This story was originally published in PLOS One Journal:
URL: https://journals.plosone.org.
This content has not been altered
Licensed under Creative Commons Attribution (CC BY) license .
url:https://journals.plos.org/plosone/s/licenses-and-copyright
(C) PLOS One [1]
------------



Pertactin contributes to shedding and transmission of Bordetella bronchiseptica

['Longhuan Ma', 'Department Of Infectious Diseases', 'College Of Veterinary Medicine', 'University Of Georgia', 'Athens', 'Georgia', 'United States Of America', 'Kalyan K. Dewan', 'Dawn L. Taylor-Mulneix', 'Shannon M. Wagner']

Date: None

Abstract Whooping cough is resurging in the United States despite high vaccine coverage. The rapid rise of Bordetella pertussis isolates lacking pertactin (PRN), a key vaccine antigen, has led to concerns about vaccine-driven evolution. Previous studies showed that pertactin can mediate binding to mammalian cells in vitro and act as an immunomodulatory factor in resisting neutrophil-mediated clearance. To further investigate the role of PRN in vivo, we examined the functions of pertactin in the context of a more naturally low dose inoculation experimental system using C3H/HeJ mice that is more sensitive to effects on colonization, growth and spread within the respiratory tract, as well as an experimental approach to measure shedding and transmission between hosts. A B. bronchiseptica pertactin deletion mutant was found to behave similarly to its wild-type (WT) parental strain in colonization of the nasal cavity, trachea, and lungs of mice. However, the pertactin-deficient strain was shed from the nares of mice in much lower numbers, resulting in a significantly lower rate of transmission between hosts. Histological examination of respiratory epithelia revealed that pertactin-deficient bacteria induced substantially less inflammation and mucus accumulation than the WT strain and in vitro assays verified the effect of PRN on the induction of TNF-α by murine macrophages. Interestingly, only WT B. bronchiseptica could be recovered from the spleen of infected mice and were further observed to be intracellular among isolated splenocytes, indicating that pertactin contributes to systemic dissemination involving intracellular survival. These results suggest that pertactin can mediate interactions with immune cells and augments inflammation that contributes to bacterial shedding and transmission between hosts. Understanding the relative contributions of various factors to inflammation, mucus production, shedding and transmission will guide novel strategies to interfere with the reemergence of pertussis.

Author summary B. pertussis strains lacking pertactin have been rising in prevalence especially in countries using acellular vaccines containing pertactin as a key, membrane-associated surface antigen. Previous in vivo studies revealed immunomodulatory properties of pertactin in conventional B. pertussis infection models in which roughly one million bacteria are delivered into lungs, leading to severe pneumonic disease and a strong immune response. However, natural infections begin in the nasopharyngeal region, progress slowly during a prolonged catarrhal stage, only later reaching the trachea and rarely involve the lungs. In this study, a more natural experimental system takes advantage of the ability of B. bronchiseptica, a closely related species, to naturally colonize mice with inocula as low as 5 colony forming units (CFU). In this system B. bronchiseptica can be observed to efficiently colonize, grow, spread within the respiratory tract, is shed from the nares, and transmits between hosts, allowing each of these steps to be measured and studied. Under these conditions, an isogenic pertactin deletion strain was indistinguishable from its parental strain in its abilities to colonize, grow in numbers and spread within the respiratory tract. However, the pertactin-deficient mutant was shed from these mice in lower numbers than wild type, and was defective in transmission between mice. These assays reveal novel roles of pertactin in the induction of inflammation, mucus production, shedding and transmission.

Citation: Ma L, Dewan KK, Taylor-Mulneix DL, Wagner SM, Linz B, Rivera I, et al. (2021) Pertactin contributes to shedding and transmission of Bordetella bronchiseptica. PLoS Pathog 17(8): e1009735. https://doi.org/10.1371/journal.ppat.1009735 Editor: Rachel M. McLoughlin, Trinity College Dublin, IRELAND Received: October 22, 2020; Accepted: June 21, 2021; Published: August 4, 2021 Copyright: © 2021 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting information files. Funding: This work was supported by grants 1R21DC018496-01A1 and 5R21AI142678-02 of the National Institutes of Health to ETH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Pertussis, or whooping cough, is an acute respiratory disease caused by the gram-negative bacterial pathogen Bordetella pertussis. Historically, pertussis had a high morbidity and mortality rate and was the predominant childhood killer before the introduction of whole cell vaccines (WCV) that greatly reduced its prevalence [1]. In highly vaccinated populations, the reduced incidence of severe disease led to attention being focused on the moderate side effects associated with WCV, including common local reactions (swelling and pain), uncommon systemic reactions (fever, irritability, drowsiness, loss of appetite and vomiting), and very rare neurological reactions (acute encephalopathy in newborns) [2]. To address these concerns, less reactive pertussis acellular vaccines (ACV) containing up to five purified, detoxified B. pertussis proteins, including pertussis toxoid, pertactin, type 2 and 3 fimbriae, and filamentous hemagglutinin, were increasingly used in the 1980s and 1990s, and eventually replaced the WCV in most developed countries [3]. Coincident with the switch to ACV, the incidence of whooping cough has been increasing in all age groups, from infants to adults [4]. Moreover, in recent years several countries have seen an increased percentage of clinical isolates that fail to express pertactin (PRN), a prominent outer membrane protein [5,6]. The loss of PRN, a prominent ACV component, has led to concern that ACV vaccines are driving the evolution of B. pertussis to evade vaccine-induced immunity [7]. The long-term consequences of this possibility are difficult to predict as we have limited knowledge of the biological functions of PRN. PRN is an autotransporter protein that appears on the outer membrane of B. pertussis [8]. This protein has been included in acellular vaccines in several countries, based on evidence that pertactin specific antibodies contribute to protection against pertussis. Previous studies revealed that PRN exerts cell binding through its RGD motif and contributes to adherence and invasion of mammalian cells [9–12]. However, the adhesion functions of pertactin were not detected in some other studies [13–17]. In vivo studies have shown an immunomodulatory role of PRN [17,18], in the context of severe pneumonic infection initiated by super-natural challenge with roughly a million CFU of B. pertussis or B. bronchiseptica delivered deep into the lungs of mice where they caused severe lung pathology. This experimental system was established to study the most extreme form of disease and has enabled the development and testing of vaccines and therapeutics to prevent and treat severe disease in the lower respiratory tract. But B. pertussis is highly infectious, suggesting very small initial inocula, and natural infections begin with the weeks-long catarrhal stage, which is limited to the nasopharyngeal region and is a highly contagious period. Thus, colonization with low initial numbers followed by weeks of growth within and shedding from the upper respiratory tract are critical aspects of the infectious cycle of B. pertussis. However, these aspects are not simulated in the standard inoculation approach that delivers as many as a million bacteria deep into the lungs and measures the consequences of the subsequent extreme pneumonic pathology [19–21]. We have focused on simulating the progression of natural infections in order to study the contributions of individual bacterial factors and host immune functions [22–25]. We use B. bronchiseptica because it naturally and highly efficiently colonizes, grows, causes pathology, is shed, and transmits between mice. Importantly, it can efficiently colonize mice when delivered to the external nares in very small numbers (<5 CFU), allowing the entire natural progression of the infection to be studied, including many aspects that are obviated by the conventional approach of washing millions of bacteria deep into the lungs [22–25]. B. pertussis and B. bronchiseptica are very closely related and shared genes are ~ 98% identical at the nucleotide level, but differ in some notable regards. While most of the best studied factors are shared, each expresses a somewhat different subset of factors [5,26–31], that are likely to explain their differences in host range and/or the diseases they cause: while B. pertussis cause an acute and severe disease in humans, B. bronchiseptica causes infection that can range in severity and often persists in the upper respiratory tract of infected animals. However, similarities far outbalance differences. Both species infect mammals and, with the notable exception of expression of the pertussis toxin, share nearly all genes implicated in interacting with their hosts, including the vaccine antigens fimbriae (FIM), filamentous hemagglutinin (FHA) and both species express PRN orthologs that are 92% similarity of PRN at the amino acid level and are likely to perform similar functions [31]. The study of the true in vivo functions of any of Bordetella spp. factors requires experimental systems in which their effects can be measured. Unfortunately, B. pertussis poorly colonizes mice, making studies of the details of natural infections challenging. Unlike B. pertussis, B. bronchiseptica efficiently colonizes mice with an infectious dose less than 5 CFU with infections progressing efficiently, allowing the study of the mechanistic basis for their complex interactions in the context of naturally progressing infection in the natural host [22–25]. We also previously described B. bronchiseptica efficiently transmitting among TLR4-deficient C3H/HeJ mice. B. bronchiseptica grows and is shed in higher numbers in these mice, allowing more efficient transmission [22–25]. This experimental model allows all the bacterial components necessary for efficient transmission to be experimentally manipulated to better understand bacterial mechanisms involved in the transmission process. In this study, to investigate the biological role(s) of PRN in the infectious process and pathogenicity of Bordetella species, we used a mutant of B. bronchiseptica with an in-frame deletion in the prn gene which was generated by allelic exchange as described by Inatsuka et al. [17]. In vivo experiments showed that prn was not necessary for efficient colonization or early growth in the nasopharynx, but was required for efficient bacterial shedding and transmission between mice. Furthermore, wild-type B. bronchiseptica induced higher levels of inflammation and more mucus secretion than the Δprn mutant, revealing a role for PRN in promoting inflammation and mucus secretion, which was supported by in vitro assays showing higher secretion levels of the pro-inflammatory cytokine TNF-α from WT infected compared to Δprn mutant infected murine macrophages. Moreover, the WT strain, but not the prn deletion strain, was recovered from splenocytes of infected mice, indicating that PRN contributes to to systemic dissemination that involves intracellular survival. Together these data suggest that PRN promotes the induction of inflammation and mucus production, mediates shedding and transmission to new hosts and is involved in the intracellular survival and systemic dissemination of the pathogen.

Discussion Previous studies have used different and unrelated clinical isolates of B. pertussis with or without PRN, demonstrating, for example, that an isolate lacking PRN sustained a longer infection in mice immunized with an acellular pertussis vaccine [40]. Such differences cannot be specifically attributed to the loss of PRN, since the genomes of these two separate isolates differ in many other genes. Moreover, because B. pertussis poorly colonizes, sheds, and transmits between mice, it has been difficult to assess the role of PRN in these critical aspects of the infectious cycle. To address these problems, we and Inatsuka et al. used an in-frame deletion of prn in B. bronchiseptica, a close evolutionary progenitor of B. pertussis that naturally infects mice, to test the roles of PRN in infections of a natural host. In the conventional Bordetella spp. experimental system in which a very high inoculation dose (5 x 105 CFU) delivered in a large volume (50 μL) to deposit bacteria deep in the lungs, PRN had a modest effect in BALB/c mice in the prior study [17]. In our high dose pneumonic challenge experiment, using different mice (C57BL/6) assessed at different time points, we did not observe measurable defects of the prn mutant. Since BALB/c and C57BL/6 mice have different immune responses to B. pertussis infection and vaccination [41–43], the different genetic backgrounds of two mouse strains and/or the different time points may explain the different observations in these two studies. Rather those extensive comparisons between experimental conditions using the conventional experimental system, which is an extreme model of severe pneumonic disease, we directed our efforts toward assays that are more sensitive to various aspects of the infection and transmission processes. To better simulate a more natural, gradually progressing Bordetella spp. infection, we used a low dose inoculation model in which an experimental dose as low as 5 CFU is delivered into the external nares of mice in a 5μl droplet that deposits bacteria only in the nose. In this more natural experimental system, PRN was not required for efficient colonization and spread within inoculated hosts. When delivered only into the nasal cavity, rather than deep into the lungs, Bb WT-inoculated mice shed significantly more bacteria and transmitted between mice much more efficiently than BbΔprn-inoculated mice (Fig 1). Bb WT also induced significantly more inflammation and mucus production than RB50Δprn. Inflammation and mucus production contribute to the rhinorrhea that facilitates shedding and transmission, as noted previously with Streptococcus pneumonia transmission [44]. The significantly higher number of neutrophils were observed in the nares of Bb WT infected mice, suggesting that PRN may play critical roles in recruitment and activation of neutrophils and/or in resistance to neutrophils-mediated clearance, as was observed by Inatsuka CS et al [17]. PRN induced secretion of TNF-α from RAW 264.7 macrophages in in-vitro, which may explain the higher inflammation levels observed in mice infected with Bb WT. The role of TNF-α in the transmission of B. bronchiseptica deserves more attention in future studies as this factor may work as a potential therapeutic target for blocking transmission of Bordetella spp. and other pathogens. PRN, as an outer surface protein with a demonstrated ability to affect adhesion to mammalian cells in vitro [9–11], is generally considered an “adhesin”. However, it is not clear that PRN contributes to adhesion to either bronchial or laryngeal cells in vivo. In this study, deleting PRN did not affect the ability of B. bronchiseptica to efficiently colonize mice with a remarkably small inoculum, indicating that PRN is not required for the efficient adherence involved in initial colonization. However, Bb WT showed significantly higher recovery from spleens of infected hosts, indicating that PRN affects the ability to get to and/or survive in the spleen. The observation of substantial numbers of Bb WT, but not BbΔprn, within splenocytes (Fig 6), suggests PRN might affect invasion of immune cells and/or phagocytic killing within splenocytes. Upon encountering invading bacteria, immune cells trigger cascades of inflammatory responses by secreting cytokines, chemokines, small lipid mediators (SLM), and antimicrobial peptides (AMPs) that can contribute to increased phagocytic capacity and bacterial clearance [45], amongst other potential effects. After phagocytosis, peripheral immune cells can carry engulfed bacteria to deeper tissues including draining lymph nodes for T cell priming [46]. Thus, systemic dissemination of Bb WT, but not BbΔprn, may result in a stronger immune response against the former, consistent with the histopathology analysis and IgG antibody titers observed (Fig 2 and S10 Fig). In addition, the role of PRN in inducing TNF-α secretion may also contribute to the higher inflammation observed in the nasal cavity of Bb WT infected mice (Fig 5). The increasing prevalence of PRN-deficient B. pertussis strains raises questions about both positive and negative (purifying) selection. It appears increasingly likely that PRN-containing ACVs select for PRN-deficient B. pertussis. It is also likely that the epidemiology of pertussis has changed as host behavior, population density, and worldwide travel affect the network of connected hosts. Even though PRN-deficient B. pertussis isolates may be shed less, a dense and well-connected population of vaccinated hosts in countries like the USA may be sufficient for a successful chain of transmission. Alternatively, the loss of PRN may have less cost in B. pertussis due to compensation by other genes. There are 15 other autotransporter genes in the genome of B. pertussis [5,31], one or more of which may compensate for the loss of PRN. Since PRN plays roles in systemic dissemination and induction of inflammation in murine transmission models (Figs 2–6), other factors facilitating systemic dissemination and promoting inflammation may compensate for its loss. Alternatively, C3H/HeJ mice may fail to mimic immunocompetent hosts in every aspect of pathogenesis or B. pertussis may be different from B. bronchiseptica in various aspects, and it is possible that the functions of PRN in these two species may differ to some extent. However, to test this, an efficient B. pertussis transmission model is needed. In addition, further studies are needed to identify factors that might compensate for the loss of PRN in current circulating strains. This might lead to the identification of alternative antigens that could be included in next generation vaccines against B. pertussis.

Material and methods Ethics statement Mouse experiments used in this study were performed in strict accordance to recommendations outlined in the Guide for Care and Use of Laboratory Animals of the National Institute of Health. Protocols were approved by the Institutional Animal Care and Use Committee at University of Georgia (A2016 02-010-Y2-A3, Bordetella-Host Interactions). Mice were closely monitored during experiments and any mouse found moribund was euthanized using CO 2 inhalation to prevent unnecessary suffering. Bacterial strains and growth B. bronchiseptica WT strain RB50 and isogenic pertactin knockout strain BbΔprn (SP5) have been previously described [11,17]. To allow mutant and WT to be distinguished in “competition assays”, SM10λpir cells carrying allelic exchange vector pEH10 was used to generate gentamicin-resistant Δprn mutant strain. The generation of pEH10 was described previously [29]. Liquid cultures were prepared using Stainer Scholte (SS) medium supplemented with 0.5% (w/v) Heptakis (2,6-di-O-methyl)-β-cyclodextrin) (Sigma H0513). Plate cultures were grown on Bordet Gengou (BG) agar supplemented with 10% (v/v) defibrinated sheep blood (Hemstat, Hemostat Laboratories) and streptomycin (20 μg/mL). Comparative growth curves were generated from triplicate cultures of bacteria grown 48 hours in SS medium at 37°C and shaking at 200 rpm. Adherence assay Adherence assays were conducted following protocols described earlier [12,47]. In brief, human epithelial lung A459 cells were seeded in triplicate in 24-well plates at a density of 2.5 × 105 cell/well in Dulbecco’s modified Eagle’s medium (DMEM) (supplemented with 10% fetal bovine serum, 10 mM glutamine, 25 mM sodium pyruvate, 10 mM HEPES). Log-phase bacteria were suspended in warm DMEM medium and added to each well at a multiplicity of infection of 10:1 (bacteria: eukaryotic cells). The plate was centrifuged at 300Xg for 10 minutes to synchronize infection and the assay plates were incubated for 5 minutes at 37°C. Unattached bacteria were then removed by washing the cells 4 times with 1 mL phosphate-buffered saline (PBS). A459 cells were lysed with 100 μL of 0.1% sodium deoxycholate for 5 minutes and released bacteria suspended in 900 μL of PBS. The bacteria were enumerated by dilution plating on BG agar plates. RB50 ΔfhaB [12], a mutant strain deleted of the gene encoding the filamentous hemagglutinin and known to be defective in adherence, was used as the negative control. Cytotoxicity assay Cytotoxicity assays were conducted on RAW 264.7 cells, using the CytoTox 96 Nonradioactive Cytotoxicity Assay Kit (Promega) following manufacturer’s protocols. In brief, 100 μL of 2.5 × 104 macrophages were seeded in triplicate in a 96-well plate followed by adding bacteria at a multiplicity of infection of 10:1. The assay plate was centrifuged at 300Xg for 10 minutes. Bacteria were incubated with the macrophages for 4 hours, following which the plate was centrifuged for 5 minutes (300Xg). 50 μL of the supernatant was placed into a fresh flat-bottomed 96-well plate and was calorimetrically assayed for lactate dehydrogenase. A noninfected group was used as a negative control. Cytokine test To assess secretion of TNF-α and IL-1β from RAW 264.7 macrophages, the supernatant of infected cells (MOI = 10 or MOI = 100) was collected at 1-or 4 hpi. To assess the levels of TNF-α and IL-1β in the noses of mice infected with Bb WT or BbΔprn, noses were collected in PBS and homogenized using a bead tissue disruptor. Cytokine levels were determined using the commercially available ELISA kits R&D Systems Mouse IL-1 beta DuoSet ELISA and TNF-alpha DuoSet ELISA following the manufacturer’s instructions. Mouse infections All work with mice was conducted following institutional guidelines. Six-week-old female C57 BL/6 or C3H/HeJ mice (Jackson Laboratories, Bar Harbor, Maine) were used for assessing the colonization and the progress of infection of the respiratory tract. As required, 5–150 CFU of bacteria was delivered in 5 μL of PBS to the nares of mice anesthetized with isoflurane/oxygen. For the colonization profiles, groups of 4 mice were inoculated with WT or mutant bacteria, and at the indicated days 4 mice of each group were euthanized with carbon dioxide (CO 2 ) and the nasal cavity, trachea, and lungs were collected in PBS and homogenized using a bead tissue disruptor. Bacterial load was enumerated by dilution plating. Transmission and shedding assay Transmission assays were conducted using the transmission permissive C3H/HeJ (TLR4 deficient) strain of mouse (Jackson Laboratory) whereby infected (index) mice were co-housed with uninfected (naive) mice [25]. In brief, mice were lightly anaesthetized with isoflurane/oxygen and inoculated with 150 CFU of bacteria delivered in 5 μL of PBS onto the nares. Inoculated (index) mice were then placed in cages with 2 uninfected (naive) mice. Transmission of B. bronchiseptica was assessed after 3 weeks of co-housing by enumerating the bacterial load in the nasal cavities of the naive mice. To monitor shedding, the external nares of the index mice were swabbed (32 swipes) with a dry Dacron polyester tipped swab at the indicated times. The swab was vortexed vigorously in 1 mL PBS for 30 seconds and bacteria enumerated on BG agar plates. Histopathology Following fixation in neutral-buffered, 10% formalin solution and subsequent decalcification in Kristensen’s solution, coronal sections were made through the nose. Tissues were subsequently processed, embedded in paraffin, sectioned at approximately 5μm, and stained with hematoxylin and eosin. Histopathological examination consisted of evaluation of the nose for the incidence (presence or absence), severity, and distribution of inflammation. Histopathologic severity scores were assigned as grades 0 (no significant histopathological alterations); 1 (minimal); 2 (mild); 3 (moderate); or 4 (severe) based on an increasing extent and/or complexity of change, unless otherwise specified. Lesion distribution was recorded as focal, multifocal, or diffuse, with distribution scores of 1, 2, or 3, respectively [48]. Flow cytometry Five mice per experimental group were euthanized by CO 2 inhalation. Nasal cavities were harvested, placed in 1mL of RPMI 1640 medium and homogenized via a syringe plunger against a 40μm cell strainer. Cell suspensions were centrifuged at 1,500rpm for 10 minutes and remaining red blood cells were lysed with ACK lysing buffer. After washing with PBS, the cells were incubated with 1μl Zombie aqua (Biolegend) for 20 minutes, washed again, and incubated with 1μl Fc Block (Biolegend) for 30 minutes. Surface marker staining was added to each sample from a master mix of antibodies. Cells were fixed, washed, and resuspended in 250μl FACS buffer. Flow cytometry (Acea Novocyte Quanteon) was performed was used to sort neutrophils (CD11b-Pe-Cy7, CD115-APC, Ly6G-AF488) and macrophages (CD11b-PE-Cy7 CD115-APC, Ly6G-AF488, F4/80-PE). In a separate panel, T cells (CD45-AF700, CD3-APC), B cells (B220-PE-Cy7), and NK cells (NK1.1-PE) were sorted via a separate gating strategy. Viable cells were gated as Zombie aqua-negative cells. The data were analysed with FlowJo 10.0. (S11 Fig). Cell isolation for intracellular survival test The spleen organs harvested from WT or mutant infected Hej mice was cut into 3–4 pieces using sterile scissors. Spleen pieces were mashed with the rough surfaces of two sterile frosted microscope slides and slide surfaces were rinsed with 1 mL (FBS-free) DMEM medium. The samples were then passed through a 70 μm cell strainer to obtain a single-cell suspension. A 100μl sample was plated on BG agar containing 20 μg/ml streptomycin to estimate the number of total bacteria. Cells were washed twice with PBS and then incubated for 1 h with 300 μg/ml gentamicin to eliminate extracellular bacteria. Cells were washed twice with PBS and then lysed with 0.1% triton X-100 for 15 minutes at room temperature. CFU counting was performed on cell lysates by plating 10-fold serial dilutions onto BG agar plates containing 20 μg/ml streptomycin to estimate the number of intracellular bacteria. Control experiments to assess the efficacy of gentamicin were performed in parallel [49]. Briefly, samples of 103, 104 or 105 bacteria were incubated with 300 μg/ml gentamicin for 1 hour at 37°C and plated on BG agar. There were 3 replicates for each group. The results showed that more than 99.9% bacteria were killed. Statistical analysis Statistical analysis of differences between the WT and mutant groups was performed using the Unpaired Student 2-tailed t test, One-way ANOVA and Two-way ANOVA test, as appropriate. P value less than 0.05 shown as *; P value less than 0.01 shown as **; P value less than 0.001 shown as ***; P value less than 0.0001 shown as ****.

Acknowledgments We acknowledge the staff of the mouse facilities at the University Research Animal Resources (URAR), University of Georgia, for facilitating the work. We also thank members of the Harvill laboratory for helpful discussions and editing of the manuscript.

[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009735

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.