Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

G Thedei Jr,M Bolean,D.P.S Leitão,A.C.C Spadaro,T.P Paulino,P Ciancaglini

doi:10.1590/s0100-879x2008001200002

Abstract

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 microM) and oligomycin (4 microg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.

Highlights

Dental caries have long been recognized as an infectious disease related to the presence of dental plaque [1]
The main objective of the present research was to determine if toluene permeabilization can affect F- and Ptype ATPase activities in S. mutans cells and membrane fractions isolated from these cells
In order to evaluate the extent of bacterial cell damage caused by toluene treatment, samples of toluenized and intact cells were submitted to electron microscopy and details of cellular structure were compared (Figure 1)

Summary

Introduction

Dental caries have long been recognized as an infectious disease related to the presence of dental plaque [1]. Dental diseases pose a distinct challenge when it comes to determining their microbial etiology. Disease occurs at sites with a pre-existing natural and diverse microflora, while even more complex but distinct consortia of microorganisms are implicated in the pathology [2]. The oral bacteria that are most closely associated with caries development are the mutans streptococci, mainly Streptococcus mutans and S. sobrinus, noted for their ability to produce acids from the fermentation of dietary carbohydrates, which are directly associated with tooth mineral www.bjournal.com.br dissolution [3]. S. mutans adheres to the tooth surface and forms dental plaque, a classic biofilm. The pH of the oral cavity is predominantly acidic; within the dental plaque, S. mutans induces an acid-tolerance response that favors its survival under low pH conditions [4]

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Methods

Results