Perspectives on computational modeling of biological systems and the significance of the SysMod community.

Motivation: In recent years, applying computational modeling to systems biology has caused a substantial surge in both discovery and practical applications and a significant shift in our understanding of the complexity inherent in biological systems. Results: In this perspective article, we briefly overview computational modeling in biology, highlighting recent advancements such as multi-scale modeling due to the omics revolution, single-cell technology, and integration of artificial intelligence and machine learning approaches. We also discuss the primary challenges faced: integration, standardization, model complexity, scalability, and interdisciplinary collaboration. Lastly, we highlight the contribution made by the Computational Modeling of Biological Systems (SysMod) Community of Special Interest (COSI) associated with the International Society of Computational Biology (ISCB) in driving progress within this rapidly evolving field through community engagement (via both in person and virtual meetings, social media interactions), webinars, and conferences. Availability and implementation: Additional information about SysMod is available at https://sysmod.info.

Genome-scale model of Rothia mucilaginosa predicts gene essentialities and reveals metabolic capabilities.

Cystic fibrosis (CF), an inherited genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, results in sticky and thick mucosal fluids. This environment facilitates the colonization of various microorganisms, some of which can cause acute and chronic lung infections, while others may positively impact the disease. Rothia mucilaginosa, an oral commensal, is relatively abundant in the lungs of CF patients. Recent studies have unveiled its anti-inflammatory properties using in vitro three-dimensional lung epithelial cell cultures and in vivo mouse models relevant to chronic lung diseases. Apart from this, R. mucilaginosa has been associated with severe infections. However, its metabolic capabilities and genotype-phenotype relationships remain largely unknown. To gain insights into its cellular metabolism and genetic content, we developed the first manually curated genome-scale metabolic model, iRM23NL. Through growth kinetics and high-throughput phenotypic microarray testings, we defined its complete catabolic phenome. Subsequently, we assessed the model's effectiveness in accurately predicting growth behaviors and utilizing multiple substrates. We used constraint-based modeling techniques to formulate novel hypotheses that could expedite the development of antimicrobial strategies. More specifically, we detected putative essential genes and assessed their effect on metabolism under varying nutritional conditions. These predictions could offer novel potential antimicrobial targets without laborious large-scale screening of knockouts and mutant transposon libraries. Overall, iRM23NL demonstrates a solid capability to predict cellular phenotypes and holds immense potential as a valuable resource for accurate predictions in advancing antimicrobial therapies. Moreover, it can guide metabolic engineering to tailor R. mucilaginosa's metabolism for desired performance.IMPORTANCECystic fibrosis (CF) is a genetic disorder characterized by thick mucosal secretions, leading to chronic lung infections. Rothia mucilaginosa is a common bacterium found in various parts of the human body, acting as a normal part of the flora. In people with weakened immune systems, it can become an opportunistic pathogen, while it is prevalent and active in CF airways. Recent studies have highlighted its anti-inflammatory properties in the lower pulmonary system, indicating the intricate relationship between microbes and human health. Herein, we have developed the first manually curated metabolic model of R. mucilaginosa. Our study examined the previously unknown relationships between the bacterium's genotype and phenotype and identified essential genes that impact the metabolism under various conditions. With this, we opt for paving the way for developing new strategies in antimicrobial therapy and metabolic engineering, leading to enhanced therapeutic outcomes in cystic fibrosis and related conditions.

Genome-scale metabolic models consistently predict in vitro characteristics of Corynebacterium striatum.

Introduction: Genome-scale metabolic models (GEMs) are organism-specific knowledge bases which can be used to unravel pathogenicity or improve production of specific metabolites in biotechnology applications. However, the validity of predictions for bacterial proliferation in in vitro settings is hardly investigated. Methods: The present work combines in silico and in vitro approaches to create and curate strain-specific genome-scale metabolic models of Corynebacterium striatum. Results: We introduce five newly created strain-specific genome-scale metabolic models (GEMs) of high quality, satisfying all contemporary standards and requirements. All these models have been benchmarked using the community standard test suite Metabolic Model Testing (MEMOTE) and were validated by laboratory experiments. For the curation of those models, the software infrastructure refineGEMs was developed to work on these models in parallel and to comply with the quality standards for GEMs. The model predictions were confirmed by experimental data and a new comparison metric based on the doubling time was developed to quantify bacterial growth. Discussion: Future modeling projects can rely on the proposed software, which is independent of specific environmental conditions. The validation approach based on the growth rate calculation is now accessible and closely aligned with biological questions. The curated models are freely available via BioModels and a GitHub repository and can be used. The open-source software refineGEMs is available from https://github.com/draeger-lab/refinegems.

An integrated systems biology approach reveals differences in formate metabolism in the genus Methanothermobacter.

Methanogenesis allows methanogenic archaea to generate cellular energy for their growth while producing methane. Thermophilic hydrogenotrophic species of the genus Methanothermobacter have been recognized as robust biocatalysts for a circular carbon economy and are already applied in power-to-gas technology with biomethanation, which is a platform to store renewable energy and utilize captured carbon dioxide. Here, we generated curated genome-scale metabolic reconstructions for three Methanothermobacter strains and investigated differences in the growth performance of these same strains in chemostat bioreactor experiments with hydrogen and carbon dioxide or formate as substrates. Using an integrated systems biology approach, we identified differences in formate anabolism between the strains and revealed that formate anabolism influences the diversion of carbon between biomass and methane. This finding, together with the omics datasets and the metabolic models we generated, can be implemented for biotechnological applications of Methanothermobacter in power-to-gas technology, and as a perspective, for value-added chemical production.

Can Genome Sequencing Coupled to Flux Balance Analyses Offer Precision Guidance for Industrial Strain Development? The Lessons from Carbon Trafficking in Corynebacterium glutamicum ATCC 21573.

Systems biology tools offer new prospects for industrial strain selection. For bacteria that are significant for industrial applications, whole-genome sequencing coupled to flux balance analysis (FBA) can help unpack the complex relationships between genome mutations and carbon trafficking. This work investigates the l-tyrosine (l-Tyr) overproducing model system Corynebacterium glutamicum ATCC 21573 with an eye to more rational and precision strain development. Using genome-wide mutational analysis of C. glutamicum, we identified 27,611 single nucleotide polymorphisms and 479 insertion/deletion mutations. Mutations in the carbon uptake machinery have led to phosphotransferase system-independent routes as corroborated with FBA. Mutations within the central carbon metabolism of C. glutamicum impaired the carbon flux, as evidenced by the lower growth rate. The entry to and flow through the tricarboxylic acid cycle was affected by mutations in pyruvate and α-ketoglutarate dehydrogenase complexes, citrate synthase, and isocitrate dehydrogenase. FBA indicated that the estimated flux through the shikimate pathway became larger as the l-Tyr production rate increased. In addition, protocatechuate export was probabilistically impossible, which could have contributed to the l-Tyr accumulation. Interestingly, aroG and cg0975, which have received previous attention for aromatic amino acid overproduction, were not mutated. From the branch point molecule, prephenate, the change in the promoter region of pheA could be an influential contributor. In summary, we suggest that genome sequencing coupled with FBA is well poised to offer rational guidance for industrial strain development, as evidenced by these findings on carbon trafficking in C. glutamicum ATCC 21573.

SBOannotator: a Python tool for the automated assignment of systems biology ontology terms.

MOTIVATION: The number and size of computational models in biology have drastically increased over the past years and continue to grow. Modeled networks are becoming more complex, and reconstructing them from the beginning in an exchangeable and reproducible manner is challenging. Using precisely defined ontologies enables the encoding of field-specific knowledge and the association of disparate data types. In computational modeling, the medium for representing domain knowledge is the set of orthogonal structured controlled vocabularies named Systems Biology Ontology (SBO). The SBO terms enable modelers to explicitly define and describe model entities, including their roles and characteristics. RESULTS: Here, we present the first standalone tool that automatically assigns SBO terms to multiple entities of a given SBML model, named the SBOannotator. The main focus lies on the reactions, as the correct assignment of precise SBO annotations requires their extensive classification. Our implementation does not consider only top-level terms but examines the functionality of the underlying enzymes to allocate precise and highly specific ontology terms to biochemical reactions. Transport reactions are examined separately and are classified based on the mechanism of molecule transport. Pseudo-reactions that serve modeling purposes are given reasonable terms to distinguish between biomass production and the import or export of metabolites. Finally, other model entities, such as metabolites and genes, are annotated with appropriate terms. Including SBO annotations in the models will enhance the reproducibility, usability, and analysis of biochemical networks. AVAILABILITY AND IMPLEMENTATION: SBOannotator is freely available from https://github.com/draeger-lab/SBOannotator/.

New workflow predicts drug targets against SARS-CoV-2 via metabolic changes in infected cells.

COVID-19 is one of the deadliest respiratory diseases, and its emergence caught the pharmaceutical industry off guard. While vaccines have been rapidly developed, treatment options for infected people remain scarce, and COVID-19 poses a substantial global threat. This study presents a novel workflow to predict robust druggable targets against emerging RNA viruses using metabolic networks and information of the viral structure and its genome sequence. For this purpose, we implemented pymCADRE and PREDICATE to create tissue-specific metabolic models, construct viral biomass functions and predict host-based antiviral targets from more than one genome. We observed that pymCADRE reduces the computational time of flux variability analysis for internal optimizations. We applied these tools to create a new metabolic network of primary bronchial epithelial cells infected with SARS-CoV-2 and identified enzymatic reactions with inhibitory effects. The most promising reported targets were from the purine metabolism, while targeting the pyrimidine and carbohydrate metabolisms seemed to be promising approaches to enhance viral inhibition. Finally, we computationally tested the robustness of our targets in all known variants of concern, verifying our targets' inhibitory effects. Since laboratory tests are time-consuming and involve complex readouts to track processes, our workflow focuses on metabolic fluxes within infected cells and is applicable for rapid hypothesis-driven identification of potentially exploitable antivirals concerning various viruses and host cell types.

Genome-scale model of Pseudomonas aeruginosa metabolism unveils virulence and drug potentiation.

Pseudomonas aeruginosa is one of the leading causes of hospital-acquired infections. To decipher the metabolic mechanisms associated with virulence and antibiotic resistance, we have developed an updated genome-scale model (GEM) of P. aeruginosa. The model (iSD1509) is an extensively curated, three-compartment, and mass-and-charge balanced BiGG model containing 1509 genes, the largest gene content for any P. aeruginosa GEM to date. It is the most accurate with prediction accuracies as high as 92.4% (gene essentiality) and 93.5% (substrate utilization). In iSD1509, we newly added a recently discovered pathway for ubiquinone-9 biosynthesis which is required for anaerobic growth. We used a modified iSD1509 to demonstrate the role of virulence factor (phenazines) in the pathogen survival within biofilm/oxygen-limited condition. Further, the model can mechanistically explain the overproduction of a drug susceptibility biomarker in the P. aeruginosa mutants. Finally, we use iSD1509 to demonstrate the drug potentiation by metabolite supplementation, and elucidate the mechanisms behind the phenotype, which agree with experimental results.

Hierarchical modelling of microbial communities.

SUMMARY: The human body harbours a plethora of microbes that play a fundamental role in the well-being of the host. Still, the contribution of many microorganisms to human health remains undiscovered. To understand the composition of their communities, the accurate genome-scale metabolic network models of participating microorganisms are integrated to construct a community that mimics the normal bacterial flora of humans. So far, tools for modelling the communities have transformed the community into various optimization problems and model compositions. Therefore, any knockout or modification of each submodel (each species) necessitates the up-to-date creation of the community to incorporate rebuildings. To solve this complexity, we refer to the context of SBML in a hierarchical model composition, wherein each species's genome-scale metabolic model is imported as a submodel in another model. Hence, the community is a model composed of submodels defined in separate files. We combine all these files upon parsing to a so-called 'flattened' model, i.e., a comprehensive and valid SBML file of the entire community that COBRApy can parse for further processing. The hierarchical model facilitates the analysis of the whole community irrespective of any changes in the individual submodels. AVAILABILITY AND IMPLEMENTATION: The module is freely available at https://github.com/manuelgloeckler/ncmw.

NCMW: A Python Package to Analyze Metabolic Interactions in the Nasal Microbiome.

The human upper respiratory tract is the reservoir of a diverse community of commensals and potential pathogens (pathobionts), including Streptococcus pneumoniae (pneumococcus), Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus, which occasionally turn into pathogens causing infectious diseases, while the contribution of many nasal microorganisms to human health remains undiscovered. To better understand the composition of the nasal microbiome community, we create a workflow of the community model, which mimics the human nasal environment. To address this challenge, constraint-based reconstruction of biochemically accurate genome-scale metabolic models (GEMs) networks of microorganisms is mandatory. Our workflow applies constraint-based modeling (CBM), simulates the metabolism between species in a given microbiome, and facilitates generating novel hypotheses on microbial interactions. Utilizing this workflow, we hope to gain a better understanding of interactions from the metabolic modeling perspective. This article presents nasal community modeling workflow (NCMW)-a python package based on GEMs of species as a starting point for understanding the composition of the nasal microbiome community. The package is constructed as a step-by-step mathematical framework for metabolic modeling and analysis of the nasal microbial community. Using constraint-based models reduces the need for culturing species in vitro, a process that is not convenient in the environment of human noses. Availability: NCMW is freely available on the Python Package Index (PIP) via pip install NCMW. The source code, documentation, and usage examples (Jupyter Notebook and example files) are available at https://github.com/manuelgloeckler/ncmw.

A Computational Model of Bacterial Population Dynamics in Gastrointestinal Yersinia enterocolitica Infections in Mice.

The complex interplay of a pathogen with its virulence and fitness factors, the host's immune response, and the endogenous microbiome determine the course and outcome of gastrointestinal infection. The expansion of a pathogen within the gastrointestinal tract implies an increased risk of developing severe systemic infections, especially in dysbiotic or immunocompromised individuals. We developed a mechanistic computational model that calculates and simulates such scenarios, based on an ordinary differential equation system, to explain the bacterial population dynamics during gastrointestinal infection. For implementing the model and estimating its parameters, oral mouse infection experiments with the enteropathogen, Yersinia enterocolitica (Ye), were carried out. Our model accounts for specific pathogen characteristics and is intended to reflect scenarios where colonization resistance, mediated by the endogenous microbiome, is lacking, or where the immune response is partially impaired. Fitting our data from experimental mouse infections, we can justify our model setup and deduce cues for further model improvement. The model is freely available, in SBML format, from the BioModels Database under the accession number MODEL2002070001.

The systems biology simulation core library.

SUMMARY: Studying biological systems generally relies on computational modeling and simulation, e.g., model-driven discovery and hypothesis testing. Progress in standardization efforts led to the development of interrelated file formats to exchange and reuse models in systems biology, such as SBML, the Simulation Experiment Description Markup Language (SED-ML) or the Open Modeling EXchange format. Conducting simulation experiments based on these formats requires efficient and reusable implementations to make them accessible to the broader scientific community and to ensure the reproducibility of the results. The Systems Biology Simulation Core Library (SBSCL) provides interpreters and solvers for these standards as a versatile open-source API in JavaTM. The library simulates even complex bio-models and supports deterministic Ordinary Differential Equations; Stochastic Differential Equations; constraint-based analyses; recent SBML and SED-ML versions; exchange of results, and visualization of in silico experiments; open modeling exchange formats (COMBINE archives); hierarchically structured models; and compatibility with standard testing systems, including the Systems Biology Test Suite and published models from the BioModels and BiGG databases. AVAILABILITY AND IMPLEMENTATION: SBSCL is freely available at https://draeger-lab.github.io/SBSCL/ and via Maven Central. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Towards the human nasal microbiome: Simulating D. pigrum and S. aureus.

The human nose harbors various microbes that decisively influence the wellbeing and health of their host. Among the most threatening pathogens in this habitat is Staphylococcus aureus. Multiple epidemiological studies identify Dolosigranulum pigrum as a likely beneficial bacterium based on its positive association with health, including negative associations with S. aureus. Carefully curated GEMs are available for both bacterial species that reliably simulate their growth behavior in isolation. To unravel the mutual effects among bacteria, building community models for simulating co-culture growth is necessary. However, modeling microbial communities remains challenging. This article illustrates how applying the NCMW fosters our understanding of two microbes' joint growth conditions in the nasal habitat and their intricate interplay from a metabolic modeling perspective. The resulting community model combines the latest available curated GEMs of D. pigrum and S. aureus. This uses case illustrates how to incorporate genuine GEM of participating microorganisms and creates a basic community model mimicking the human nasal environment. Our analysis supports the role of negative microbe-microbe interactions involving D. pigrum examined experimentally in the lab. By this, we identify and characterize metabolic exchange factors involved in a specific interaction between D. pigrum and S. aureus as an in silico candidate factor for a deep insight into the associated species. This method may serve as a blueprint for developing more complex microbial interaction models. Its direct application suggests new ways to prevent disease-causing infections by inhibiting the growth of pathogens such as S. aureus through microbe-microbe interactions.

High-Quality Genome-Scale Reconstruction of Corynebacterium glutamicum ATCC 13032.

Corynebacterium glutamicum belongs to the microbes of enormous biotechnological relevance. In particular, its strain ATCC 13032 is a widely used producer of L-amino acids at an industrial scale. Its apparent robustness also turns it into a favorable platform host for a wide range of further compounds, mainly because of emerging bio-based economies. A deep understanding of the biochemical processes in C. glutamicum is essential for a sustainable enhancement of the microbe's productivity. Computational systems biology has the potential to provide a valuable basis for driving metabolic engineering and biotechnological advances, such as increased yields of healthy producer strains based on genome-scale metabolic models (GEMs). Advanced reconstruction pipelines are now available that facilitate the reconstruction of GEMs and support their manual curation. This article presents iCGB21FR, an updated and unified GEM of C. glutamicum ATCC 13032 with high quality regarding comprehensiveness and data standards, built with the latest modeling techniques and advanced reconstruction pipelines. It comprises 1042 metabolites, 1539 reactions, and 805 genes with detailed annotations and database cross-references. The model validation took place using different media and resulted in realistic growth rate predictions under aerobic and anaerobic conditions. The new GEM produces all canonical amino acids, and its phenotypic predictions are consistent with laboratory data. The in silico model proved fruitful in adding knowledge to the metabolism of C. glutamicum: iCGB21FR still produces L-glutamate with the knock-out of the enzyme pyruvate carboxylase, despite the common belief to be relevant for the amino acid's production. We conclude that integrating high standards into the reconstruction of GEMs facilitates replicating validated knowledge, closing knowledge gaps, and making it a useful basis for metabolic engineering. The model is freely available from BioModels Database under identifier MODEL2102050001.

An updated genome-scale metabolic network reconstruction of Pseudomonas aeruginosa PA14 to characterize mucin-driven shifts in bacterial metabolism.

Mucins are present in mucosal membranes throughout the body and play a key role in the microbe clearance and infection prevention. Understanding the metabolic responses of pathogens to mucins will further enable the development of protective approaches against infections. We update the genome-scale metabolic network reconstruction (GENRE) of one such pathogen, Pseudomonas aeruginosa PA14, through metabolic coverage expansion, format update, extensive annotation addition, and literature-based curation to produce iPau21. We then validate iPau21 through MEMOTE, growth rate, carbon source utilization, and gene essentiality testing to demonstrate its improved quality and predictive capabilities. We then integrate the GENRE with transcriptomic data in order to generate context-specific models of P. aeruginosa metabolism. The contextualized models recapitulated known phenotypes of unaltered growth and a differential utilization of fumarate metabolism, while also revealing an increased utilization of propionate metabolism upon MUC5B exposure. This work serves to validate iPau21 and demonstrate its utility for providing biological insights.

COVID19 Disease Map, a computational knowledge repository of virus-host interaction mechanisms.

We need to effectively combine the knowledge from surging literature with complex datasets to propose mechanistic models of SARS-CoV-2 infection, improving data interpretation and predicting key targets of intervention. Here, we describe a large-scale community effort to build an open access, interoperable and computable repository of COVID-19 molecular mechanisms. The COVID-19 Disease Map (C19DMap) is a graphical, interactive representation of disease-relevant molecular mechanisms linking many knowledge sources. Notably, it is a computational resource for graph-based analyses and disease modelling. To this end, we established a framework of tools, platforms and guidelines necessary for a multifaceted community of biocurators, domain experts, bioinformaticians and computational biologists. The diagrams of the C19DMap, curated from the literature, are integrated with relevant interaction and text mining databases. We demonstrate the application of network analysis and modelling approaches by concrete examples to highlight new testable hypotheses. This framework helps to find signatures of SARS-CoV-2 predisposition, treatment response or prioritisation of drug candidates. Such an approach may help deal with new waves of COVID-19 or similar pandemics in the long-term perspective.

Genome-Scale Metabolic Model of Infection with SARS-CoV-2 Mutants Confirms Guanylate Kinase as Robust Potential Antiviral Target.

The current SARS-CoV-2 pandemic is still threatening humankind. Despite first successes in vaccine development and approval, no antiviral treatment is available for COVID-19 patients. The success is further tarnished by the emergence and spreading of mutation variants of SARS-CoV-2, for which some vaccines have lower efficacy. This highlights the urgent need for antiviral therapies even more. This article describes how the genome-scale metabolic model (GEM) of the host-virus interaction of human alveolar macrophages and SARS-CoV-2 was refined by incorporating the latest information about the virus's structural proteins and the mutant variants B.1.1.7, B.1.351, B.1.28, B.1.427/B.1.429, and B.1.617. We confirmed the initially identified guanylate kinase as a potential antiviral target with this refined model and identified further potential targets from the purine and pyrimidine metabolism. The model was further extended by incorporating the virus' lipid requirements. This opened new perspectives for potential antiviral targets in the altered lipid metabolism. Especially the phosphatidylcholine biosynthesis seems to play a pivotal role in viral replication. The guanylate kinase is even a robust target in all investigated mutation variants currently spreading worldwide. These new insights can guide laboratory experiments for the validation of identified potential antiviral targets. Only the combination of vaccines and antiviral therapies will effectively defeat this ongoing pandemic.

SysMod: the ISCB community for data-driven computational modelling and multi-scale analysis of biological systems.

Computational models of biological systems can exploit a broad range of rapidly developing approaches, including novel experimental approaches, bioinformatics data analysis, emerging modelling paradigms, data standards and algorithms. A discussion about the most recent advances among experts from various domains is crucial to foster data-driven computational modelling and its growing use in assessing and predicting the behaviour of biological systems. Intending to encourage the development of tools, approaches and predictive models, and to deepen our understanding of biological systems, the Community of Special Interest (COSI) was launched in Computational Modelling of Biological Systems (SysMod) in 2016. SysMod's main activity is an annual meeting at the Intelligent Systems for Molecular Biology (ISMB) conference, which brings together computer scientists, biologists, mathematicians, engineers, computational and systems biologists. In the five years since its inception, SysMod has evolved into a dynamic and expanding community, as the increasing number of contributions and participants illustrate. SysMod maintains several online resources to facilitate interaction among the community members, including an online forum, a calendar of relevant meetings and a YouTube channel with talks and lectures of interest for the modelling community. For more than half a decade, the growing interest in computational systems modelling and multi-scale data integration has inspired and supported the SysMod community. Its members get progressively more involved and actively contribute to the annual COSI meeting and several related community workshops and meetings, focusing on specific topics, including particular techniques for computational modelling or standardisation efforts.

Curating and comparing 114 strain-specific genome-scale metabolic models of Staphylococcus aureus.

Staphylococcus aureus is a high-priority pathogen causing severe infections with high morbidity and mortality worldwide. Many S. aureus strains are methicillin-resistant (MRSA) or even multi-drug resistant. It is one of the most successful and prominent modern pathogens. An effective fight against S. aureus infections requires novel targets for antimicrobial and antistaphylococcal therapies. Recent advances in whole-genome sequencing and high-throughput techniques facilitate the generation of genome-scale metabolic models (GEMs). Among the multiple applications of GEMs is drug-targeting in pathogens. Hence, comprehensive and predictive metabolic reconstructions of S. aureus could facilitate the identification of novel targets for antimicrobial therapies. This review aims at giving an overview of all available GEMs of multiple S. aureus strains. We downloaded all 114 available GEMs of S. aureus for further analysis. The scope of each model was evaluated, including the number of reactions, metabolites, and genes. Furthermore, all models were quality-controlled using MEMOTE, an open-source application with standardized metabolic tests. Growth capabilities and model similarities were examined. This review should lead as a guide for choosing the appropriate GEM for a given research question. With the information about the availability, the format, and the strengths and potentials of each model, one can either choose an existing model or combine several models to create models with even higher predictive values. This facilitates model-driven discoveries of novel antimicrobial targets to fight multi-drug resistant S. aureus strains.