<p>Bone marrow (A-B and E-F) or peritoneally-recruited (C-D and G-J) neutrophils were isola... more <p>Bone marrow (A-B and E-F) or peritoneally-recruited (C-D and G-J) neutrophils were isolated from <i>B</i>. <i>burgdorferi</i>-infected and mock-infected C57BL/6 (A, C, E, G) and C3H/HeN (B, D, F, H-J) mice at 4 weeks post-infection (5–6 weeks of sustained hyperglycemia), and coincubated with complement-opsonized <i>E</i>. <i>coli</i> (A-D) or <i>B</i>. <i>burgdorferi</i> (E-J). <i>E</i>. <i>coli</i> survival (A-D) and <i>B</i>. <i>burgdorferi</i> uptake by neutrophils (E-H) were measured by comparing numbers of <i>E</i>. <i>coli</i> CFUs and intact <i>B</i>. <i>burgdorferi</i> following neutrophil co-incubation with values for complement-opsonized input bacteria. (I-J) <i>B</i>. <i>burgdorferi</i> killing was measured by LIVE-DEAD staining. Panel I shows sample images for bacteria incubated in the absence (no PMN) and presence (+PMN) of neutrophils. Panel J shows quantification of <i>B</i>. <i>burgdorferi</i> killing. Summary values: mean ±SEM. N = 4–6 mice/group. Experimental groups: No neutrophil (PMN) control: opsonized bacteria incubated in absence of neutrophils, normoglycemic mock-infected (Vh), hyperglycemic mock-infected (STZ), normoglycemic <i>B</i>. <i>burgdorferi</i>-infected (Vh+<i>Bb</i>), hyperglycemic <i>B</i>. <i>burgdorferi</i>-infected (STZ+<i>Bb</i>). Statistical analysis: Two-way ANOVA with Holm-Sidak post-tests. * indicates p<0.05 vs. Vehicle; # indicates p<0.05 vs. normoglycemic <i>B</i>. <i>burgdorferi</i>-infected (Vh+<i>Bb</i>) mice; † indicates p<0.05 vs. hyperglycemic mock-infected mice (STZ); & indicates p<0.05 vs no PMN control (panel J only).</p
<p>(A, B) Mean ±SEM numbers of circulating neutrophils in blood of C57BL/6 (A) and C3H/HeN ... more <p>(A, B) Mean ±SEM numbers of circulating neutrophils in blood of C57BL/6 (A) and C3H/HeN mice (B). Neutrophils were enumerated by particle counting with a Coulter counter in blood drawn by cardiac puncture from <i>B</i>. <i>burgdorferi</i>-infected and mock-infected mice at 4 weeks post-infection (5–6 weeks of sustained hyperglycemia). N = 6–9 mice/group. Experimental groups: Normoglycemic mock-infected (Vh), hyperglycemic mock-infected (STZ), normoglycemic <i>B</i>. <i>burgdorferi</i>-infected (Vh+<i>Bb</i>), hyperglycemic <i>B</i>. <i>burgdorferi</i>-infected (STZ+<i>Bb</i>). Statistical analysis: Two-way ANOVA with Holm-Sidak post-tests. * indicates p<0.05 vs. Vehicle; # indicates p<0.05 vs. normoglycemic <i>B</i>. <i>burgdorferi</i>-infected (Vh+<i>Bb</i>) mice.</p
<p>(A-C) Non-fasting blood glucose levels in STZ- and vehicle-treated C57BL/6 (A) and C3H/H... more <p>(A-C) Non-fasting blood glucose levels in STZ- and vehicle-treated C57BL/6 (A) and C3H/HeN (B) mice, and in Akita mice and age-matched wild type C57BL/6 mice (C). (D-F) Body weight of STZ- and vehicle-treated C57BL/6 (D) and C3H/HeN (E) mice, and of Akita mice and age-matched wild type C57BL/6 mice (F). T<sub>0</sub> stands for baseline values (before STZ treatment), T<sub>i</sub> corresponds to time of infection, and T<sub>f</sub> stands for time of sacrifice. N = 17–21 mice per group. Statistical analysis: (A-B, D-E) Two-way ANOVA with Holm-Sidak post-tests. (C, F) Two-tailed t-test. * indicates p<0.05 vs. T<sub>0</sub> within group; † indicates p<0.05 vs. Vehicle within time point. ^ indicates p<0.05 vs wild-type.</p
<p>(A-B) Cardiac cellularity in C57BL/6 (A) and C3H/HeN (B) mice. Experimental groups: norm... more <p>(A-B) Cardiac cellularity in C57BL/6 (A) and C3H/HeN (B) mice. Experimental groups: normoglycemic mock-infected (Vh), hyperglycemic mock-infected (STZ), normoglycemic infected (Vh+<i>Bb</i>), hyperglycemic infected (STZ+<i>Bb</i>). The numbers of nuclei in five 100 mm<sup>2</sup> regions of interest in 2–3 matched H&E-stained sagittal sections per heart were enumerated using a counting grid. Nuclei were counted in each atrium and ventricle and the heart apex, and the majority of tissue included in each region of interest was derived from the myocardium. Summary values are shown for the average numbers of nuclei/section/mouse. Tukey box plots represent the 25–75% range, line and plus symbols (+) correspond to medians and means, respectively, and error bars span minimum to maximum values. N = 11–15 mice per group. Statistics: Two-way ANOVA with Holm-Sidak post-test. * indicates p<0.05 vs mock-infected within vehicle- or STZ-treated groups. (C) Representative H&E-stained sagittal heart sections of C3H/HeN mice. Scale bar: 100 μm. (D) Arthritis scoring in C3H/HeN mice. Scoring of arthritis severity was performed in a blinded fashion by a pathologist using the following scoring system: 0: no pathology, 1: mild pathology, 2: severe pathology. N = 10–11 mice/group. Shown are mean ±SEM severity scores. Statistics: Kruskal-Wallis ANOVA with Dunn’s post-test. * indicates p<0.05 vs. mock-infected within vehicle- or STZ-treated groups. (E) Representative H&E-stained tibiotarsal joint sections of C3H/HeN mice. Scale bars: 100–200 μm.</p
<p>(A, C and E) Percentage of tissues/mouse positive for <i>B</i>. <i>bur... more <p>(A, C and E) Percentage of tissues/mouse positive for <i>B</i>. <i>burgdorferi flaB</i> DNA in infected normoglycemic and hyperglycemic mice at 4 weeks post-infection. Percentage of qPCR-positive tissues/mouse in C57BL/6 (A), C3H/HeN (C), and Akita (E) mice are shown. (B, D and F) Median <i>B</i>. <i>burgdorferi flaB</i> copy number/μg total DNA in indicated tissues and in all tissues combined (ALL) in infected normoglycemic and hyperglycemic mice at 4 weeks post-infection. Shown are individual values and medians (bars) in tissues of C57BL/6 (B), C3H/HeN (D) and Akita (F) mice. Values are plotted on a log scale to facilitate visualization of a large range of values. Dotted lines in each graph indicate the cutoff point (1 <i>flaB</i> copy/μg DNA) below which tissues were considered negative. Statistical analysis: Kruskal-Wallis ANOVA with Dunn’s post-test. For all panels, N = 10–13 mice per experimental group and strain. Fold differences in medians are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158019#pone.0158019.t002" target="_blank">Table 2</a>. * indicates p<0.05 vs. normoglycemic controls.</p
<p><b>(A)</b> Left—Tertiary structure of Tp0751_78A with the lipocalin core col... more <p><b>(A)</b> Left—Tertiary structure of Tp0751_78A with the lipocalin core colored marine and the unique N-terminal helix grey. Key residues of SCR1 and SCR3 are colored orange with side chains shown as sticks and highlighted by an orange box. Right—Nitrophorin 4 (PDB ID 4KOI) shown in the same orientation and color scheme as Tp0751_78A. N- and C-terminal disulfide bonds are shown as sticks and highlighted by gold starbursts. <b>(B)</b> Left—SCR1 and SCR3 sequences from Tp0751 and other lipocalins: Nitrophorin 4 (PDB ID 1KOI), Triabin (PDB ID 1AVG), MPI (metalloproteinase inhibitor, PDB ID 1SMP), and hNGAL (Human neutrophil gelatinase-associated lipocalin, PDB ID 1NGL). Key conserved residues are colored orange. For the motif, H is hydrophobic and P is polar. Right—Sigma-A weighted 2Fo-Fc electron density map contoured at 1.2 sigma around the SCR1 and SCR3 motif Trp127 and Arg226 residues of Tp0751_78A. Dashed lines indicate 2.8 Å hydrogen bonds. (<b>C</b>) Orthogonal views of the Tp0751_78A hydrophobic core (lime green). Residues that stack on the hydrophobic core at the open end are shown as sticks in the right panel with the Trp-Met stack anchoring Loop 7 indicated by the gold arrow. Note the lack of a pocket in the open end.</p
<p><b>(A)</b> Thirteen overlapping Tp0751 synthetic 24-mer peptides were evalua... more <p><b>(A)</b> Thirteen overlapping Tp0751 synthetic 24-mer peptides were evaluated for binding to immobilized fibrinogen, fibronectin, collagen type I, and collagen type IV. Average absorbance readings (600 nm) from three wells are presented with bars indicating standard error and the results are representative of two independent experiments. For statistical analyses, attachment of each ECM-binding Tp0751 peptide (p4, p6, p10, p11 and p10scr1) to each host protein was individually compared to the level of binding individually exhibited by peptides p1, p2, p3, p5, p7, p8, p9, p12, p13, p4scr and p6scr to each ECM component, using the Student’s two-tailed <i>t</i>-test. Tp0751 peptides p4, p6, p11, and exhibited statistically significant levels of binding to all four host proteins (*<i>p</i>≤0.0113). Peptide p10 showed significant binding to fibrinogen, fibronectin, and collagen IV (<i>p</i>≤0.0006) but not to collagen I. <b>(B)</b> Dose-dependent binding assays with Tp0751 peptides p4, p6, p10, p11, p10scr1, and p10scr2 to fibrinogen (Fg—upper panel) and fibronectin (Fn—lower panel). Average absorbance readings (600 nm) from three wells are presented with bars indicating standard error and the results are representative of two independent experiments. Apparent <i>K</i><sub>d</sub> values, calculated using GraphPad Prism, were as follows: p4 = 6.9 ± 0.4 μM (Fg) / 2.4 ± 0.1 μM (Fn); p6 = 10.5 ± 2.0 μM (Fg) / 4.5 ± 0.9 μM (Fn); p10 = 1.6 ± 0.1 μM (Fg) / 1.6 ± 0.2 μM (Fn); p11 = 9.2 ± 1.0 μM (Fg) / 5.8 ± 0.3 μM (Fn); p10scr = 1.5 ± 0.2 μM (Fg) / 1.3 ± 0.1 μM (Fn), and p10scr2 = 11.0 ± 1.3 μM (Fg) / 7.9 ± 0.7 μM (Fn) <b>(C)</b> Peptides p4 (yellow), p6 (purple), p10 (blue/cyan) and p11 (cyan/green) are mapped onto the Tp0751 lipocalin domain. The strand colored in cyan reflects the overlap of p10 and p11. Color coded sequences of peptides p4, p6, p10, p10scr1, and p10scr2 are shown. Arginine residues in the p10 peptides are indicated by underlining.</p
<p><b>(A)</b> Schematic showing the experimental design of the competitive inhi... more <p><b>(A)</b> Schematic showing the experimental design of the competitive inhibition assay. Confluent HUVEC monolayers seeded on artificial basement membrane were preincubated with synthetic Tp0751 peptides (p4, p6, p10, p11, p4scr, p6scr, and p10scr1) to allow for peptide-endothelial cell interactions. HUVECs were then co-incubated with parent (negative control) or Tp0751-expressing <i>B</i>. <i>burgdorferi</i> strains to assess the competitive inhibition capacity of the synthetic Tp0751 peptides. After washing to remove non-adherent bacteria, <i>B</i>. <i>burgdorferi</i> adhesion to HUVECs was quantified using fluorescence microscopy to count the number of adherent bacteria per field of view (FOV) at 400x magnification. <b>(B)</b> Bar graph illustrating the number of adherent <i>B</i>. <i>burgdorferi</i> per FOV. Mean counts ± SEM from ten FOV for each biological replicate are presented with standard error bars. For statistical analyses, attachment by strain <i>Bb</i>-Tp0751 to HUVEC monolayers preincubated with peptides p4, p6, and p10 was compared to the attachment of strain <i>Bb</i>-Tp0751 in the presence of corresponding scrambled peptides, using the Student’s two-tailed <i>t</i>-test. Strain <i>Bb</i>-Tp0751 exhibited statistically significant lower levels of binding to HUVEC monolayers preincubated with peptide p10 (*<i>p</i>≤0.005) when compared to the levels of binding when preincubated with the scrambled version of peptide 10 (p10scr1). <i>Bb</i>-Tp0751 adhered significantly more to endothelia than the parent under untreated conditions and when monolayers were pre-incubated with all other peptides (p˂0.05). The parent and <i>Bb</i>-Tp0751 strains are indicated by clear white bars and striped colored bars, respectively. <b>(C)</b> Line graph representing the effect of increasing concentrations (0 nM, 0.54 nM, 5.45 nM, 54.5 nM, and 545 nM) of p10 and negative control peptide p8 on adherence of Parent and <i>Bb</i>-Tp0751 to HUVECs. Mean counts ± SEM from ten FOV for each biological replicate are presented with standard error bars. Statistical analysis, was performed using the Student’s two-tailed <i>t</i>-test. Strain <i>Bb</i>-Tp0751 exhibited significantly lower levels of binding to HUVEC monolayers when preincubated with ≥ 5.45 nM p10 compared to the levels of binding when preincubated with ≥ 5.45 nM p8 (*<i>p</i>≤0.005). A non-linear regression curve was fitted and the IC50 for p10 inhibition of HUVEC binding by <i>Bb</i>-Tp0751 was calculated (IC50 = 17 nM) using GraphPad Prism data analysis software (San Diego, CA).</p
<p>Left–<i>T</i>. <i>pallidum</i> (purple bacterium) initially inte... more <p>Left–<i>T</i>. <i>pallidum</i> (purple bacterium) initially interacts with ECM components. Inset: Tp0751 (grey surface; dotted line represents a predicted disordered N-terminus embedded in the membrane by a lipid anchor) presented on the outer membrane (OM) mediates attachment to host endothelium via a large surface consisting of p4, p6, p10, and p11 (red surface) forming promiscuous contacts with ECM components. Middle—Defined adhesion between <i>T</i>. <i>pallidum</i> and endothelial cells mediated via a region of Tp0751 (inset) isolated to p10 (blue surface) and a specific, unidentified host cell receptor (Receptor X). Right—Transmigration of <i>T</i>. <i>pallidum</i> during extravasation.</p
<p><b>(A)</b> Schematic depicting Tp0751 expression construct in <i>B<... more <p><b>(A)</b> Schematic depicting Tp0751 expression construct in <i>B</i>. <i>burgdorferi</i> including 1) constitutive <i>flaB</i> promoter (P<sub><i>flaB</i></sub>), 2) Tp0751 lipoprotein localization signal sequence and Tp0751 coding sequence, and 3) a C-terminal 3X-FLAG tag. Inserts were cloned into XhoI and NotI sites of a pCE320 (<i>cp32</i>)-derived shuttle vector (resulting plasmid pCC_3–1), and transformed into a non-infectious, adhesion-attenuated B31-A-derived GFP-expressing parent strain (GCB706). Strain names and details are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005919#ppat.1005919.s004" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005919#ppat.1005919.s005" target="_blank">S2</a> Tables. (<b>B-D</b>) Fluorescence-activated cell sorting (FACS) analysis of Tp0751 localization on the surface of <i>B</i>. <i>burgdorferi</i>. Strains were mock-treated with phosphate based saline buffer or permeabilized with methanol before probing with antibodies to the FLAG tag (part of BBK32 and Tp0751 fusion proteins), or to constitutively expressed periplasmic (non-surface-localized) flagellin B protein (FlaB). (<b>B</b>) Representative histogram depicting fluorescence intensities of indicated mock-treated <i>B</i>. <i>burgdorferi</i> strains probed with anti-FLAG antibodies. The mean ± SEM percentages of bacteria that were FLAG-positive for each strain in all replicates are indicated. (<b>C-D</b>) Relative expression levels per bacterium of FLAG-tagged fusion proteins Tp0751 and BBK32 (<b>C</b>) and FlaB (<b>D</b>) in mock-treated and methanol-permeabilized <i>B</i>. <i>burgdorferi</i>. Mean ± SEM fluorescence intensities for each strain were expressed as a proportion of mean fluorescence intensity for <i>B</i>. <i>burgdorferi</i> expressing BBK32-FLAG. N = 8 independent cultures per strain analyzed in two FACS experiments. Statistical analysis: One-way Kruskal-Wallis ANOVA with Dunn’s post-test. * indicates p<0.05 vs. permeabilized BBK32-FLAG control within treatment (mock or permeabilized).</p
Obesity is a major global public health concern. Immune responses implicated in obesity also cont... more Obesity is a major global public health concern. Immune responses implicated in obesity also control certain infections. We investigated the effects of high-fat diet-induced obesity (DIO) on infection with the Lyme disease bacterium Borrelia burgdorferi in mice. DIO was associated with systemic suppression of neutrophil-and macrophage-based innate immune responses. These included bacterial uptake and cytokine production, and systemic, progressive impairment of bacterial clearance, and increased carditis severity. B. burgdorferi-infected mice fed normal diet also gained weight at the same rate as uninfected mice fed high-fat diet, toll-like receptor 4 deficiency rescued bacterial clearance defects, which greater in female than male mice, and killing of an unrelated bacterium (Escherichia coli) by bone marrow-derived macrophages from obese, B. burgdorferi-infected mice was also affected. Importantly, innate immune suppression increased with infection duration and depended on cooperative and synergistic interactions between DIO and B. burgdorferi infection. Thus, obesity and B. burgdorferi infection cooperatively and progressively suppressed innate immunity in mice.
Syphilis is a chronic disease caused by the bacterium Treponema pallidum subsp. pallidum. Trepone... more Syphilis is a chronic disease caused by the bacterium Treponema pallidum subsp. pallidum. Treponema pallidum disseminates widely throughout the host and extravasates from the vasculature, a process that is at least partially dependent upon the ability of T. pallidum to interact with host extracellular matrix (ECM) components. Defining the molecular basis for the interaction between T. pallidum and the host is complicated by the intractability of T. pallidum to in vitro culturing and genetic manipulation. Correspondingly, few T. pallidum proteins have been identified that interact directly with host components. Of these, Tp0751 (also known as pallilysin) displays a propensity to interact with the ECM, although the underlying mechanism of these interactions remains unknown. Towards establishing the molecular mechanism of Tp0751-host ECM attachment, we first determined the crystal structure of Tp0751 to a resolution of 2.15 Å using selenomethionine phasing. Structural analysis revealed...
Insulin-insufficient type 1 diabetes is associated with attenuated bactericidal function of neutr... more Insulin-insufficient type 1 diabetes is associated with attenuated bactericidal function of neutrophils, which are key mediators of innate immune responses to microbes as well as pathological inflammatory processes. Neutrophils are central to immune responses to the Lyme pathogen Borrelia burgdorferi. The effect of hyperglycemia on host susceptibility to and outcomes of B. burgdorferi infection has not been examined. The present study investigated the impact of sustained obesity-independent hyperglycemia in mice on bacterial clearance, inflammatory pathology and neutrophil responses to B. burgdorferi. Hyperglycemia was associated with reduced arthritis incidence but more widespread tissue colonization and reduced clearance of bacterial DNA in multiple tissues including brain, heart, liver, lung and knee joint. B. burgdorferi uptake and killing were impaired in neutrophils isolated from hyperglycemic mice. Thus, attenuated neutrophil function in insulin-insufficient hyperglycemia was...
Background. Bacterial pathogens causing systemic infections disseminate from the initial infectio... more Background. Bacterial pathogens causing systemic infections disseminate from the initial infection focus to the target organs usually through the blood vasculature. To be able to colonize various organs, bacteria need to adhere to the endothelial cells of the vascular wall, and the adhesion must be strong enough to resist the shear force of the blood flow. Borrelia burgdorferi sensu lato spirochetes, the causative agents of the tick-borne disease Lyme borreliosis, disseminate hematogenously from the tick bite site to the joints, the heart, and the central nervous system of the patient. Methods. We used both wild-type and genetically modified B. burgdorferi s. l. bacteria, recombinant borrelia adhesins, and an array of adhesion assays carried out both under stationary and flow conditions to investigate the molecular mechanisms of borrelial adhesion to human endothelial cells. Results. Borrelia garinii, a member of the B. burgdorferi s. l. complex, adhered to biglycan expressed by human endothelial cells in a flow-tolerant manner. The adhesion was mediated by the decorin-binding protein A (DbpA) and DbpB surface molecules of B. garinii. Conclusions. The proteoglycan biglycan is a receptor molecule for flow-resistant adhesion of the bacterial pathogen B. garinii on human endothelial cells.
Similar to circulating tumour and immune cells, many blood-borne microbes preferentially “home” t... more Similar to circulating tumour and immune cells, many blood-borne microbes preferentially “home” to specific vascular sites and tissues during hematogenous dissemination 1–5. For many pathogens, the “postal codes” and mechanisms responsible for tissue-specific vascular tropism are unknown and have been challenging to unravel. Members of the Lyme disease Borreliella burgdorferi species complex infect a broad range of mammalian tissues and exhibit complex strain-, species- and host-specific tissue tropism patterns. Intravenous perfusion experiments and intravital microscopy studies suggest that heterogeneous tissue tropism properties may depend on tissue-specific differences in host and microbial molecules supporting vascular interaction and extravasation. However, interpreting these studies can be complicated because of the immune-protective moonlighting (multitasking) properties of many B. burgdorferi adhesins. Here, we investigated whether B. burgdorferi vascular interaction propert...
Proceedings of the National Academy of Sciences of the United States of America, Apr 10, 2017
Bacterial dissemination via the cardiovascular system is the most common cause of infection morta... more Bacterial dissemination via the cardiovascular system is the most common cause of infection mortality. A key step in dissemination is bacterial interaction with endothelia lining blood vessels, which is physically challenging because of the shear stress generated by blood flow. Association of host cells such as leukocytes and platelets with endothelia under vascular shear stress requires mechanically specialized interaction mechanisms, including force-strengthened catch bonds. However, the biomechanical mechanisms supporting vascular interactions of most bacterial pathogens are undefined. Fibronectin (Fn), a ubiquitous host molecule targeted by many pathogens, promotes vascular interactions of the Lyme disease spirochete Borrelia burgdorferi Here, we investigated how B. burgdorferi exploits Fn to interact with endothelia under physiological shear stress, using recently developed live cell imaging and particle-tracking methods for studying bacterial-endothelial interaction biomechani...
<p>Bone marrow (A-B and E-F) or peritoneally-recruited (C-D and G-J) neutrophils were isola... more <p>Bone marrow (A-B and E-F) or peritoneally-recruited (C-D and G-J) neutrophils were isolated from <i>B</i>. <i>burgdorferi</i>-infected and mock-infected C57BL/6 (A, C, E, G) and C3H/HeN (B, D, F, H-J) mice at 4 weeks post-infection (5–6 weeks of sustained hyperglycemia), and coincubated with complement-opsonized <i>E</i>. <i>coli</i> (A-D) or <i>B</i>. <i>burgdorferi</i> (E-J). <i>E</i>. <i>coli</i> survival (A-D) and <i>B</i>. <i>burgdorferi</i> uptake by neutrophils (E-H) were measured by comparing numbers of <i>E</i>. <i>coli</i> CFUs and intact <i>B</i>. <i>burgdorferi</i> following neutrophil co-incubation with values for complement-opsonized input bacteria. (I-J) <i>B</i>. <i>burgdorferi</i> killing was measured by LIVE-DEAD staining. Panel I shows sample images for bacteria incubated in the absence (no PMN) and presence (+PMN) of neutrophils. Panel J shows quantification of <i>B</i>. <i>burgdorferi</i> killing. Summary values: mean ±SEM. N = 4–6 mice/group. Experimental groups: No neutrophil (PMN) control: opsonized bacteria incubated in absence of neutrophils, normoglycemic mock-infected (Vh), hyperglycemic mock-infected (STZ), normoglycemic <i>B</i>. <i>burgdorferi</i>-infected (Vh+<i>Bb</i>), hyperglycemic <i>B</i>. <i>burgdorferi</i>-infected (STZ+<i>Bb</i>). Statistical analysis: Two-way ANOVA with Holm-Sidak post-tests. * indicates p<0.05 vs. Vehicle; # indicates p<0.05 vs. normoglycemic <i>B</i>. <i>burgdorferi</i>-infected (Vh+<i>Bb</i>) mice; † indicates p<0.05 vs. hyperglycemic mock-infected mice (STZ); & indicates p<0.05 vs no PMN control (panel J only).</p
<p>(A, B) Mean ±SEM numbers of circulating neutrophils in blood of C57BL/6 (A) and C3H/HeN ... more <p>(A, B) Mean ±SEM numbers of circulating neutrophils in blood of C57BL/6 (A) and C3H/HeN mice (B). Neutrophils were enumerated by particle counting with a Coulter counter in blood drawn by cardiac puncture from <i>B</i>. <i>burgdorferi</i>-infected and mock-infected mice at 4 weeks post-infection (5–6 weeks of sustained hyperglycemia). N = 6–9 mice/group. Experimental groups: Normoglycemic mock-infected (Vh), hyperglycemic mock-infected (STZ), normoglycemic <i>B</i>. <i>burgdorferi</i>-infected (Vh+<i>Bb</i>), hyperglycemic <i>B</i>. <i>burgdorferi</i>-infected (STZ+<i>Bb</i>). Statistical analysis: Two-way ANOVA with Holm-Sidak post-tests. * indicates p<0.05 vs. Vehicle; # indicates p<0.05 vs. normoglycemic <i>B</i>. <i>burgdorferi</i>-infected (Vh+<i>Bb</i>) mice.</p
<p>(A-C) Non-fasting blood glucose levels in STZ- and vehicle-treated C57BL/6 (A) and C3H/H... more <p>(A-C) Non-fasting blood glucose levels in STZ- and vehicle-treated C57BL/6 (A) and C3H/HeN (B) mice, and in Akita mice and age-matched wild type C57BL/6 mice (C). (D-F) Body weight of STZ- and vehicle-treated C57BL/6 (D) and C3H/HeN (E) mice, and of Akita mice and age-matched wild type C57BL/6 mice (F). T<sub>0</sub> stands for baseline values (before STZ treatment), T<sub>i</sub> corresponds to time of infection, and T<sub>f</sub> stands for time of sacrifice. N = 17–21 mice per group. Statistical analysis: (A-B, D-E) Two-way ANOVA with Holm-Sidak post-tests. (C, F) Two-tailed t-test. * indicates p<0.05 vs. T<sub>0</sub> within group; † indicates p<0.05 vs. Vehicle within time point. ^ indicates p<0.05 vs wild-type.</p
<p>(A-B) Cardiac cellularity in C57BL/6 (A) and C3H/HeN (B) mice. Experimental groups: norm... more <p>(A-B) Cardiac cellularity in C57BL/6 (A) and C3H/HeN (B) mice. Experimental groups: normoglycemic mock-infected (Vh), hyperglycemic mock-infected (STZ), normoglycemic infected (Vh+<i>Bb</i>), hyperglycemic infected (STZ+<i>Bb</i>). The numbers of nuclei in five 100 mm<sup>2</sup> regions of interest in 2–3 matched H&E-stained sagittal sections per heart were enumerated using a counting grid. Nuclei were counted in each atrium and ventricle and the heart apex, and the majority of tissue included in each region of interest was derived from the myocardium. Summary values are shown for the average numbers of nuclei/section/mouse. Tukey box plots represent the 25–75% range, line and plus symbols (+) correspond to medians and means, respectively, and error bars span minimum to maximum values. N = 11–15 mice per group. Statistics: Two-way ANOVA with Holm-Sidak post-test. * indicates p<0.05 vs mock-infected within vehicle- or STZ-treated groups. (C) Representative H&E-stained sagittal heart sections of C3H/HeN mice. Scale bar: 100 μm. (D) Arthritis scoring in C3H/HeN mice. Scoring of arthritis severity was performed in a blinded fashion by a pathologist using the following scoring system: 0: no pathology, 1: mild pathology, 2: severe pathology. N = 10–11 mice/group. Shown are mean ±SEM severity scores. Statistics: Kruskal-Wallis ANOVA with Dunn’s post-test. * indicates p<0.05 vs. mock-infected within vehicle- or STZ-treated groups. (E) Representative H&E-stained tibiotarsal joint sections of C3H/HeN mice. Scale bars: 100–200 μm.</p
<p>(A, C and E) Percentage of tissues/mouse positive for <i>B</i>. <i>bur... more <p>(A, C and E) Percentage of tissues/mouse positive for <i>B</i>. <i>burgdorferi flaB</i> DNA in infected normoglycemic and hyperglycemic mice at 4 weeks post-infection. Percentage of qPCR-positive tissues/mouse in C57BL/6 (A), C3H/HeN (C), and Akita (E) mice are shown. (B, D and F) Median <i>B</i>. <i>burgdorferi flaB</i> copy number/μg total DNA in indicated tissues and in all tissues combined (ALL) in infected normoglycemic and hyperglycemic mice at 4 weeks post-infection. Shown are individual values and medians (bars) in tissues of C57BL/6 (B), C3H/HeN (D) and Akita (F) mice. Values are plotted on a log scale to facilitate visualization of a large range of values. Dotted lines in each graph indicate the cutoff point (1 <i>flaB</i> copy/μg DNA) below which tissues were considered negative. Statistical analysis: Kruskal-Wallis ANOVA with Dunn’s post-test. For all panels, N = 10–13 mice per experimental group and strain. Fold differences in medians are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158019#pone.0158019.t002" target="_blank">Table 2</a>. * indicates p<0.05 vs. normoglycemic controls.</p
<p><b>(A)</b> Left—Tertiary structure of Tp0751_78A with the lipocalin core col... more <p><b>(A)</b> Left—Tertiary structure of Tp0751_78A with the lipocalin core colored marine and the unique N-terminal helix grey. Key residues of SCR1 and SCR3 are colored orange with side chains shown as sticks and highlighted by an orange box. Right—Nitrophorin 4 (PDB ID 4KOI) shown in the same orientation and color scheme as Tp0751_78A. N- and C-terminal disulfide bonds are shown as sticks and highlighted by gold starbursts. <b>(B)</b> Left—SCR1 and SCR3 sequences from Tp0751 and other lipocalins: Nitrophorin 4 (PDB ID 1KOI), Triabin (PDB ID 1AVG), MPI (metalloproteinase inhibitor, PDB ID 1SMP), and hNGAL (Human neutrophil gelatinase-associated lipocalin, PDB ID 1NGL). Key conserved residues are colored orange. For the motif, H is hydrophobic and P is polar. Right—Sigma-A weighted 2Fo-Fc electron density map contoured at 1.2 sigma around the SCR1 and SCR3 motif Trp127 and Arg226 residues of Tp0751_78A. Dashed lines indicate 2.8 Å hydrogen bonds. (<b>C</b>) Orthogonal views of the Tp0751_78A hydrophobic core (lime green). Residues that stack on the hydrophobic core at the open end are shown as sticks in the right panel with the Trp-Met stack anchoring Loop 7 indicated by the gold arrow. Note the lack of a pocket in the open end.</p
<p><b>(A)</b> Thirteen overlapping Tp0751 synthetic 24-mer peptides were evalua... more <p><b>(A)</b> Thirteen overlapping Tp0751 synthetic 24-mer peptides were evaluated for binding to immobilized fibrinogen, fibronectin, collagen type I, and collagen type IV. Average absorbance readings (600 nm) from three wells are presented with bars indicating standard error and the results are representative of two independent experiments. For statistical analyses, attachment of each ECM-binding Tp0751 peptide (p4, p6, p10, p11 and p10scr1) to each host protein was individually compared to the level of binding individually exhibited by peptides p1, p2, p3, p5, p7, p8, p9, p12, p13, p4scr and p6scr to each ECM component, using the Student’s two-tailed <i>t</i>-test. Tp0751 peptides p4, p6, p11, and exhibited statistically significant levels of binding to all four host proteins (*<i>p</i>≤0.0113). Peptide p10 showed significant binding to fibrinogen, fibronectin, and collagen IV (<i>p</i>≤0.0006) but not to collagen I. <b>(B)</b> Dose-dependent binding assays with Tp0751 peptides p4, p6, p10, p11, p10scr1, and p10scr2 to fibrinogen (Fg—upper panel) and fibronectin (Fn—lower panel). Average absorbance readings (600 nm) from three wells are presented with bars indicating standard error and the results are representative of two independent experiments. Apparent <i>K</i><sub>d</sub> values, calculated using GraphPad Prism, were as follows: p4 = 6.9 ± 0.4 μM (Fg) / 2.4 ± 0.1 μM (Fn); p6 = 10.5 ± 2.0 μM (Fg) / 4.5 ± 0.9 μM (Fn); p10 = 1.6 ± 0.1 μM (Fg) / 1.6 ± 0.2 μM (Fn); p11 = 9.2 ± 1.0 μM (Fg) / 5.8 ± 0.3 μM (Fn); p10scr = 1.5 ± 0.2 μM (Fg) / 1.3 ± 0.1 μM (Fn), and p10scr2 = 11.0 ± 1.3 μM (Fg) / 7.9 ± 0.7 μM (Fn) <b>(C)</b> Peptides p4 (yellow), p6 (purple), p10 (blue/cyan) and p11 (cyan/green) are mapped onto the Tp0751 lipocalin domain. The strand colored in cyan reflects the overlap of p10 and p11. Color coded sequences of peptides p4, p6, p10, p10scr1, and p10scr2 are shown. Arginine residues in the p10 peptides are indicated by underlining.</p
<p><b>(A)</b> Schematic showing the experimental design of the competitive inhi... more <p><b>(A)</b> Schematic showing the experimental design of the competitive inhibition assay. Confluent HUVEC monolayers seeded on artificial basement membrane were preincubated with synthetic Tp0751 peptides (p4, p6, p10, p11, p4scr, p6scr, and p10scr1) to allow for peptide-endothelial cell interactions. HUVECs were then co-incubated with parent (negative control) or Tp0751-expressing <i>B</i>. <i>burgdorferi</i> strains to assess the competitive inhibition capacity of the synthetic Tp0751 peptides. After washing to remove non-adherent bacteria, <i>B</i>. <i>burgdorferi</i> adhesion to HUVECs was quantified using fluorescence microscopy to count the number of adherent bacteria per field of view (FOV) at 400x magnification. <b>(B)</b> Bar graph illustrating the number of adherent <i>B</i>. <i>burgdorferi</i> per FOV. Mean counts ± SEM from ten FOV for each biological replicate are presented with standard error bars. For statistical analyses, attachment by strain <i>Bb</i>-Tp0751 to HUVEC monolayers preincubated with peptides p4, p6, and p10 was compared to the attachment of strain <i>Bb</i>-Tp0751 in the presence of corresponding scrambled peptides, using the Student’s two-tailed <i>t</i>-test. Strain <i>Bb</i>-Tp0751 exhibited statistically significant lower levels of binding to HUVEC monolayers preincubated with peptide p10 (*<i>p</i>≤0.005) when compared to the levels of binding when preincubated with the scrambled version of peptide 10 (p10scr1). <i>Bb</i>-Tp0751 adhered significantly more to endothelia than the parent under untreated conditions and when monolayers were pre-incubated with all other peptides (p˂0.05). The parent and <i>Bb</i>-Tp0751 strains are indicated by clear white bars and striped colored bars, respectively. <b>(C)</b> Line graph representing the effect of increasing concentrations (0 nM, 0.54 nM, 5.45 nM, 54.5 nM, and 545 nM) of p10 and negative control peptide p8 on adherence of Parent and <i>Bb</i>-Tp0751 to HUVECs. Mean counts ± SEM from ten FOV for each biological replicate are presented with standard error bars. Statistical analysis, was performed using the Student’s two-tailed <i>t</i>-test. Strain <i>Bb</i>-Tp0751 exhibited significantly lower levels of binding to HUVEC monolayers when preincubated with ≥ 5.45 nM p10 compared to the levels of binding when preincubated with ≥ 5.45 nM p8 (*<i>p</i>≤0.005). A non-linear regression curve was fitted and the IC50 for p10 inhibition of HUVEC binding by <i>Bb</i>-Tp0751 was calculated (IC50 = 17 nM) using GraphPad Prism data analysis software (San Diego, CA).</p
<p>Left–<i>T</i>. <i>pallidum</i> (purple bacterium) initially inte... more <p>Left–<i>T</i>. <i>pallidum</i> (purple bacterium) initially interacts with ECM components. Inset: Tp0751 (grey surface; dotted line represents a predicted disordered N-terminus embedded in the membrane by a lipid anchor) presented on the outer membrane (OM) mediates attachment to host endothelium via a large surface consisting of p4, p6, p10, and p11 (red surface) forming promiscuous contacts with ECM components. Middle—Defined adhesion between <i>T</i>. <i>pallidum</i> and endothelial cells mediated via a region of Tp0751 (inset) isolated to p10 (blue surface) and a specific, unidentified host cell receptor (Receptor X). Right—Transmigration of <i>T</i>. <i>pallidum</i> during extravasation.</p
<p><b>(A)</b> Schematic depicting Tp0751 expression construct in <i>B<... more <p><b>(A)</b> Schematic depicting Tp0751 expression construct in <i>B</i>. <i>burgdorferi</i> including 1) constitutive <i>flaB</i> promoter (P<sub><i>flaB</i></sub>), 2) Tp0751 lipoprotein localization signal sequence and Tp0751 coding sequence, and 3) a C-terminal 3X-FLAG tag. Inserts were cloned into XhoI and NotI sites of a pCE320 (<i>cp32</i>)-derived shuttle vector (resulting plasmid pCC_3–1), and transformed into a non-infectious, adhesion-attenuated B31-A-derived GFP-expressing parent strain (GCB706). Strain names and details are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005919#ppat.1005919.s004" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005919#ppat.1005919.s005" target="_blank">S2</a> Tables. (<b>B-D</b>) Fluorescence-activated cell sorting (FACS) analysis of Tp0751 localization on the surface of <i>B</i>. <i>burgdorferi</i>. Strains were mock-treated with phosphate based saline buffer or permeabilized with methanol before probing with antibodies to the FLAG tag (part of BBK32 and Tp0751 fusion proteins), or to constitutively expressed periplasmic (non-surface-localized) flagellin B protein (FlaB). (<b>B</b>) Representative histogram depicting fluorescence intensities of indicated mock-treated <i>B</i>. <i>burgdorferi</i> strains probed with anti-FLAG antibodies. The mean ± SEM percentages of bacteria that were FLAG-positive for each strain in all replicates are indicated. (<b>C-D</b>) Relative expression levels per bacterium of FLAG-tagged fusion proteins Tp0751 and BBK32 (<b>C</b>) and FlaB (<b>D</b>) in mock-treated and methanol-permeabilized <i>B</i>. <i>burgdorferi</i>. Mean ± SEM fluorescence intensities for each strain were expressed as a proportion of mean fluorescence intensity for <i>B</i>. <i>burgdorferi</i> expressing BBK32-FLAG. N = 8 independent cultures per strain analyzed in two FACS experiments. Statistical analysis: One-way Kruskal-Wallis ANOVA with Dunn’s post-test. * indicates p<0.05 vs. permeabilized BBK32-FLAG control within treatment (mock or permeabilized).</p
Obesity is a major global public health concern. Immune responses implicated in obesity also cont... more Obesity is a major global public health concern. Immune responses implicated in obesity also control certain infections. We investigated the effects of high-fat diet-induced obesity (DIO) on infection with the Lyme disease bacterium Borrelia burgdorferi in mice. DIO was associated with systemic suppression of neutrophil-and macrophage-based innate immune responses. These included bacterial uptake and cytokine production, and systemic, progressive impairment of bacterial clearance, and increased carditis severity. B. burgdorferi-infected mice fed normal diet also gained weight at the same rate as uninfected mice fed high-fat diet, toll-like receptor 4 deficiency rescued bacterial clearance defects, which greater in female than male mice, and killing of an unrelated bacterium (Escherichia coli) by bone marrow-derived macrophages from obese, B. burgdorferi-infected mice was also affected. Importantly, innate immune suppression increased with infection duration and depended on cooperative and synergistic interactions between DIO and B. burgdorferi infection. Thus, obesity and B. burgdorferi infection cooperatively and progressively suppressed innate immunity in mice.
Syphilis is a chronic disease caused by the bacterium Treponema pallidum subsp. pallidum. Trepone... more Syphilis is a chronic disease caused by the bacterium Treponema pallidum subsp. pallidum. Treponema pallidum disseminates widely throughout the host and extravasates from the vasculature, a process that is at least partially dependent upon the ability of T. pallidum to interact with host extracellular matrix (ECM) components. Defining the molecular basis for the interaction between T. pallidum and the host is complicated by the intractability of T. pallidum to in vitro culturing and genetic manipulation. Correspondingly, few T. pallidum proteins have been identified that interact directly with host components. Of these, Tp0751 (also known as pallilysin) displays a propensity to interact with the ECM, although the underlying mechanism of these interactions remains unknown. Towards establishing the molecular mechanism of Tp0751-host ECM attachment, we first determined the crystal structure of Tp0751 to a resolution of 2.15 Å using selenomethionine phasing. Structural analysis revealed...
Insulin-insufficient type 1 diabetes is associated with attenuated bactericidal function of neutr... more Insulin-insufficient type 1 diabetes is associated with attenuated bactericidal function of neutrophils, which are key mediators of innate immune responses to microbes as well as pathological inflammatory processes. Neutrophils are central to immune responses to the Lyme pathogen Borrelia burgdorferi. The effect of hyperglycemia on host susceptibility to and outcomes of B. burgdorferi infection has not been examined. The present study investigated the impact of sustained obesity-independent hyperglycemia in mice on bacterial clearance, inflammatory pathology and neutrophil responses to B. burgdorferi. Hyperglycemia was associated with reduced arthritis incidence but more widespread tissue colonization and reduced clearance of bacterial DNA in multiple tissues including brain, heart, liver, lung and knee joint. B. burgdorferi uptake and killing were impaired in neutrophils isolated from hyperglycemic mice. Thus, attenuated neutrophil function in insulin-insufficient hyperglycemia was...
Background. Bacterial pathogens causing systemic infections disseminate from the initial infectio... more Background. Bacterial pathogens causing systemic infections disseminate from the initial infection focus to the target organs usually through the blood vasculature. To be able to colonize various organs, bacteria need to adhere to the endothelial cells of the vascular wall, and the adhesion must be strong enough to resist the shear force of the blood flow. Borrelia burgdorferi sensu lato spirochetes, the causative agents of the tick-borne disease Lyme borreliosis, disseminate hematogenously from the tick bite site to the joints, the heart, and the central nervous system of the patient. Methods. We used both wild-type and genetically modified B. burgdorferi s. l. bacteria, recombinant borrelia adhesins, and an array of adhesion assays carried out both under stationary and flow conditions to investigate the molecular mechanisms of borrelial adhesion to human endothelial cells. Results. Borrelia garinii, a member of the B. burgdorferi s. l. complex, adhered to biglycan expressed by human endothelial cells in a flow-tolerant manner. The adhesion was mediated by the decorin-binding protein A (DbpA) and DbpB surface molecules of B. garinii. Conclusions. The proteoglycan biglycan is a receptor molecule for flow-resistant adhesion of the bacterial pathogen B. garinii on human endothelial cells.
Similar to circulating tumour and immune cells, many blood-borne microbes preferentially “home” t... more Similar to circulating tumour and immune cells, many blood-borne microbes preferentially “home” to specific vascular sites and tissues during hematogenous dissemination 1–5. For many pathogens, the “postal codes” and mechanisms responsible for tissue-specific vascular tropism are unknown and have been challenging to unravel. Members of the Lyme disease Borreliella burgdorferi species complex infect a broad range of mammalian tissues and exhibit complex strain-, species- and host-specific tissue tropism patterns. Intravenous perfusion experiments and intravital microscopy studies suggest that heterogeneous tissue tropism properties may depend on tissue-specific differences in host and microbial molecules supporting vascular interaction and extravasation. However, interpreting these studies can be complicated because of the immune-protective moonlighting (multitasking) properties of many B. burgdorferi adhesins. Here, we investigated whether B. burgdorferi vascular interaction propert...
Proceedings of the National Academy of Sciences of the United States of America, Apr 10, 2017
Bacterial dissemination via the cardiovascular system is the most common cause of infection morta... more Bacterial dissemination via the cardiovascular system is the most common cause of infection mortality. A key step in dissemination is bacterial interaction with endothelia lining blood vessels, which is physically challenging because of the shear stress generated by blood flow. Association of host cells such as leukocytes and platelets with endothelia under vascular shear stress requires mechanically specialized interaction mechanisms, including force-strengthened catch bonds. However, the biomechanical mechanisms supporting vascular interactions of most bacterial pathogens are undefined. Fibronectin (Fn), a ubiquitous host molecule targeted by many pathogens, promotes vascular interactions of the Lyme disease spirochete Borrelia burgdorferi Here, we investigated how B. burgdorferi exploits Fn to interact with endothelia under physiological shear stress, using recently developed live cell imaging and particle-tracking methods for studying bacterial-endothelial interaction biomechani...
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