RESUMO
SARS-CoV-2 entry into host cells is facilitated by the interaction between the receptor-binding domain of its spike protein (CoV2-RBD) and host cell receptor, ACE2, promoting viral membrane fusion. The virus also uses endocytic pathways for entry, but the mediating host factors remain largely unknown. It is also unknown whether mutations in the RBD of SARS-CoV-2 variants promote interactions with additional host factors to promote viral entry. Here, we used the GST pull-down approach to identify novel surface-located host factors that bind to CoV2-RBD. One of these factors, SH3BP4, regulates internalization of CoV2-RBD in an ACE2-independent but integrin- and clathrin-dependent manner and mediates SARS-CoV-2 pseudovirus entry, suggesting that SH3BP4 promotes viral entry via the endocytic route. Many of the identified factors, including SH3BP4, ADAM9, and TMEM2, show stronger affinity to CoV2-RBD than to RBD of the less infective SARS-CoV, suggesting SARS-CoV-2-specific utilization. We also found factors preferentially binding to the RBD of the SARS-CoV-2 Delta variant, potentially enhancing its entry. These data identify the repertoire of host cell surface factors that function in the events leading to the entry of SARS-CoV-2.
Assuntos
Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Domínios Proteicos , Células HEK293 , COVID-19/metabolismo , COVID-19/virologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Interações Hospedeiro-PatógenoRESUMO
Twenty-seven rosmarinic acid derivatives were synthesized, among which compound RA-N8 exhibited the most potent antibacterial ability. The minimum inhibition concentration of RA-N8 against both S. aureus (ATCC 29213) and MRSA (ATCC BAA41 and ATCC 43300) was found to be 6 µg/mL, and RA-N8 killed E. coli (ATCC 25922) at 3 µg/mL in the presence of polymyxin B nonapeptide (PMBN) which increased the permeability of E. coli. RA-N8 exhibited a weak hemolytic effect at the minimum inhibitory concentration. SYTOX Green assay, SEM, and LIVE/DEAD fluorescence staining assay proved that the mode of action of RA-N8 is targeting bacterial cell membranes. Furthermore, no resistance in wildtype S. aureus developed after incubation with RA-N8 for 20 passages. Cytotoxicity studies further demonstrated that RA-N8 is non-toxic to the human normal cell line (HFF1). RA-N8 also exerted potent inhibitory ability against biofilm formation of S. aureus and even collapsed the shaped biofilm.
Assuntos
Antibacterianos , Staphylococcus aureus Resistente à Meticilina , Humanos , Antibacterianos/química , Staphylococcus aureus , Ácido Rosmarínico , Escherichia coli , Relação Estrutura-Atividade , Testes de Sensibilidade Microbiana , BiofilmesRESUMO
We successfully developed a fluorescent drug sensor from clinically relevant New Delhi metallo-ß-lactamase-1 (NDM-1). The F70 residue was chosen to be replaced with a cysteine for conjugation with thiol-reactive fluorescein-5-maleimide to form fluorescent F70Cf, where "f" refers to fluorescein-5-maleimide. Our proteolytic studies of unlabeled F70C and labeled F70Cf monitored by electrospray ionization-mass spectrometry (ESI-MS) revealed that fluorescein-5-maleimide was specifically linked to C70 in 1:1 mole ratio (F70C:fluorophore). Our drug sensor (F70Cf) can detect the ß-lactam antibiotics cefotaxime and cephalothin by giving stronger fluorescence in the initial binding phase and then declining fluorescence signals as a result of the hydrolysis of the antibiotics into acid products. F70Cf can also detect non-ß-lactam inhibitors (e.g., l-captopril, d-captopril, dl-thiorphan, and thanatin). In all cases, F70Cf exhibits stronger fluorescence due to inhibitor binding and subsequently sustained fluorescence signals in a later stage. Native ESI-MS results show that F70Cf can bind to all four inhibitors. Moreover, our drug sensor is compatible with a high-throughput microplate reader and has the capability to perform in vitro drug screening.