Effect of Iron Depletion on Protein Profiles of Bilophila wadsworthia

Abstract

Pathogenicity of microorganisms is a multifactorial phenomenon. Host environment plays a role in the development of the infectious process. The presence of key nutrients, such as iron, may be important for the expression of virulence factors. Iron is an essential component of a wide variety of biochemical processes in microorganisms, and its availability is of major importance in bacterial pathogenesis. In response to a low level of available iron in vivo, the creation of high-affinity iron-uptake systems and of a number of iron-regulated membrane proteins is induced. Although relationships between iron and virulence factors of aerobes have been better studied, a few such studies have been carried out with regard to anaerobes (mainly Bacteroides fragilis) [ 1, 2]. As discussed by Duerden [3], there is as yet no clear understanding of many of the virulence factors of anaerobes. Little is known about the mechanisms of iron uptake used by anaerobic bacteria, and nothing about those of Bilophila wadsworthia. The purpose of this work was to study the properties of this recently recognized anaerobe in relation to iron metabolism. B. wadsworthia is an anaerobic, gram-negative, asaccharolytic, bileresistant, and strongly catalase-positive bacillus [4]. This bacterium was first isolated from patients with gangrenous and perforative appendicitis. This study sought to determine whether B. wadsworthia expressed iron-regulated outer-membrane proteins (OMPs) in an iron-depleted environment and whether these proteins were immunogenic. Iron-free reagents and materials were used to prepare the ironfree medium. In this work, all glassware was deferrated. Chemicals used to prepare the medium were of highest purity. The medium was one previously described, to which oxgall (2%) and pyruvate (10 mM) were added [5]. The medium components were deferrated with Chelex-100 resin (Bio-Rad Laboratories, Hercules, CA), except for MgSO4, amino acids, and vitamins. Medium without iron contained <0.04 tM Fe+++. Iron was added to a concentration of 100 iM in preparation of the ironcontaining medium. The final pH was adjusted to 7.0-7.1. Four strains of B. wadsworthia originally isolated from appendicitis case specimens were studied, including the reference strain ATCC 49260. They were all incubated for 14 days in 200-mL flask cultures (in media with and without iron) at 37?C in an anaerobic atmosphere. Detection of siderophores in culture supernatants was attempted by means of the method described by Schwyn and Neilands [6]. Preparation of the outer membrane was accomplished according to the method described by Sprott et al. [7]. Cells harvested by centrifugation were disrupted ultrasonically. Unbroken cells were discarded by centrifugation. The supernatant was treated with 2% Triton