Staphylococcus aureus (S. aureus), a Gram-positive facultative anaerobic bacterium, has the potential to become a highly virulent opportunistic pathogen, causing a broad spectrum of infections, including severe life-threatening diseases like pneumonia, sepsis, and endocarditis. The clinical threat posed by S. aureus is exacerbated by its capacity to develop resistance to multiple antibiotics. A prime example of this is the acquisition of the mecA gene, which encodes penicillin-binding protein 2a (PBP2a), a variant with a significantly lower affinity for β-lactam antibiotics. Such a capacity allows the bacterium to continue synthesizing its cell wall even in the presence of these antibiotics, leading to the emergence of methicillin-resistant S. aureus (MRSA) (Peacock and Paterson, 2015, Wielders et al., 2002). The pathogenicity of S. aureus is attributed to a variety of virulence factors, including surface proteins that promote adherence to host tissues, secreted toxins that mediate tissue damage, and enzymes that facilitate immune evasion. The widespread prevalence of MRSA strains, particularly in healthcare settings, underscores the importance of stringent infection control measures and the prudent use of antibiotics to mitigate the spread of these resistant pathogens (Taylor and Unakal, 2025). Further, besides being a member of the human microbiome, S. aureus is a member of highly virulent and antibiotic-resistant bacteria – a group so-called ESKAPE (Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp) pathogens.

MRSA can cause community-acquired and predominantly healthcare (hospital)-acquired infections ranging from mild superficial to invasive life-threatening (Ma et al., 2020, Taylor and Unakal, 2025). As a formidable pathogen, MRSA is capable of causing severe infections such as necrotizing pneumonia, sepsis, osteomyelitis, and infective endocarditis, particularly in previously healthy individuals (Shoaib et al., 2022). Recent efforts to control MRSA have focused on enhancing infection prevention, improving diagnostic capabilities, and developing novel therapeutic strategies, including de novo antibiotics and vaccines. In this line, therapeutically, new antibiotics like linezolid, tigecycline, and daptomycin have been introduced to combat MRSA infections (Shoaib et al., 2022). Investigations into antibiotic resistance have further highlighted the critical need for alternative treatments such as phage therapy and antimicrobial peptides to combat multidrug-resistant S. aureus strains (Michalik et al., 2025). A meta-analysis focusing on low- and middle-income countries has demonstrated that while the overall colonization rates of S. aureus are similar to those in high-income regions, the incidence of resistance and virulence factors is disproportionately higher (Locke et al., 2025). Studies in specific population groups suggest that although identifying as a man who has sex with men is not an independent risk factor, engaging in high-risk behaviors can promote the spread of community-acquired MRSA (de Jong et al., 2025).

Moreover, immunization approaches using killed whole-cell vaccine formulations with appropriate adjuvants have elicited strong immune responses, underscoring their potential in preventing serious S. aureus infections (Bagherzadeh et al., 2025), which highlights the importance of novel vaccine development.

Notably, MRSA has been a leading cause of nosocomial (hospital-acquired) infections, significantly contributing to patient morbidity, mortality, and healthcare costs worldwide. In 2017, the U.S. recorded an estimated 119,247 S. aureus bloodstream infections and 19,832 related deaths. From 2005–2012, hospital-onset MRSA infections declined by 17.1 % annually, but this decline slowed between 2013 and 2016. Community-onset MRSA dropped by 6.9 % annually from 2005 to 2016. Hospital-onset MSSA rates remained stable (p = 0.11), while community-onset MSSA infections rose by 3.9 % annually from 2012 to 2017 (Kourtis et al., 2019). Globally, MRSA accounts for nearly 20 % of S. aureus infections, with prevalence rates varying by region, i.e., higher in North America and parts of Asia compared to Northern Europe (Lee et al., 2018). S. aureus-origin bacteremia has a mortality rate of greater than 25 % within three months (Bai et al., 2022). Recent data shows that MRSA bacteremia is associated with a 90-day mortality rate of 18.3 %, with a significantly higher risk of mortality compared to methicillin-susceptible S. aureus, reflected by a pooled odds ratio of 2.35 and hazard ratio of 1.61 (Adeiza and Aminul, 2024). In a study, heterogeneous IgG and IgA responses to 79 S. aureus antigens in 996 individuals were quantified using a seven-order-of-magnitude multiplex array and found that colonization and host factors only partially explain the extensive variability underpinning potential disease-specific diagnostic signatures (Meyer et al., 2021).

Broker group recently reported that S. aureus serine protease-like proteins (especially SplA and SplE) and the iron-regulated surface protein IsdB enhance inflammatory responses in keratinocytes and innate immune cells (via IL-8 induction and TLR4/inflammasome activation respectively), highlighting their roles in immune modulation and implications for vaccine development against this multidrug-resistant pathogen (De Donato et al., 2024, Gonzalez et al., 2024). Despite the observed immunoprotective efficacy of anti-S. aureus vaccines in animal models, there has been limited success documented in human clinical trials. Notably, S. aureus superantigens (SAg), as highly potent immune activators, play a key role in allergic diseases, nosocomial infections, and severe chronic airway inflammation (Abdurrahman et al., 2020). Conspicuously, vaccine development targets key SAgs and virulence factors, which may offer a promising strategy to prevent invasive infections and reduce MRSA-related deaths. Further, it is hypothesized that forthcoming vaccine developments might leverage gene-editing technologies (Omidi et al., 2022). This could involve the inactivation of pivotal virulence factors, facilitating the creation of engineered live attenuated vaccines (A. Salemi et al., 2021), nucleic acid-based vaccines, and novel vaccine design and delivery strategies (Jin et al., 2023, Pourseif et al., 2022).

In recent scientific investigations, S. aureus proteome has been explored through the application of network-based reverse screening methodologies. Central to this endeavor is the emphasis on analyzing undirected protein-protein interaction networks (PPINs) characterized by (i) nodes, representing individual proteins, and (ii) edges, denoting pairwise interactions between these proteins. As a result, PPINs are counted as prevailing tools to model biological complexes (Huang et al., 2018, Pizzuti and Rombo, 2014).

Thereby, the significant measures of PPINs (e.g., degree, betweenness, bottleneck, etc.) can be calculated to predict potential vaccine candidates (PVCs) by deciphering important physical and/or functional interplays, pathogenesis-related pathways/mechanisms, and core functional clusters in different cellular compartments of an organism (Cheng et al., 2018, Cho et al., 2016). In the field of network interactomics, a significant emphasis is placed on the identification and isolation of proteins that exhibit high degrees of connectivity, which are often referred to as "hub nodes". Some proteins also serve as crucial connecting points within the network, which are termed "bottlenecks." Both hub nodes and bottlenecks play pivotal roles in ensuring the structural and functional integrity of PPINs (Yu et al., 2007), which often represent the most vital proteins involved in bacterial survival and pathogenesis machinery. High-throughput interactomics can be used not only for the deep categorization of complex PPINs into the two subsets of intracellular and extracellular proteins but also for the quantitative sorting of labeled proteins based on their function in bacterial pathogenesis. Vital cellular proteins typically function through interactions with other proteins based on the "guilt-by-association" principle, where the role of an unknown protein in a direct protein-protein interaction (PPI) can be inferred from its well-annotated neighbors (Z. C. Li et al., 2016), especially in the co-clustered sub-networks (Masoudi-Sobhanzadeh et al., 2023). These less-known proteins after a comprehensive pathway and functional enrichment may be identified as novel vaccine targets. Within this context, the integration of complementary gene ontology and clustering analysis serves to provide a more precise annotation of the proteome and reduces possible biases.

Several in silico studies have used whole genome and/or proteome data of pathogens to predict PVCs (Hajialibeigi et al., 2021; J. Li et al., 2021). Rawal et al. developed a web-based platform to predict pathogen’s PVCs using 17 different bioinformatics and immunoinformatics parameters (Rawal et al., 2021). However, here we have developed a novel platform to reversely screen the interactome network of the S. aureus reference proteome with the overall aim of identifying PVCs. In addition, to improve the reliability of the computational measures, the experimental findings relating to the S. aureus PVCs were employed as quantified values. In the current study, to identify PVCs, the S. aureus proteome was interpreted as a complex system of interrelated interactions that can accomplish hub proteins. As proof of the effectiveness of the overall approach, we followed our analysis with the measurement of various network centralities, high-throughput gene ontology, and interactome clustering of the network elements to rank high degree (i.e., hub) and high betweenness (i.e., bottleneck) nodes. Then, the explored proteins were subjected to the second round of customized screenings based on the parameters, including pathogen-host interactions (PHIs), essentiality, virulence, antigenicity, allergenicity, similarity to the hosts’ proteome, strain-specific sequence variability (by multiple sequence alignment) and expression level in 12 most common S. aureus strains (by Blastp tool).

All in all, in this study, network-based interactomics (Aysan Salemi, 2020), as a promising platform, was exploited for high-throughput proteome enrichment and the discovery of some less-known/unknown proteins with possible characteristics of becoming PVCs against S. aureus. One notable limitation of this approach may lie in its inherent dependence on annotated functional data, which inevitably leads to the exclusion of proteins with unknown or poorly characterized functions. This study, while being informative, may not fully capture the complexity of in vivo immune responses, and hence, experimental validation and broader genome-based strategies are essential to further validate the initial computational framework.