) in the presence of TG, and subjected to SDS-PAGE in 10% slab gels. Incorporation of BPA was detected with horseradish peroxidase-conjugated streptavidin. Loaded recombinant proteins were detected by Western blotting with a horseradish peroxidase-conjugated anti-6× His tag antibody. Data are representative of at least three independent experiments.</p

TG-dependent polymerization of drosocrystallin <i>in vitro</i> and <i>in vivo</i>.

<p><b>(</b>A) Wild-type drosocrystallin (WT) or the KR mutant (KR) was incubated with TG, and subjected to SDS-PAGE in 10% TGX FastCast gels (Bio-Rad Laboratories). These recombinants were detected by Western blotting with a horseradish peroxidase-conjugated anti-6 × His tag antibody. Monodansylcadaverine (MDC) was used as an inhibitor of protein-protein crosslinking. Data are representative of at least three independent experiments. <b>(</b>B) Gut extracts from systemic <i>TG-</i>RNAi flies (<i>Da>TG IR</i>) and their counterparts (<i>Da>+</i>) were subjected to SDS-PAGE in 12% slab gels. Native drosocrystallin in the extracts was detected by Western blotting with an anti-drosocrystallin antibody (Anti-Dcy). Asterisks indicate unknown cross-reacted proteins. Data are representative of three independent experiments.</p

TG-dependent protection against <i>P</i>. <i>entomophila</i> infection in the gut.

<p>(A) Survival analysis of gut-specific <i>TG-</i>RNAi flies (<i>NP1>TG IR</i>) and their counterparts (<i>NP1>+</i>) upon oral infection with <i>P</i>. <i>entomophila</i> (<i>Pe</i>) or <i>Ecc15</i>. Statistical analysis was performed using a log-rank test. At least 50 flies were used. N.S., not significant. (B) Cell-death was quantified by propidium iodide staining. Results represent the percentage of dead cells (propidium iodide-positive nuclei) in the midguts of flies infected for 4 h with <i>P</i>. <i>entomophila</i> (<i>Pe</i>). Results represent the mean of 10 independent experiments. Statistical analysis was performed by one-way analysis of variance followed by Bonferroni correction for multiple comparisons to evaluate the pairwise difference. UC, unchallenged. (C) A schematic model of the TG-mediated peritrophic matrix formation. TG crosslinks drosocrystallin (Dcy) on the peritrophic matrix (PM). Crosslinked drosocrystallin is not digested by AprA, and the crosslinked drosocrystallin strengthens the peritrophic matrix to function as a physical barrier against exotoxins of pathogenic microbes.</p

Covalently crosslinked drosocrystallin fibers are resistant to proteolytic digestion and trap a pore-forming exotoxin, monalysin.

<p>(A) A schematic of the assay for the fiber formation of drosocrystallin. Briefly, wild-type drosocrystallin (Dcy) was incubated on cover glass in the presence or absence of TG and Ca²⁺ or EDTA for 1 h. The culture supernatant from <i>P</i>. <i>entomophila</i> (<i>Pe</i> sup) containing protease AprA was added on the cover glass with or without monalysin (Mnl). After incubation, proteins on the cover glass were fixed with paraformaldehyde and detected by immunofluorescence. (B) Wild-type drosocrystallin was incubated on coverslip glass and observed by immunofluorescence microscopy. Wild-type drosocrystallin was detected using an anti-His tag antibody and CF488-conjugated secondary antibody (green). Representative data from at least five experiments are shown. (C) Wild-type drosocrystallin was incubated with or without TG on coverslip glass in the presence of Ca²⁺, and then 2-fold serial dilutions of the culture supernatant from <i>P</i>. <i>entomophila</i> (<i>Pe</i> sup) were added. The concentration of the culture supernatant is indicated. Bars indicate the mean and standard deviations of the mean gray values from four independent experiments (upper panel). Lower panels show representative data of drosocrystallin fibers formed in the presence or absence of TG and <i>Pe</i> sup. Wild-type drosocrystallin was detected by anti-His tag antibody and CF568-conjugated secondary antibody (red). (D) Wild-type drosocrystallin was incubated with or without TG on coverslip glass in the presence of Ca²⁺, and then wild-type monalysin was added in the presence (<i>Pe</i> sup +) or absence (<i>Pe</i> sup −) of the culture supernatant from <i>P</i>. <i>entomophila</i>. Wild-type drosocrystallin was detected by anti-His tag antibody and CF568-conjugated secondary antibody (red), and wild-type monalysin was detected by anti-histidine affinity tag (HAT) antibody and CF488-conjugated secondary antibody (green). One representative experiment from at least five independent experiments is shown.</p

Polymerized drosocrystallin protects against AprA.

<p>(A) Wild-type drosocrystallin (WT) was incubated at 37°C for 30 min with or without TG, and then the culture supernatant from <i>P</i>. <i>entomophila</i> (<i>Pe</i>) was added, and the mixture was subjected to SDS-PAGE in 10% TGX FastCast gels. Open arrowhead, the monomeric recombinant; closed arrowhead, the crosslinked recombinant. Data are representative of at least three independent experiments. (B) Wild-type drosocrystallin was subjected to SDS-PAGE in 10% slab gels and detected by Western blotting after incubating with each fraction obtained by gel filtration of the culture supernatant from <i>P</i>. <i>entomophila</i>. (C) Fraction No. 27 from the gel filtration was subjected to SDS-PAGE in 15% slab gels, and proteases in this fraction were identified by liquid chromatography tandem mass spectroscopy analysis. (D) Wild-type drosocrystallin was incubated with purified AprA at 25°C or 37°C and analyzed by SDS-PAGE in 10% slab gels, and detected by Western blotting using anti-6 × His tag antibody (upper panel). Western blotting data are representative of four independent experiments. The relative intensity of each band compared to that of the untreated protein (0 min) was calculated using ImageJ software (lower panel). (E) Wild-type drosocrystallin was incubated with the culture supernatant from <i>P</i>. <i>entomophila</i> (<i>Pe</i>) or the <i>AprA</i>-knockout strain (<i>Pe</i>^{<i>ΔaprA</i>}), analyzed by SDS-PAGE in 10% slab gels, and detected by Western blotting using anti-6 × His tag antibody. Western blotting data are representative of three independent experiments (upper panel). The relative intensity of each band compared to that of the untreated protein (0 min) was calculated using ImageJ software (lower panel).</p

Sequence identities among CCP, VWF and SP domains of TtC2/Bf-1 and TtC2/Bf-2.

<p>CCP, complement control protein domain; VWF, von Willebrand factor domain; SP, serine protease domain.</p

Mg²⁺-dependent activation of TtC2/Bf-1 and TtC2/Bf-2.

<p>Microbes were incubated with dialyzed HDP at 37°C for 30 min in the presence (CaCl₂ = 10 mM, MgCl₂ = 50 mM) or absence of divalent cations. Microbes were removed by centrifugation, and 20 µl of the supernatants were subjected to Western blotting, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036783#pone-0036783-g002" target="_blank">Figure 2A</a>. Each experiment was performed at least three times. Representative blots are shown. A cross-reacting protein with 68 kDa against the anti-TtC2/Bf-2-SP antibody by Western blotting is shown by (*).</p

Effects of plasma lectins on the deposition of TtC3b on microbes.

<p>Microbes were incubated with HDP, and the deposition of TtC3b on the microbial surface was analyzed by flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036783#pone-0036783-g007" target="_blank">Figure 7A</a> (blue line). The dotted line indicates autonomous fluorescence. The red line indicates flow-cytometric data obtained by pretreatment of HDP with the anti-TtCRP-1 (<i>Tachypleus tridentatus</i> C-reactive protein-1) antibody (A), anti-TL-5A (Tachylectin-5A) antibody (B), or anti-TL-1 (Tachylectin-1) antibody (C) at 10 µg/ml for 1 hour at 4°C. Mean fluorescence intensity of <i>E. coli</i>: (A) AF, 7.2; HDP, 266; HDP+antibody, 276; (B) AF, 6.83; HDP, 110; HDP+antibody, 155; (C) AF, 8.96; HDP, 201; HDP+antibody, 79. MFI of <i>S. aureus</i>: (A) AF, 7.2; HDP, 66; HDP+antibody, 26; (B) AF, 7.2; HDP, 66; HDP+antibody, 18; (C) AF, 3.6; HDP, 42; HDP+antibody, 25. Mean fluorescence intensity of <i>P. pastoris</i>: (A) AF, 5.8; HDP, 96; HDP+antibody, 160; (B) AF, 5.8; HDP, 96; HDP+antibody, 150; (C) AF, 6.04; HDP, 50; HDP+antibody, 16. In (A), (B), and (C), one representative experiment out of three repeats is shown.</p

Effects of Mg²⁺ and Ca²⁺ on the deposition of TtC3b on microbes.

<p>(A) Microbes were incubated with HDP. The deposition of TtC3b on the microbial surfaces was analyzed by flow cytometry using the Alexa 488-conjugated anti-TtC3 antibody (blue line). The dotted line indicates autonomous fluorescence (AF) of microbes. (B) Dialyzed HDP was incubated with various microbes, and the deposition of TtC3b on the microbial surfaces was analyzed in the presence or absence of cations, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036783#pone-0036783-g007" target="_blank">Figure 7A</a>. Blue line, dialyzed HDP; red line, dialyzed HDP+10 mM Ca²⁺; green line, dialyzed HDP+50 mM Mg²⁺; orange line, dialyzed HDP+10 mM Ca²⁺ and 50 mM Mg²⁺. Mean fluorescence intensity of <i>E. coli</i>: AF, 5.9; HDP, 250; dialyzed HDP, 266; dialyzed HDP+Ca²⁺, 236; dialyzed HDP+Mg²⁺, 286; dialyzed HDP+Ca²⁺ and Mg²⁺, 376. MFI of <i>S. aureus</i>: AF, 6.8; HDP, 116; dialyzed HDP, 21; dialyzed HDP+Ca²⁺, 62; dialyzed HDP+Mg²⁺, 220; dialyzed HDP+Ca²⁺ and Mg²⁺, 231. MFI of <i>P. pastoris</i>: AF, 6.1; HDP, 57; dialyzed HDP, 20; dialyzed HDP+Ca²⁺, 28; dialyzed HDP+Mg²⁺, 60; dialyzed HDP+Ca²⁺ and Mg²⁺, 139. In (A) and (B), one representative experiment out of three repeats is shown.</p

Proposed mechanism for deposition of TtC3b on the surfaces of microbes.

<p>On the surface of Gram-negative bacteria, the LPS-sensitive protease factor C is activated through the interaction with LPS. Activated factor C then acts as a C3 convertase, and the resulting TtC3b is deposited. On the Gram-positive bacteria, Ca²⁺-dependent lectins such as TtCRP-1 (<i>Tachypleus tridentatus</i> C-reactive protein-1) and TL-5A (Tachylectin-5A) recruit the complex (C3-B), TtC3-TtC2/Bf-1, or TtC3-TtC2/Bf-2. The second C3 convertase (C3b-Bb), TtC3b-TtC2/Bf-1b or TtC3b-TtC2/Bf-2b complex, may be formed by an unidentified protease on the surfaces of Gram-positive bacteria and fungi. TL-1 (Tachylectin-1) and possibly TPL-1 (<i>Tachypleus</i> plasma lectin-1) also promote TtC3b deposition on the three types of microbes, especially on fungi.</p