###### Abstract  
Methicillin-resistant _Staphylococcus aureus_ (MRSA) is a leading cause of life-threatening endovascular infections. Endothelial cell (EC) damage is a key factor in the pathogenesis of these syndromes. However, genetic factors related to the EC damage have not been well studied. This study aims to identify genetic determinants that impact human EC damage by screening the genome-wide Nebraska Transposon Mutant Library (NTML). A well-established MTT assay was used to test the in vitro damage of human EC cell line (HMEC-1) caused by each mutant strain in the NTML. We first confirmed some global regulators and genes positively impact the EC damage, which is consistent with published results. These data support the utility of the high-throughput approach. Importantly, we demonstrated 317 mutants significantly decreased the EC damage, while only 6 mutants enhanced the EC damage vs. parental JE2 strain. The majority of these genes have not been previously defined to affect human EC damage. Interestingly, many of these newly identified genes are involved in metabolism, genetic and environmental information processing, and cellular processes. These results advance our knowledge of staphylococcal genetic factors related to human EC damage which may provide novel targets for the development of effective agents against MRSA endovascular infection.  
M 2022 1136  
we have recently demonstrated a positive correlation between in vitro human EC damage and virulence, as well as vancomycin treatment persistent outcome in an experimental endocarditis model caused by clinical MRSA isolates [5]. However, little is known about the genetic factors involved in the EC damage in _S. aureus_.  
The Nebraska Transposon Mutant Library (NTML) consists of 1920 sequence-defined transposon insertion mutants of non-essential genes in a community-associated (CA) MRSA USA300 strain, JE2 [6]. This library has been used for screening several biological phenotypes, including hemolysis, proteolysis, carotenoid pigment formation, antibiotic susceptibility, and biofilm formation [6; 7; 8]. These investigations demonstrate that the NTML may serve as a valuable genetic tool to study host-pathogen interaction.  
Numerous investigations have used human umbilical vein EC (HUVECs) to study microbial-EC interactions. However, the use of HUVECs requires a constant supply of umbilical cords, and there are significant donor-to-donor variations in these ECs. To overcome these difficulties, immortalized ECs, including human microvascular EC (HMEC-1), have been developed. These cell lines have better availability and less variability [9]. In addition, we previously compared _S. aureus_ EC damage with HMEC-1 cell line and HUVECs, and found HMEC-1 cells were more susceptible to damage caused by _S. aureus_ vs. HUVECs [10]. In addition, the HMEC-1 cell line has been used to study the EC interactions with multiple microorganisms, including _S. aureus_[10; 11; 12]. Thus, in the current investigation, the HMEC-1 cell line was employed to test the impact of all the mutant strains in the NTML on its damage.  
In the current study, we aimed to identify staphylococcal genes associated with the EC damage by performing an unbiased genome-wide screening of all mutations in the NTML. This study will remarkably advance our understanding of staphylococcal genetic factors related to human EC damage which may provide novel targets for the development of effective compounds against MRSA endovascular infections.