Inactivation of a fibronectin-binding TonB-dependent protein increases adhesion properties of Bacteroides fragilis

Abstract

Bacteroides fragilis is the Gram-negative strictly anaerobic bacterium most frequently isolated from clinical infections, including intra-abdominal abscess and bacteraemia. A number of factors can contribute to its virulence, including the expression of adhesins. Some of them are already characterized and can recognize and bind to extracellular matrix components, such as fibronectin. One of the molecules responsible for fibronectin-binding is an outer-membrane protein previously described by our group, which belongs to the TonB-dependent family. The aim of the present work was to characterize this protein. Initially, it was confirmed by fluorescence and electron microscopy that the fibronectin-binding molecules were located in the bacterial surface, but the distribution of these molecules on the surface was not uniform. To further evaluate the role of this protein, the gene bf1991, responsible for encoding this protein, was inactivated by a suicide vector and the mutant strains generated were used in several experiments to verify possible phenotypical alterations. In adherence assays with fibronectin immobilized on latex beads an increased adhesion was observed with the mutant strains compared with the wild-type strain. Western blot analysis in the mutant strain revealed the absence of the 120 kDa TonB-dependent outer-membrane protein and an alteration in the expression of an unknown 30 kDa protein. Killing assays using peritoneal macrophages were performed to evaluate the role of this protein as a virulence attribute and it was observed that the mutant strains were more efficiently internalized than the wild-type strains, with more internalization in the samples covered with fibronectin than in the samples not covered with it.

TonB Energy Transduction Systems of Riemerella anatipestifer Are Required for Iron and Hemin Utilization

“…The TonB2 system, which is located 16 kb downstream of the TonB1 system, is flanked by an upstream gene transcribed in the same direction that codes for a leucyl-tRNA synthetase and a downstream gene transcribed in the same direction that codes for an phosphate ABC transporter substrate-binding protein ( Fig 1C ). In addition, genes coding for an ABC transport related protein and fibronectin, which may have some connection with TonB [ 44 ], are closely located to the TonB2 system. The third tonB gene, coding for a 31-kDa protein, is located far apart from the other two tonB gene clusters and is flanked by two genes that code for a permease-family protein and a ATPase ( Fig 1D ).…”