Clinical MicrobiologyAnaerobe/aerobe environmental flux determines protein expression profiles of Bacteroides fragilis, a redox pathogen
Introduction
Abrupt displacement of Bacteroides fragilis from an intestinal reducing to a peritoneal oxidizing environment can result in peritonitis. This clinical observation suggested that B. fragilis would respond to changes in oxidation-reduction (redox) conditions. Previous attempts to clarify this observation based on redox levels, designated by Eh in millivolts (mV) adjusted to pH 7 (Eh7), have revealed a potential pathogenic characteristic. Three-dimensional (3D) confocal laser scanning microscopy (CLSM) indicated that B. fragilis penetrated into Hela cell monolayers [1]. This redox effect occurred for the bacteria poised for growth under oxidizing but not reducing conditions. When B. fragilis bacteria were cultured under oxidizing conditions, examination by scanning electron microscopy had shown that the bacteria were widely dispersed, but under reducing conditions they were densely aggregated [2]. Using appropriate software, an adapted algorithm was applied that separated the Hela cell image into regions of nucleus and cytoplasm [3]. CLSM examination allowed the necessary subtle information to be extracted thus shaping and limiting the cytoplasmic area for treatment of consecutive laser cuts which enabled this image to be reassembled in 3D [4]. Then, it could be demonstrated that B. fragilis bacteria grown under oxidizing conditions were located inside the Hela cell.
In earlier CLSM studies, the B. fragilis bacteria in preparation for interaction with Hela cells were poised for growth at either frankly reducing, mildly oxidizing or relatively oxidizing conditions by varying the concentration of cysteine as the redox reagent [5]. Coupling metabolic energy to membrane potential results in the fine tuning of metabolism to environmental changes. These are perceived to control gene expression when presented with changes in redox environment [6]. Hence, B. fragilis cells will undergo significant alterations in their physiology while growing in aerobic versus anaerobic environments. Both input and internal signals explain how signaling molecules, which are based on communication modules that send and receive protein phosphorylation signals, enable bacteria to transduce adaptive responses [6], [7]. When grown under frankly reducing conditions (Eh7 ca. - 60 mV), no bacteria were evident within Hela cells; in contrast under mildly oxidizing conditions (Eh7 ca. + 20 mV) bacterial cells were observed to project from the Hela cell surface, and by comparison under relatively oxidizing conditions (Eh7 ca. + 100 mV), B. fragilis bacteria penetrated into the Hela cells. Thus, when responding to particular redox conditions [1], [3], this extracellular microorganism of the intestinal flora produces an unexpected biological response: distinct bacterial penetration (Diagram 1a).
The same procedures were followed for bacterial cultures grown under oxidizing and reducing conditions. The most recent results suggested that extracts of bacterial outer membrane proteins (OMP) expressed at the cell surface of B. fragilis were influenced by the Eh [8]. Gel electrophoresis (SDS-PAGE) of extracted OMP preparations would be expected to separate many protein components. The protein components were silver stained to assess, by densitometry, the areas under the curves and differences in the protein profiles for the bacteria using the XY Extract Graph Digitizer Program [9]. The “xyExtract” software is used for the extraction of data from a 2D graph contained in a graphic file, while “xyExtract” converts the graph back to the xy data file (up to 1500 points).
Section snippets
Bacterial strains
Three clinical strains of B. fragilis (deposited at the Cell Culture Collection of the Anaerobic Bacteria Laboratory, Instituto de Microbiologia Prof. Paulo de Góes – IMPPG, UFRJ, Brasil) were used in the present studies. These strains were 1081, taken from blood hemoculture, MC2 from osteomyelitis and RBG-A from vaginal tract (normal microbiota).
Culturing of B. fragilis, preparation and electrophoresis (i.e. SDS-PAGE) of bacterial outer membrane protein (OMP) extracts and densitometry analysis of electrophoresis separations [8]
Culture conditions - The strains were first grown in brain heart infusion (BHI, Sigma Co) under anaerobic conditions [2], [10], consisting of previously
Analaysis of the OMP extracts for B. fragilis strains 1081, MC2 and RBG-A under oxidizing and reducing conditions
The SDS-PAGE profiles of the OMP extracts for the three strains of B. fragilis, 1081, MC2 and RBG-A grown under oxidizing and reducing conditions are shown in Fig. 1A, B and C. The corresponding densitometry values are given for the three strains in Fig. 2A, B and C, while their respective (XY extraction) analyses are shown in Table 1B, Table 1C, Table 1A.
Graphical curves and Regions analyzed by the XY Extract Graph Digitizer Program [9]
The gel electrophoresis (GE series), i.e. SDS-PAGE, of OMP preparations separated many components resembling those already reported for B.
Oxidizing and reducing comparisons
The discerning OMP analysis and densitometry results (Fig. 1A, B and C; Table 1B, Table 1C, Table 1A) based on the graphical curves reflected the differences exerted between oxidizing and reducing conditions of B. fragilis bacterial growth. These involved the two infectious strains (1081, MC2) and the one non-infectious strain (RBG-A). The findings were influenced by the recent gel electrophoresis result on separated OMP bands [8] from the ATCC strain 43859 cultured under oxidizing and reducing
Conclusion
The anaerobe/aerobe environmental flux during the process of an anaerobic infection could be significant (e.g. B. fragilis). Outer membrane protein (OMP) profiles from bacterial extracts revealed that the overall profiles of OMP for the infectious strains were statistically different when comparing oxidizing and reducing conditions but this difference was marginal for the non-infectious strain. The effect of the redox level thus displays some differentiation in the bacterial OMP extract.
Acknowledgement
The authors wish to express their gratitude for the technical assistance given by Joaquim dos Santos (Laboratory of Medical Microbiology, Rio do Janeiro, Brasil), Ilario Corna (Brock University, St. Catharines, Canada) and a special thank you to Darren Peters (Brock University, St. Catharines, Canada) for his detailed calculations. The authors also extend special thanks to Mathematics Professor Henryk Fuks, Brock University, for directing attention to the XY Extract Graph Digitizer Program, and
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