Natural flavonoid morin showed anti-bacterial activity against Vibrio cholera after binding with cell division protein FtsA near ATP binding site

Highlights

• Morin, a bioflavonoid, shows antibacterial activities with low cytotoxicity.

• Morin targets a major cell division protein, FtsA, in Vibrio cholerae.

• Morin binds to FtsA close to ATP binding site affecting the ATPase activity of FtsA and inhibiting its polymerization.

• Inhibition of FtsA polymerization affects the cell division of Vibrio cholerae altering its morphology to an elongated one.

• Morin could be a potent anti-bacterial agent against Vibrio cholerae.

Abstract

Background

Increasing antibiotic-resistance in bacterial strains has boosted the need to find new targets for drug delivery. FtsA, a major bacterial divisome protein can be a potent novel drug-target.

Methods and results

This study finds, morin (3,5,7,2′,4′-pentahydroxyflavone), a bio-available flavonoid, had anti-bacterial activities against Vibrio cholerae, IC₅₀ (50 μM) and MIC (150 μM). Morin (2 mM) kills ~20% of human lung fibroblast (WI38) and human intestinal epithelial (HIEC-6) cells in 24 h in-vitro. Fluorescence studies showed morin binds to VcFtsA (FtsA of V. cholerae) with a K_d of 4.68 ± 0.4 μM, inhibiting the protein's polymerization by 72 ± 7% at 25 μM concentration. Morin also affected VcFtsA's ATPase activity, recording ~80% reduction at 20 μM concentration. The in-silico binding study indicated binding sites of morin and ATP on VcFtsA had overlapping amino acids. Mant-ATP, a fluorescent ATP-derivative, showed increased fluorescence on binding to VcFtsA in absence of morin, but in its presence, Mant-ATP fluorescence decreased. VcFtsA-S40A mutant protein did not bind to morin.

Conclusions

VcFtsA–morin interaction inhibits the polymerization of the protein by affecting its ATPase activity. The destabilized VcFtsA assembly in-turn affected the cell division in V. cholerae, yielding an elongated morphology.

General significance

Collectively, these findings explore the anti-bacterial effect of morin on V. cholerae cells targeting VcFtsA, encouraging it to become a potent anti-bacterial agent. Low cytotoxicity of morin against human cells (host) is therapeutically advantageous. This study will also help in synthesizing novel derivatives that can target VcFtsA more efficiently.

Introduction

Cholera, caused by a gram-negative bacterium, Vibrio cholerae, is an acute infectious disease, characterized by profuse watery diarrhea. According to WHO reports (WHO media center report on Cholera), there are many serogroups of V. cholerae, but of them only two, O1 and O139, cause outbreaks across the globe. Previously, only V. cholerae serogroup O1 was considered to be the cause of epidemic outbreaks. However, in 1993, a new serogroup designated as V. cholerae serogroup O139 was first isolated from the Indian subcontinent region [1]. By routine treatment of a community with broad-spectrum antibiotics, or mass chemoprophylaxis, little effect was achieved on the control of the spread of cholera. This was due to the rapid surfacing of resistant strains [2]. The emergence of resistance against various antibiotics in V. cholerae is the major problem faced nowadays. It is thus necessary to find some potential drugs that can bind novel targets thereby blocking the spread of cholera with prolonging sensitivity.

FtsA is one of the major proteins present in the cell divisome complex of bacteria. It functions by anchoring the Z-ring (formed by FtsZ) to the cell membrane at mid-cell and facilitates the Z-ring constriction leading to effective septum formation during cytokinesis phase of cell division in bacteria [3]. The proteins of the divisome complex are potent anti-microbial targets because the hampering of cell division stalls the spread of virulent bacterial strains. In the past, few studies have aimed at targeting FtsZ (another major divisome protein) by screening potential small molecules [[4], [5], [6]]. This lacks in the case of FtsA. The in-detail study of FtsA from Vibrio cholerae (VcFtsA) [7] from our group has led us to think that this protein can be a novel target for new-generation drugs to control the spread of V. cholerae.

Morin (3,5,7,2′,4′-pentahydroxyflavone), a yellow natural bioflavonoid, is a major component of traditional medicinal plants like almond (Prunus dulcis), fig (Chlorophora tinctoria) and also other members of Moraceae family fruits like Indian guava (Psidium guajava). Morin and its derivatives are known as anti-microbial agents [[8], [9], [10]]. On the other hand, morin has low cytotoxicity [11,12]. Reports have also mentioned the protective effects of the flavonoid against chemotoxicity [11]. All these properties made morin a potent candidate, which can be tested for its anti-microbial effect targeting the divisome complex proteins. Taking advantage of the fact that there are no previous studies of morin and its effect on cell division proteins, we got interested in elucidating the mechanism of action of morin on V. cholerae N16961 cells. A comprehensive study on the effect of morin on VcFtsA has been made by us here. The binding parameters of the interaction and studying the nature of the binding site will help us in the future to synthesize new derivatives of morin that will have higher potency in targeting VcFtsA hence showing better efficacy in controlling the spread of Vibrio cholerae.