Genome-Wide Fitness Signatures of MRSA Across Skin, Systemic, and Blood Infections

Staphylococcus aureus (particularly methicillin-resistant S. aureus (MRSA) is a leading cause of severe community and hospital-acquired infections worldwide. Its remarkable capacity to adapt to diverse host environments and resist multiple antimicrobials poses an urgent public health challenge. Understanding the genetic determinants that underpin MRSA fitness across distinct infection niches is essential for developing new therapeutic strategies. This thesis combines methodological innovation with in vivo and ex vivo functional genomics to map the metabolic rewiring and context-specific vulnerabilities of epidemic USA300 MRSA. The first study (Paper I) focused on optimizing the construction of a highly saturated transposon mutant (tn-mutant) library of MRSA, a prerequisite for robust transposon insertion sequencing (TIS) studies. Using a Himar1-based two-plasmid system, we enhanced phage ϕ11 transduction efficiency via chloramphenicol pretreatment and an in-house enriched medium and implemented an optimized plasmid curing protocol. These improvements yielded a library with over 400,000 unique insertions, the most saturated to date in S. aureus, providing comprehensive genome coverage for downstream applications. In the second study (Paper II), we applied Transposon-Directed Insertion Site Sequencing (TraDIS) in murine models of skin, kidney, and spleen infection to identify tissue-specific fitness genes. A three-step filtering approach identified 46, 76, and 69 genes required for fitness in skin, kidney, and spleen, respectively. The gluconeogenesis genes fbp was essential across all tissues, while pckA (kidney) and gapB (spleen) displayed organ-specific essentiality. Skin infection uniquely depended on oxidative stress and DNA repair genes (ahpC, ahpF, dps, uvrC, xseA), whereas systemic infection relied on branched-chain amino acid catabolism (bkdAB), lipid metabolism (SAUSA300_0355), and putative polyamine biosynthesis (SAUSA300_0458). The third study (Accepted Manuscript III) investigated MRSA adaptation to the intravascular environment using freshly collected human blood. TraDIS identified 76 essential genes. As proof of concept, competition assays in blood confirmed the fitness defects of purA, purB, fbp, hssR, and aroA2 genes. Interestingly, disruption of certain regulators (saeRS, σ^B) and adhesins (fnbA, clfA) genes conferred a competitive advantage, revealing a potential trade-off between virulence regulation and survival in blood. Collectively, the findings of this thesis establish a genome-scale framework of MRSA fitness requirements across tissue and blood environments, highlight conserved and niche-specific metabolic dependencies, and identify novel vulnerabilities for therapeutic targeting. By integrating methodological advances in tn-mutant library construction with high-resolution in vivo and ex vivo functional genomics, this work expands our understanding of MRSA adaptation and paves the way for targeted interventions against this formidable pathogen.

Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA), is a major cause of severe community- and hospital-acquired infections worldwide. Its ability to adapt to diverse host environments and resist multiple antimicrobials represents a critical public health challenge. Defining the genetic determinants that sustain MRSA fitness across infection niches is essential for developing new therapeutic strategies. This thesis integrates methodological innovation with in vivo and ex vivo functional genomics to define metabolic rewiring and context-specific vulnerabilities of the epidemic USA300 lineage. Paper I optimized construction of a highly saturated transposon mutant library, a prerequisite for robust transposon insertion sequencing. Using a Himar1 two-plasmid system, we enhanced phage ϕ11 transduction through chloramphenicol pretreatment, enriched medium, and improved plasmid curing. The library contained >400,000 unique insertions, achieving high genome saturation in S. aureus and enabling fitness profiling. Paper II applied TraDIS in murine skin, kidney, and spleen infection models. A three-step filtering strategy identified 46, 76, and 69 fitness genes in skin, kidney, and spleen, respectively. The gluconeogenic gene fbp was required across tissues, whereas pckA (kidney) and gapB (spleen) showed organ-specific essentiality. Skin infection depended on oxidative stress and DNA repair pathways (ahpCF, dps, uvrC, xseA), while systemic tissues relied on branched-chain amino acid catabolism (bkdAB), lipid metabolism (SAUSA300_0355), and putative polyamine biosynthesis (SAUSA300_0458). Paper III examined MRSA adaptation to human blood. TraDIS identified 76 fitness determinants. Competition assays validated defects in purA, purB, fbp, hssR, and aroA2. Disruption of regulators (saeRS, σB) and adhesins (fnbA, clfA) conferred competitive advantage, suggesting a trade-off between virulence regulation and intravascular survival. Collectively, this work establishes a genome-scale framework of MRSA fitness across tissues and blood, revealing conserved metabolic backbones and niche-specific therapeutic vulnerabilities