Inhibition of Major Virulence Pathways of Streptococcus mutans by Quercitrin and Deoxynojirimycin: A Synergistic Approach of Infection Control

Khan AU (2014) Inhibition of Major Virulence Pathways of Streptococcus mutans by Quercitrin and Deoxynojirimycin: A Synergistic Approach of Infection Control. PLoS ONE 9(3): e91736. doi:10.1371/journal.pone.0091736 Inhibition of Major Virulence Pathways of Streptococcus mutans by Quercitrin and Deoxynojirimycin: A Synergistic Approach of Infection Control Sadaf Hasan 0 Kunal Singh 0 Mohd Danisuddin 0 Praveen K. Verma 0 Asad U. Khan 0 Gunnar F. Kaufmann, The Scripps Research Institute and Sorrento Therapeutics, Inc., United States of America 0 1 Medical Microbiology and Molecular Biology Lab, Interdisciplinary Biotechnology Unit, Aligarh Muslim University , Aligarh , India , 2 National Institute of Plant Genome Research, Aruna Asaf Ali Marg , New Delhi , India Objectives: To evaluate the synergistic effect of Quercitrin and Deoxynojirimycin (DNJ) together with their individual inhibitory effect against virulence pathways of Streptococcus mutans. Methodology: MICs of both the compounds were determined by the microdilution method, followed by their in vitrosynergy using checkerboard and time kill assay. The nature of interaction was classified as synergistic on the basis of fractional inhibitory concentration index (FICI) value of #0.5. Furthermore, the activity of Quercitrin and DNJ was evaluated individually and in combination against various cariogenic properties of S. mutans UA159 such as acidogenesis, aciduracity, glucan production, hydrophobicity, biofilm and adherence. Moreover, expression of virulent genes in S. mutans was analysed by quantitative RT- PCR (qRT-PCR) and inhibition of F1F0-ATPase, lactate dehydrogenase and enolase was also evaluated. Finally, scanning electron microscopy (SEM) was used to investigate structural obliteration of biofilm. Results: The in vitro synergism between Quercitrin and DNJ was observed, with a FICI of 0.313. Their MIC values were found to be 64 mg/ml and 16 mg/ml respectively. The synergistic combination consistently showed best activity against all the virulence factors as compared to Quercitrin and DNJ individually. A reduction in glucan synthesis and biofilm formation was observed at different phases of growth. The qRT-PCR revealed significant downregulation of various virulent genes. Electron micrographs depicted the obliteration of biofilm as compared to control and the activity of cariogenic enzymes was also inhibited. Conclusions: The whole study reflects a prospective role of Quercitrin and DNJ in combination as a potent anticariogenic agent against S. mutans. - Funding: This study was supported by internal funds of Interdisciplinary Biotechnology Unit, Aligarh Muslim University and Indian Councel of Medical Research grant no. 59/6/2009/BMS/TRM to AUK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Dental caries is a multifactorial infection, is characterized by progressive destruction of tooth enamel [1].S. mutans, a member of the oral micro flora, is considered to be the primary causative agent of dental caries (or tooth decay) and is one of the best known biofilm forming bacterium [2]. It has also been reported that S. mutans adhere to damaged cardiac tissues which is marked as a significant event in the pathogenesis of chronic infective endocarditis [3], with a death rate of up to 50% in spite of antibiotic treatments [4]. The aetiology of dental caries is associated with (i) bacterial fermentation of dietary carbohydrates resulting in acid production (ii) its ability to produce glucosyltransferases (GTFs), which leads to the synthesis of intracellular polysaccharides (IPS) and extracellular polysaccharides (EPS) and (iii) its attachment to the tooth pellicle mediated by glucans [5,6]. The combination of these characteristic mechanisms acquired by S. mutans aid its effective colonization in the oral cavity and regulate the transformation from non-pathogenic to extremely cariogenic dental plaque biofilms [7]. Other fundamental cariogenic properties of S. mutans include the membrane-bound F1F0-ATPase system, lactate dehydrogenase and enolase. The membrane bound F1F0-ATPasesystem protects S. mutans against environmental acid stress by regulating pH homeostasis. This attribute determines the aciduracity or acid tolerance capability in S. mutans [8]. Lactate dehydrogenase (LDH) responsible for producing lactic acid and enolase (a glycolytic enzyme) produces phosphoenolpyruvate (PEP), which is a key component of the PEP:carbohydrate phosphotransferase system (PTS), further contributes to the promotion of cariogenecity by S. mutans [9].Several studies have shown that mutants of S. mutans which lack aforementioned virulence factors, are less cariogenic being more susceptible to different environmental stresses as compared to the wild strain [10,11].There is an abundance of Indian medicinal herbs that are employed for the treatment of dental caries as they bear low or no toxicity, albeit the pure compounds have mostly reported to show better activity than the crude extracts. Supportingly, a study demonstrated that purified compound of M. alba showed an 8-fold greater reduction of MIC against S. mutans than the crude extract [12]. The quest for plants with medicinal properties will continue to receive attention but taking into consideration the recent emergence of microbes which are unaffected by most of the antimicrobial drugs and posing a challenge for the treatment of infections [13], there is an urgent need to come up with new antimicrobials which are less toxic and more efficient in combating such microorganisms. Another effective approach is described by combinational therapy which has been proved to be an effective alternative to monotherapy [14,15]. Moreover, studies have demonstrated that those compounds which failed initially as antimicrobials, drastically enhanced the effectiveness ofother chemotherapeutic agent [16].Therefore, we have conducted this study using purified plant based compounds Quercitrin and Deoxynojirimycin (DNJ) in order to meet the need of efficiency with low toxicity levels [12,17].Earlier studies have reported the significance of Quercitrin and DNJ in medicine. Quercitrin (quercetin 3- O-alpha- L-rhamnopyranoside), a flavonoid glycoside has been reported by many researchers for its wide array of pharmacological activities such as, anti-inflammatory [18], antileishmanial [19], antimelanogenic [20],prevention oflipid peroxidation [21] and protectiveagainst UVB-induced oxidative damage of skin [22].However, its activity against cariogenic properties of S. mutans has not been explored.Moreover, DNJis known to prevent diet-induced obesity [23], hepatitis C virus [24], modulate glucose metabolism and has anti-diabetic effects [25].DNJ, nevertheless, is known for its anti-biofilm effect but its activity and mechanism of action as pure co (...truncated)