## 3 Discussion  
It is well recognized that EC damage plays a crucial role in the pathogenesis of _S. aureus_ endovascular infection [5; 13; 14]. For instance, we have demonstrated a positive correlation between in vitro EC damage and virulence, as well as antibiotic treatment persistent outcome in an experimental endocarditis model caused by clinical MRSA isolates [5]. In addition, we also noticed that clinical MRSA strains collected from patients with persistent bacteremia cause significantly greater EC damage compared to clinical resolving MRSA isolates [15]. Moreover, the inactivation of _agr_, _saeR_, and _arlSR_ has been proved significantly reduce EC damage as compared to their respective parental strains [13; 16]. However, these studies only focused on a few virulence factors in _S. aureus_. Thus, the current study was designed to broadly define genetic determiners in _S. aureus_ which involve in human EC damage using a high-throughput approach to screen a transposon mutant library containing 1920 non-essential gene mutants in MRSA USA300 JE2 background. In the current study, we first verified the reliability of our high-throughput screening system. Consistent with previous reports [13; 16], we demonstrated that the inactivation of global regulators such as _agr_, _arlRS_, or _saeRS_ significantly decreases EC damage. In  
Figure 2: Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the mutant strains significantly decreasing HMEC-1 damage rate: (**A**) genes were identified in the KEGG database and belonged to four major KEGG pathways; (**B**) the sub-pathway enrichment analysis of the genes in the metabolism pathway; and (**C**) the sub-pathway enrichment analysis of the genes in the other three pathways. addition, consistent results were obtained between 384-well and 24-well plates assays, which validated the improvement of testing significantly more samples each time. Several interesting and important observations emerged from the present investigations. Overall, over 320 mutants had a significant impact on the EC damage. The majority of these mutants significantly reduced EC damage vs. JE2 parental strain. Using KEGG pathway analysis, mutant strains were classified into four categories, including metabolism, genetic information processing, environmental information processing, and cellular processes (Figure 3). Only six mutants were found with significantly increased EC damage vs. JE2 parental strain. Importantly, many of these genes are not previously defined to impact human EC damage in _S. aureus_. Many staphylococcal genetic factors related to metabolism were shown to intimately impact the EC damage. For instance, several gene mutants related to carbohydrate metabolism including tricarboxylic acid (TCA) cycle (e.g., _pdhA_, and _lpdA_) showed significantly decreased EC damage. Inactivation of _pdhA_ or _lpdA_ was reported to be associated with slower growth [17; 18]. Since the TCA cycle processes produce the main energy resources for cellular activities [19], inactivation of corresponding TCA genes may result in lack of energy which may subsequently cause slower growth and decrease EC damage. In addition, mutants with genes related to energy metabolism (e.g., _cyoE_, and _aptH_) also displayed lower EC damage rates vs. parental strain JE2. It has been reported that _cyoE_ encoding a proteome IX farnesyltransferase is essential for processing heme into the electron transport chain and plays a critical role in cytolytic toxins production in _S. aureus_. Deletion of _cyoE_ in _S. aureus_ significantly decreases the expression of cytolytic toxins [20]. [PERSON] et al. reported that mutation of _aptH_ (associated with ATP synthase) had attenuated virulence and less invasiveness in vivo [21]. These results suggest that genetic factors associated with energy metabolism have activities on EC damage that may link to virulence. Figure 3: Genetic factors in MRSA JE2 strain contribute to the HMEC-1 damage by KEGG analysis.