Inhibitory activity of curcumin against helicobacter pylori biofilms : the role of flagellar genes and comparative proteomics analysis

Abstract

Helicobacter pylori is a leading etiologic agent causing peptic ulcer and gastric cancer. The bacterium is shown to have alternate life style as a biofilm, which facilitates bacterial survival in the hazardous environments. The antimicrobial as well as anti-adhesive activities of curcumin (diferuloymethane) against H. pylori have been widely described. Objectives of this study were to investigate inhibitory effect of curcumin on H. pylori biofilm and mechanisms or genes, including flagellar genes that regulate the biofilm formation. The roles of flagellar genes, including flaA, flgR, and fliQ genes, in H. pylori were investigated by the construction of isogenic mutants using inverse PCR mutagenesis. The effect of curcumin was investigated on H. pylori wild type, flagellar mutants and ATCC43504 against biofilm formation both qualitatively by pellicle assay and quantitatively by crystal violet staining. Three-dimensional structure of biofilm was imaged by scanning electron microscopy (SEM). The effect of curcumin on H. pylori adherence to HEp-2 cells was also investigated. Six flagellar mutants were obtained, including PA315 and NA2 (flaA mutants), PR611 and NR2 (flgR mutants), and PQ and NQ (fliQ mutants). The flgR mutants formed a significant reduction of biofilm level compared to their wild types (ATCC26695 and N6) and other mutants. Additionally, an ability to adhere to the HEp-2 cells of both flgR and fliQ mutants was significantly decreased. Sub-minimum inhibitory concentrations (sub-MICs) of curcumin inhibited H. pylori biofilm in dose dependent manner. However, H. pylori could restore ability to form biofilm during extended time of incubation. SEM revealed a dense mature biofilm characterized by a presence of cells encased in extracellular polymeric matrix and connected together forming multicellular layers. The less amorphous matrix, slow of morphological conversion to coccoid form with cell damage was demonstrated after curcumin treatment. Curcumin significantly decreased the ability of H. pylori to adhere to the HEp-2 cells. A proteomics analysis was performed in order to investigate the difference in protein profile expression between biofilm and planktonic counterparts with an absence and presence of sub-MICs of curcumin. Proteins involving in biofilm formation in H. pylori belonged to chemotaxis and motility, chaperone, stress response, electron transport, nitrogen and carbohydrate metabolism, and metabolic intermediate biosynthesis. With a presence of 1/4 MIC curcumin, a chaperone group protein was up-regulated both in biofilm and planktonic counter parts. This is the first report showing genes, including flgL, flgE, flgD, fliD, flaA, tufA, tig, hsp60, grpE, efp, katA, trxB, tsaA, porB, fldA, ureA, ureB, acnB, gltA, and fadA, which being possibly involved in mechanism of H. pylori biofilm formation. The advantages of curcumin to inhibit biofilm formation and adherence by H. pylori, making it as an alternative complimentary medicine for curing of H. pylori-biofilm related infections.