Figure 1. Scientific work package and status at the end of the second year of the study.
The overall goal of INFECT is to advance our understanding of the pathophysiological mechanisms, prognosis, and diagnosis of the multifactorial highly lethal necrotizing soft tissue infections (NSTIs). NSTI’s are rapidly spreading infections that may cause extensive soft tissue or limb loss, multiorgan failure and are associated with a considerable fatality rate. It is undisputed that rapid diagnosis and prompt intervention is directly related to survival. The initial presentation may be limited to unspecific symptoms such as tenderness, swelling, erythema and pain. Thus, diagnosis and management are difficult due to heterogeneity in clinical presentation, in co-morbidities and in microbiological aetiology. There is an urgent need for novel diagnostic and therapeutic strategies in order to improve outcome of NSTIs. To achieve this, a comprehensive and integrated knowledge of diagnostic features, causative microbial agent, treatment strategies, and pathogenic mechanisms (host and bacterial disease traits and their underlying interaction network) is required. INFECT is designed to obtain such insights through an integrated systems biology approach in patients and different clinically relevant experimental models.
Specific objectives of INFECT are to:
Unravel specific mechanisms underlying diseases responses through a bottom-up systems approach applied to clinically relevant experimental modelsApply a top-down systems biology approach to NSTI patient samples to pin-point key host and pathogen factors involved in the onset and development of infectionIdentify and quantify disease signatures and underlying networks that contribute to disease outcomeExploit identified disease traits for the innovation of optimized diagnostic toolsTranslate the advanced knowledge generated into evidence-based guidelines for classification and management, and novel therapeutic strategies
Summary of the project for the reporting period
During this reporting period, the main objective of WP1 was to identify QTLs in the optimized NSTI BXD mice model that we had developed in the last reporting period (partner 15). We have accomplished this task and identified a highly significant QTL under mouse chromosome 2 that is associated with differences in survival of BXD mouse strains infected with Streptococcus pyogenes 5448. A list of host susceptibility genes has been identified under this highly significant QTL and this gene list has been shared with partners 9 and 11 for further evaluation in WP4. In addition, we have conducted experiments with the INFECT clinical isolate 2006 (S. pyogenes), which is currently under investigation for host susceptibility/outcomes. Also, we have collected skin tissue samples from the infected mice and will continue to collect these samples to send them to partner 8 for RNAseq analyses (WP3) and to partner 1 (WP5). Experiments have been done to optimize the Staphylococcus aureus NSTI model by using INFECT isolates SA6005, SA6006, and SA6008. However, the BXD mice are not developing as severe tissue lesions as those seen in patients or with S. pyogenes. We are currently exploring possibilities to increase the severity by use of different isolates and different interventions. However, as evident by the clinical database (WP2, partners 2-6), S. aureus NSTI is predominantly noted in severely co-morbid patients, whereas S. pyogenes is commonly seen in immunocompetent individuals; thus, the obtained results in the BXD mice might reflect this requirement for comorbidities.
During this reporting period, major achievements of WP2 include: 1) Secured the enrolments of patients and data, isolates, tissue, and blood sample collection by all teams (partners 2-6), and 2) Isolates, blood and tissue biopsies are being preserved and handled immediately according to the agreed standardized operating procedures (SOPs) developed and agreed to during 2013 (partners 2-6), and stored in the biobank. Shipments of biological material have been established and SOP’s on shipping proceedures developed among all clinical partners. In total, 196 NSTI patients have been enrolled at the 5 clinical sites uring the first two years of sampling. This is in line with the tentative estimates made in the annex I of approximately 100 patients yearly. It so follows that the WP2 group comply with its outmost important task of creating the necessary clinical data for subsequent analysis and development of treatment guidelines. By means of extensive laboratory analysis (WP3, WP5), these patient samples will be linked to detailed clinical information collected in the clinical databases. The clinical database will be continuously utilized to ensure that the experimental work and evaluations are clinically relevant.
The over all aim of WP3 is to identify specific pathogen and host signatures associated with disease development. One objective was the application of various standard typing methods as well as specialized PCR-based approaches and microarray analyses to establish a database containing virulence specific information of bacterial pathogens involved in NSTI (partners 8 and 10). The analyses provided new insights in the exotoxin gene distribution of streptococcal NSTI isolates, verified the predominance of Streptococcus pyogenes serotype M1 besides some Streptococcus dysgalactiae ssp. equisimilis strains and showed genes encoding Panton Valentine Leucocidin to be over-represented in severe skin infections. The second objective was the identification of bacterial factors responsible for the development of a severe NSTI mainly through comparative whole-genome analysis on streptococcal and staphylococcal NSTI- and non-NSTI isolates (partners 8 and 10). Comparative whole genome analysis of M1 strains indicated that a combination of prophage encoded factors and a dysregulation of virulence related genes may discriminate between a systemic bacteremia and local tissue infections like NSTI. Comparative genome analysis of S. aureus isolates allowed todiscriminate between NSTI isolates and bacteremia isolates (partner 10). A third objective was the identification of pathogen traits associated with the outcome of NSTI mainly through the analysis of host-pathogen interactions on the cellular level (partners 1, 8). Indications for the different strategies used were now obtained for all three groups of bacteria targeted. The fourth objective was the identification of host (and pathogen) factors influencing the development of a necrotizing soft tissue infection through an interlinked transcriptomic, proteomic and metabolomic approach. To this end the role of the adaptive immune system for the development of an NSTI was investigated by an extensive serology screening approach, which revealed a significant susceptibility only of NSTI cases against corresponding bacterial toxins as well as the bacterial isolate. A reliable transcriptomic workflow was developed and first samples have successfully been analyzed (partner 8).
The overall goal of WP4 is identifying major molecular interaction networks between human host and infectious bacteria in NSTI through (i) integration, analyses, and modeling of data from WP1-3 and WP 5-6; and (ii) model-driven design of experiments to determine mechanistic actions relevant to novel therapeutic strategies (WP6) and to further explore the pathogenesis at local tissue-specific infection sites (WP5). During this second period WP4 has maintained and curated the standardized data handling procedure that has been developed in year 1 to ensure proper data management, analysis and integration and developed computational tools for visualizing signalling/metabolic pathways and modules in NSTI disease specific context (partners 9, 11, 14). WP4 has developed an innovative platform for bacterial genome annotation based on sematic web technology (SAPP) to guarantee high-quality annotation data. This tool has been applied to generate data that allowed the analysis of >120 Streptococcus strains from a metabolic point of view. WP4 carried out fundamental research to deploy a genome-scale metabolic model (GSMM) for the mammalian host (mouse, human cell lines, biopsies), as well as modeling of specific regulatory aspects of the host, to predict and then identify key pathogen-host interactions. A predictive disease and pathogen-specific model has been realized that will be used to generate valuable predictions that can be tested and validated experimentally. WP4 has also been involved in the development of new statistical techniques for network inference and in the refinement and assessment of established data analysis techniques.
The overall aim of WP5 is to validate results obtained in WP1-4, and to identify mechanistic action of novel therapeutic strategies. This will be done by analyses of host-microbe interaction at the local site of infection, i.e. in patient tissue biopsies (partner 1). Work achieved during the reporting period has included analyses of tissue biopsies from NSTI patients for bacterial load and host inflammatory mediators hypothesized to be involved in NSTIs. A large panel of tissue biopsies from patients with NSTI caused by S. pyogenes, S. aureus or group G streptococcus has been analyzed. The analyses provided important insight into bacterial load, bacterial and host factors indicating differences in pathogenic mechanisms between different pathogens. Among others it shows differences in HMGB-1 responses and neutrophil infiltration in the tissue. This has been used to phrase working hypothesis that will now be tested in WP4 and validated by further analyses of patient samples (partners 8, 10 -WP3, and partner 1 - WP5) as well as in our experimental models (partner 15-WP1 and partner 1-WP6).
The over all aim of WP6 is to establish a clinically relevant, robust and reproducible multicellular system, a so called organotypic model, resembling normal human skin tissue that can used for experimental infections and test of therapeutic strategies (partner 1). During the 2nd period, partner 1 focused the efforts on establishing infections of the standardized organotypic model of human skin tissue using INFECT bacterial isolates, including S. pyogenes, Streptococcus dysgalactiae subspecies equisimilis (SDSE), and Staphylococcus aureus. Infections were performed with all three bacterial species, and models were infected for various length of time to follow the progress of infection. Analyses of gene and protein expression after infection revealed differential gene expression of both host (structural and inflammatory components) and bacterial (regulatory components and virulence factors), as well as altered host protein expression patterns. The results from these experiments have been compiled into two separate manuscripts, one focusing on S pyogenes and another focusing on SDSE infection. Experiments with the INFECT 2006 isolate (S pyogenes) have also been performed to generate infected and non-infected tissue model samples for dualRNA sequencing analysis (partner 8) from which data will be integrated in the data driven computational modelling platform (partners 9, 11, 14). Over all, the progress of work is well in line with the objectives and tasks of this work package and the deliverables and milestones have been fully met.
In WP7, the work accomplished by partner 12 so far is the initial specification of diagnostics tests, done together with clinical partners 2-6, for early pathogen detection (based on compact sequencing) and inflammation and organ monitoring (based on compact profiling) to support clinical decisions for diagnosis and during treatment of NSTIs. These specifications had been defined as deliverables. Prototypes of tests have been (further) developed and verified against specifications, which is defined as a milestone. Initial clinical validations have been undertaken both for early pathogen detection in aspirates and blood (samples from partner 6) and monitoring (samples collected from partners 2-6). Over all, the progress of work is ahead of schedule with respect to the objectives and tasks of this work package and the deliverables and milestones have been fully met.
The ultimate objectives of WP8 is to ensure an efficient dissemination and exploitation of knowledge generated within the INFECT project using established communication methods directed at professionals, by disseminating clinical guidelines developed on basis of knowledge present towards the end of the project period, and providing updated information material for patients, society in large, and medical staff (all partners). An educational program for medical staff is under development within the project period. During the second reporting period main task have been dissemination of information of the INFECT consortium and project and dissemination of knowledge generated in INFECT. This has been achieved by several means, and over 80 dissemination activities have been completed out of which 43 during period 2. Our external open access website containing updated information of scientific advances is one important platform, which is used not only to provide information on the INFECT project but also updates on new insights obtained in the project. The partners have also actively disseminated advances made through presentations nationally and internationally, both in the scientific community (all partners), healthcare settings (mainly partner 1, clinical partners 2-6, partner 13) as well as for policy makers where the patient organization has been a key player (partner 13).
WP9 aims to provide an efficient project management for INFECT and its 14 partners. Main activities during this period have focused on ensure efficient communication, data sharing and integration between the participants in the project, as well as to ensure full compliance with all legal and EC requirements. One important platform for this purpose, is the user-friendly access restricted internal website where documents and data are uploaded and shared among all partners of INFECT. The internal website also contains the important links including the EU portal and the SPARQL (WP4 data modulation tool). Furthermore, instruments such as data integration panel reports and templates for reports have been created and distributed among the partners. In addition, there has been extensive scientific contact within the consortium including a workshop in Braunschweig January 2014 with a focus on data generation, integration and sharing (partners 1, 8-14), smaller WP meetings betweer partners, and the annual meeting in Braunschweig January 2015. In addition, an ethical review of the work conducted during the second period has been conducted by the external advisor.