Plasmin complexed with C3(b) and Sbi or Efb degrades C3 and C3b within the complex.

<p>Plasmin degraded C3/C3b within the Sbi:plasmin:C3/C3b or Efb:plasmin:C3/C3b complexes (lane 1 and 4) and C3/C3b cleavage products appeared (marked with arrows). Bacterial proteins were immobilized (equimolar) and plasminogen was added together with C3 (A) or C3b (B). The activator SAK was applied and C3/C3b cleavage was followed by Western blot analysis using anti-C3-HRP (Fab). The mobility of the α (α ´) and β chains of C3/C3b and the cleavage products are indicated by arrows. CRASP-5 and HSA had no effect on cleavage (lane 7 and 8). Data shown are representative of three independent experiments. (C) Proposed schematic model of the C3 cleavage products generated by plasmin modified after <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047638#pone.0047638-Seya1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047638#pone.0047638-Nagasawa1" target="_blank">[32]</a>. The C3 dg cleavage product is not recognized by the C3-antibodies used in this study.</p

Plasminogen is recruited by Sbi and Efb.

<p>(A) Plasminogen bound to Sbi and Efb (lane 1 and 2). The accuracy of the recombinant staphylococcal proteins was confirmed as both, Sbi and Efb, bound to C3 (lane 3 and 4) and Sbi also bound IgG. Recombinant Sbi or Efb was separated by SDS-PAGE and either blotted to a membrane (lane 1–6) or silver stained (lane 7–8). Membranes were incubated with biotinylated PLG (PLG_b), C3, or HRP- coupled IgG and bound proteins were detected as indicated. (B) Binding of plasminogen to Sbi or Efb was confirmed by ELISA. Bacterial proteins (equimolar) were immobilized and PLG_b was applied and detected using streptavidin-HRP. Plasminogen binding to Sbi and Efb was comparable to plasminogen binding to the borrelial protein CRASP-5. Plasminogen did not bind to HSA or the plate (buffer). Data represent mean values ± standard deviations from three independent experiments. (C) Plasminogen was recruited to Sbi and Efb. Non-labeled plasminogen was applied to immobilized bacterial proteins. Bound proteins were eluted, separated by SDS-PAGE and plasminogen (92 kDa) was detected by Western blot analysis. CRASP-5 bound plasminogen in contrast to HSA and buffer. A representative experiment out of three is shown.</p

Characterization of the Plasminogen interactions with Sbi and Efb-C.

<p>(A+B) Plasminogen (PLG) bound to the fragments 3–4 of Sbi (A) and the C-terminus of Efb (B). Sbi 3–4 or Efb-C was immobilized and increasing amounts of plasminogen were added. Bound plasminogen was detected with specific antisera. Plasminogen concentrations showing saturated binding are marked by arrows. (C+D) Plasminogen (solid lines) associated with Sbi 3–4 (C) or Efb-C (D) was determined by surface plasmon resonance. Sbi 3–4 or Efb-C was immobilized and plasminogen was applied in fluid phase. Binding of plasminogen to Sbi 3–4 or Efb-C was inhibited by εACA (dashed line). (E+F) Plasminogen (black columns) and C3 (grey columns) bound simultaneously to Sbi 3–4 (E) and Efb-C (F). Sbi 3–4 or Efb-C was immobilized and C3 and plasminogen were added either alone or together (200 nM C3:200 nM PLG  = 1∶1 for EfbC, 200 nM C3:400 nM PLG = 1∶2 for Sbi3-4). Representative experiments out of three independent experiments are shown.</p

Plasmin mediated cleavage of C3 is enhanced by Sbi and Efb.

<p>(A) In the presence of Sbi and Efb (lane 1 and 2) the C3 α-chain decreased and C3 cleavage products (indicated by arrows) appeared. CRASP-5 did not show this effect. C3 was incubated with SAK-activated plasmin (PL_SAK) in the presence of Sbi, Efb, CRASP-5, or HSA. Samples were separated by SDS-PAGE and C3 cleavage products were visualized using Western blot analysis. (B) A similar assay was performed with uPa as activator and this time the fragments Sbi 1–2, Sbi 3–4, and Efb-C were assayed. Sbi 3–4 (lane 5 and 6) and Efb-C (lane 8 and 9) enhanced the plasmin mediated C3 cleavage as seen by the appearance of small cleavage products. The IgG-binding Sbi 1–2 (mobility of 17 kDa) did not affect the C3 cleavage pattern (lane 2 and 3). Polyclonal C3 antiserum was used for detection. (C) In the presence of Sbi 3–4 and Efb-C the plasmin mediated C3 proteolysis was enhanced as measured by ELISA. C3 was immobilized and plasmin (activated by uPa or SAK) together with Sbi 3–4 or Efb-C were added. C3 deposition was detected after 3 h with C3a antiserum. CRASP-5 and HSA did not influence plasmin cleavage of C3. (D) Sbi 3–4 and Efb-C significantly enhanced C3 degradation by plasmin in a dose dependent manner. The C3 amount after incubation with plasmin was set to 100%. Data in C and D are mean values of three independent experiments; error bars indicate standard deviations. *<i>p</i><0.05; **<i>p</i><0.01 and ***<i>p</i><0.001.</p

Plasmin degrades C3a.

<p>(A) C3a was completely degraded by plasmin; plasminogen had a weak degrading effect. <i>S. aureus</i> was incubated with C3a in the presence of plasmin (PLG+SAK) (lane 4) or plasminogen alone (lane 6). The supernatants were analyzed by SDS-PAGE and Western blot analysis. (B) Plasmin increased the survival of <i>S. aureus</i> to 85% and plasminogen alone to 50%. In parallel <i>S. aureus</i> treated with C3a and or plasmin(ogen) was cultivated overnight on LB agar plates. CFUs were counted and the survival without C3a was set to 100% (white columns). Data represent mean values ± standard deviations from three independent experiments. ***<i>p</i><0.001.</p

Plasminogen bound to Sbi or Efb is converted to plasmin.

<p>(A) Plasmin (PL) bound to Sbi, Efb, and to the borrelial CRASP-5 converted a chromogenic substrate as followed by measuring absorbance at 405 nm after 24 h. Equimolar amounts of bacterial proteins were immobilized and incubated with plasminogen (PLG). After washing, the activator uPA was applied together with the chromogenic substrate S-2251. Human serum albumin (HSA) had no effect. (B+C) Plasmin generation of plasminogen bound to Sbi (B) and Efb (C) by uPa or staphylokinase (SAK). The plasmin activators uPa and SAK or no activator (w/o) were applied to plasminogen bound to Sbi or Efb together with the chromogenic substrate. Conversion of the chromogenic substrate was determined at various time points. Data represent mean values ± standard deviations from three independent experiments.</p

Plasmin cleavage of C3b is enhanced by Sbi and Efb.

<p>(A) Sbi 3–4 (lane 5–6) and Efb-C (lane 8–9) enhanced cleavage of C3b by plasmin, as seen by the accumulation of C3b cleavage products. Sbi 1–2 did not influence C3b degradation (lane 2–3). C3b was cleaved by plasmin in the presence of Sbi 1–2, Sbi 3–4, and Efb-C. (B+C) Sbi 3–4 and Efb-C enhanced the anti-opsonic activity of plasmin. C3b was deposited on <i>S. aureus</i> and Sbi 1–2, Sbi 3–4, Efb-C, or HSA was incubated in the absence or presence of plasmin. Remaining C3b fragments on <i>S. aureus</i> were measured by flow-cytometry. The C3b amount in the absence of plasmin was set to 100%. Data represent mean values ± standard deviations of five independent experiments. *<i>p</i><0.05. (C) In parallel the supernatants were separated by SDS-PAGE and analyzed by Western blotting using anti-C3-HRP (Fab). Sbi 1–2 did not influence the amount of C3b-fragments after plasmin degradation (lane 1). Sbi 3–4 (lane 2) and Efb-C (lane 4) increased the plasmin degradation and more C3b cleavage products were detected.</p

Clinical <i>Photorhabdus</i> isolates are able to survive exposure to higher temperatures than most non-clinical isolates.

<p>The optical density achieved by representative strains after overnight growth in static conditions (at 28°C in LB medium) after prior 18 h exposure to a range of temperatures. A range of clinical (N. American and Australian) and non-clinical (European) strains of <i>P</i>. <i>asymbiotica (Pa)</i> were tested, and the well-studied <i>P</i>. <i>luminescens</i> strain (<i>Pl</i>^TT01) was included for comparison. Green stars and red diamonds indicate thermal tolerance and intolerance respectively. <i>Pa</i> strain designations are indicated as superscripts.</p

A schematic summarising some key differences in metabolism at 37°C compared to 28°C, centred on glutamate/asparagine metabolism and the TCA cycle.

<p>This model is predicted by integrating data from the RNA-seq, proteomics and phenotype microarray studies. Intermediates (boxes) and pathways (arrows) predicted to be down regulated at 37°C are in red while those up regulated are in green. Data suggests TCA cycle intermediates (back boxes) would be relatively isolated from glutamate/asparagine metabolism and could be maintained via the conversion of L-serine into citrate via pyruvate. Black arrows indicate certain potential enzyme pathways that are present and predicted to be unchanged at 37°C. The data suggests a central role for imported peptides and amino acids in metabolism at 37°C. Opp/Dpp represent oligo- and di-peptide importers, TCT represents tricarboxylic acid and PEP is Phosphoenolpyruvate.</p

The genus <i>Photorhabdus</i> contains three predominant species.

<p>A stylized representation of a previous six gene MLST phylogeny (<i>adk</i>, <i>ghd</i>, <i>mdk</i>, <i>ndh</i>, <i>pgm</i> and <i>recA</i>) of <i>Photorhabdus</i> (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144937#pone.0144937.ref005" target="_blank">5</a>]) is shown. The grey areas indicate species that consist of multiple strains, the majority of which are unable to grow above 34°C, with only a few <i>P</i>. <i>luminescens</i> strains capable of growth at temperatures up to 37°C. Example strains are <i>P</i>. <i>luminescens</i>^TT01 and <i>P</i>. <i>temperata</i>^K122. The clinical strains adapted to 37°C are boxed. The stars and circles indicate the potential historical timing of temperature adaptation, which could have occurred ancestrally (star) or independently (circles) in different geographical isolates.</p