Adipose tissue is the first colonization site of <i>Leptospira interrogans</i> in subcutaneously infected hamsters

<div><p>Leptospirosis is one of the most widespread zoonoses in the world, and its most severe form in humans, “Weil’s disease,” may lead to jaundice, hemorrhage, renal failure, pulmonary hemorrhage syndrome, and sometimes,fatal multiple organ failure. Although the mechanisms underlying jaundice in leptospirosis have been gradually unraveled, the pathophysiology and distribution of leptospires during the early stage of infection are not well understood. Therefore, we investigated the hamster leptospirosis model, which is the accepted animal model of human Weil’s disease, by using an <i>in vivo</i> imaging system to observe the whole bodies of animals infected with <i>Leptospira interrogans</i> and to identify the colonization and growth sites of the leptospires during the early phase of infection. Hamsters, infected subcutaneously with 10⁴ bioluminescent leptospires, were analyzed by <i>in vivo</i> imaging, organ culture, and microscopy. The results showed that the luminescence from the leptospires spread through each hamster’s body sequentially. The luminescence was first detected at the injection site only, and finally spread to the central abdomen, in the liver area. Additionally, the luminescence observed in the adipose tissue was the earliest detectable compared with the other organs, indicating that the leptospires colonized the adipose tissue at the early stage of leptospirosis. Adipose tissue cultures of the leptospires became positive earlier than the blood cultures. Microscopic analysis revealed that the leptospires colonized the inner walls of the blood vessels in the adipose tissue. In conclusion, this is the first study to report that adipose tissue is an important colonization site for leptospires, as demonstrated by microscopy and culture analyses of adipose tissue in the hamster model of Weil’s disease.</p></div

<i>Leptospira</i> distribution in skin and subcutaneous tissue.

<p>Representative light field (A, C) and fluorescence images (B, D) of the skin and subcutaneous tissue (A, B) or adipose tissue (C, D) around the injection sites of M1307 collected from infected hamsters at phase 4. Fluorescence images (B, D) showing cell nuclei stained with DAPI (blue), autofluorescence of the skin and subcutaneous tissue (green, not shown in panel D), and leptospires stained with rabbit polyclonal antiserum and Cy5-conjugated anti-rabbit monoclonal antibody (red). The framed area in (B) is enlarged at the upper right. Scale bars: 100 μm (A, B), 500 μm (C, D).</p

Bioluminescence dissemination of <i>Leptospira</i> in hamsters.

<p>(A) The survival rate of Golden Syrian hamsters (n = 8) infected subcutaneously with 10⁴ <i>L</i>. <i>interrogans</i> strain M1307 into the right inguinal region, and representative ventral view photographic images tracking the hamster infections on different days post-infection. Images depict photographs overlaid with color representations of luminescence intensity, measured in photons/second/cm²/sr as indicated on the scales, where red is the most intense (3×10⁵) and purple is the least intense (3×10⁴). (B,C) Average luminescence intensities in each ROI of injection site (B) and abdominal center (C) at different days post-infection. Data are expressed as the means ± SEM of total flux in photons/second in each ROI in eight infected hamsters (●) and two uninfected controls (◦). <i>p</i> values (*<i>p</i><0.05), between groups.</p

Bioluminescence changes in hamster organs.

<p>Representative bioluminescence images (ventral view) from M1307-infected hamsters at each phase. Images represent subcutaneous tissues after skin incision and organs after laparotomy, as well as <i>ex vivo</i> organs (blood plus liver and kidney cross sections). The scale is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172973#pone.0172973.g001" target="_blank">Fig 1</a>.</p

Transmission electron microscopy of adipose tissue blood vessels.

<p>Representative transmission electron microscope images of subcutaneous adipose tissue blood vessels around the injection sites of <i>Leptospira</i>-infected hamsters at phase 4. The framed area in (A) is enlarged in (B). The scale bars represent 5 μm (A) and 1 μm (B). The arrowheads point to <i>Leptospira</i> and the arrows show the red blood cells.</p

Kawasaki Disease-Specific Molecules in the Sera Are Linked to Microbe-Associated Molecular Patterns in the Biofilms

<div><p>Background</p><p>Kawasaki disease (KD) is a systemic vasculitis of unknown etiology. The innate immune system is involved in its pathophysiology at the acute phase. We have recently established a novel murine model of KD coronary arteritis by oral administration of a synthetic microbe-associated molecular pattern (MAMP). On the hypothesis that specific MAMPs exist in KD sera, we have searched them to identify KD-specific molecules and to assess the pathogenesis.</p><p>Methods</p><p>We performed liquid chromatography-mass spectrometry (LC-MS) analysis of fractionated serum samples from 117 patients with KD and 106 controls. Microbiological and LC-MS evaluation of biofilm samples were also performed.</p><p>Results</p><p>KD samples elicited proinflammatory cytokine responses from human coronary artery endothelial cells (HCAECs). By LC-MS analysis of KD serum samples collected at 3 different periods, we detected a variety of KD-specific molecules in the lipophilic fractions that showed distinct m/z and MS/MS fragmentation patterns in each cluster. Serum KD-specific molecules showed m/z and MS/MS fragmentation patterns almost identical to those of MAMPs obtained from the biofilms formed <i>in vitro</i> (common MAMPs from <i>Bacillus cereus</i>, <i>Yersinia pseudotuberculosis</i> and <i>Staphylococcus aureus</i>) at the 1^st study period, and from the biofilms formed <i>in vivo</i> (common MAMPs from <i>Bacillus cereus</i>, <i>Bacillus subtilis/Bacillus cereus/Yersinia pseudotuberculosis</i> and <i>Staphylococcus aureus</i>) at the 2^nd and 3^rd periods. The biofilm extracts from <i>Bacillus cereus</i>, <i>Bacillus subtilis</i>, <i>Yersinia pseudotuberculosis</i> and <i>Staphylococcus aureus</i> also induced proinflammatory cytokines by HCAECs. By the experiments with IgG affinity chromatography, some of these serum KD-specific molecules bound to IgG.</p><p>Conclusions</p><p>We herein conclude that serum KD-specific molecules were mostly derived from biofilms and possessed molecular structures common to MAMPs from <i>Bacillus cereus, Bacillus subtilis</i>, <i>Yersinia pseudotuberculosis and Staphylococcus aureus</i>. Discovery of these KD-specific molecules might offer novel insight into the diagnosis and management of KD as well as its pathogenesis.</p></div

LC-MS chromatograms and MS/MS fragmentation patterns of serum KD-specific molecules at the 1^st study period.

<p>A–E: Each left upper panel: LC-MS chromatograms of KD-specific molecules (A: m/z 1531.8, B: m/z 1414.3, C: m/z 790.9, D: m/z 779.8, and E: m/z 695.0), Each left lower panel: LC-MS chromatograms of biofilm extracts (or initial culture supernatants) from <i>Y. pseudotuberculosis</i> and <i>S. aureus</i> (A) and <i>B. cereus</i> (B–E). U: Total ion current chromatograms, M: Extracted-ion chromatograms at m/z 1500–1600 (A), m/z 1400–1500 (B), m/z 700–800 (C and D), and m/z 600–700 (E), L: Extracted-ion chromatograms at m/z 1531.8 (A), m/z 1414.3 (B), m/z 790.9 (C), m/z 779.8 (D), and m/z 695.0 (E). Arrows indicate peaks of target molecules. Each right upper panel: MS/MS fragmentation patterns of KD-specific molecules (A: m/z 1531.8, B: m/z 1414.3, C: m/z 790.9, D: m/z 779.8, and E: m/z 695.0), Each right lower panel: MS/MS fragmentation patterns of biofilm extracts (or initial culture supernatants) from <i>Y. pseudotuberculosis</i> and <i>S. aureus</i> (A) and <i>B. cereus</i> (B–E). As for the molecule at m/z 779.8, cellobiose lipid shows a MS/MS fragmentation pattern similar to that of KD sera (D, right lowest panel). The intensity is shown by relative abundance. F: The detection rates of each molecule in NC (N = 5), DC (N = 41) or KD (N = 43) sera are shown. Twenty-one (48.8%) of 43 are positive at m/z 1531.8 (a), 13 (30.2%) of 43 at m/z 1414.3 (b), 17 (39.5%) of 43 at m/z 790.9 (c), 4 (9.3%) of 43 at m/z 779.8 (d) and 15 (34.9%) of 43 at m/z 695.0 (e) when the intensity above 1×10³ is considered to be significant. The overall detection rate was 76.7% (33 of 43). <i>P</i><0.0001 (a, b, c and e); <i>P</i> = 0.0364 (d) (Fisher's exact test).</p

The detection rates of serum KD-specific MAMPs at each study.

<p>At the 2^nd and 3^rd studies, both <i>in vivo</i> biofilms and serum samples were simultaneously collected. <i>In vivo</i> biofilms samples were searched for MAMPs common to those in serum samples by LC-MS and MS/MS analyses. SBA: simultaneous biofilm analysis, *: overall % positive (overall positive numbers). DC samples at the 1^st (<i>n</i> = 41), 2^nd (<i>n</i> = 30) and 3^rd (<i>n</i> = 30) study periods were all negative for all KD MAMPs. The detection rates of KD-specific serum MAMPs between KD samples (<i>n</i> = 43) and DC samples (<i>n</i> = 41) at the 1^st study showed statistically significant differences at m/z 1531.8, m/z 1414.3, m/z 790.9, m/z 695.0, and overall (<i>P</i><0.0001), but not at m/z 779.8 (<i>P</i> = 0.1164) by Fisher's exact test. The detection rates between SBA period KD samples (<i>n</i> = 12) and DC samples (<i>n</i> = 30) at the 2^nd study showed statistically significant differences at m/z 906.8 (<i>P</i> = 0.0044), m/z 695.0 (<i>P</i> = 0.0002) and overall (<i>P</i><0.0001), but not at m/z 1171.4 (<i>P</i> = 0.0767) and m/z 1169.4 (<i>P</i> = 0.0767). The detection rates between SBA period KD samples (<i>n</i> = 11) and DC samples (<i>n</i> = 30) at the 3^rd study showed statistically significant differences at all 3 molecules and overall (<i>P</i><0.0001).</p><p>The detection rates of serum KD-specific MAMPs at each study.</p

KD <i>in vivo</i> biofilms contain MAMPs common to serum KD-specific molecules (3^rd study period).

<p>Three serum KD-specific molecules (m/z 667.4, 619.4 and 409.3) at the 3^rd study showed the same m/z with MS/MS fragmentation patterns similar to MAMPs from <i>in vivo</i> biofilm extracts (Table S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113054#pone.0113054.s001" target="_blank">File S1</a>) and <i>in vitro</i> bacterial biofilm extracts. A: The molecule at m/z 667.4 was common in KD serum, tongue biofilm extracts and <i>in vitro</i> biofilm extracts from <i>S. aureus</i>. B: The molecule at m/z 619.4 was common in KD serum, teeth and tongue biofilm extracts, and <i>in vitro</i> biofilm extracts from <i>B. subtilis</i>, <i>B. cereus</i> and <i>Y. pseudotuberculosis</i>. C: The molecule at m/z 409.3 was common in KD serum, and teeth and tongue biofilm extracts.</p

LC-MS chromatograms of IgG sepharose-binding molecules.

<p>A. Representative LC-MS chromatograms of a IgG sepharose-binding molecule (m/z 1414.3) are shown in a KD patient and a DC control. TIC: Total ion current chromatograms, XIC: Extracted-ion chromatograms at m/z 1400–1500, and extracted-ion chromatograms at m/z 1414.3. (1) Human polyclonal IgG-conjugated sepharose 6 Fast (2) Inactivated CNBr Sepharose 4B control column. B. Binding of a KD-specific molecule to various affinity columns: Columns used are described in ONLINE METHODS. +: The binding quantities of a KD-specific molecule analyzed by LC-MS were equal or larger than those to human polyclonal IgG column, ±: smaller than 20% of those to human polyclonal IgG column, -: no binding. We performed the experiments 3 times.</p