Effect of Vitamin K3 Inhibiting the Function of NorA Efflux Pump and Its Gene Expression on Staphylococcus aureus.

Saulo R Tintino,Gabriel C A Da Hora,Maria C P Lima,Cícera Datiane De M Oliveira-Tintino,José G M Da Costa,José P Siqueira-Junior,Henrique D M Coutinho,Irwin R A Menezes,Pedro S Pereira,Fabíola F G Rodrigues,Veruska C A De Souza,Antonio Pereira-Neves,Tereza C Leal-Balbino,Valdir Q Balbino,Julia M A Da Silva

doi:10.3390/membranes10060130

Saulo R Tintino, Gabriel C A Da Hora + Show 13 more

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https://doi.org/10.3390/membranes10060130

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Abstract

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus, is synthesized by the expression of the norA gene. Menadione, also known as vitamin K3, is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

Highlights

Antibiotics used for several years were effective against countless infectious diseases caused by several different microorganisms, such as Staphylococcus aureus [1]
The objective of the present study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition, and to show how its action on the cell membrane is associated with the inhibition of this pump using transmission electron microscopy
The results showed the norA gene had its expression significantly diminished in the presence of menadione (Figure 4/Table 2)

Summary

Introduction

Antibiotics used for several years were effective against countless infectious diseases caused by several different microorganisms, such as Staphylococcus aureus [1]. Staphylococcus aureus is an important cause of community bacterial infections and is related to infections in hospitals [2]. After introduction of these agents into clinical large-scale use, the emergence of fluoroquinolone-resistant S. aureus was observed [5]. One of the main mechanisms related to antibiotic resistance involves membrane proteins that pump drugs from the bacterial cell interior out of the cell. It is believed that the drugs are “accidental substrates” of these transporters These proteins can be found in Gram-positive and Gram-negative bacteria. They are found in eukaryotic cells [7]

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