Establishing a direct role for the Bartonella bacilliformis invasion-associated locus B (IalB) protein in human erythrocyte parasitism

The invasion-associated locus A and B genes (ialAB) of Bartonella bacillifo rmis were previously shown to confer an erythrocyte-invasive phenotype upon Escherichia coli, indirectly implicating their role in virulence. We repor t the first direct demonstration of a role for ialB as a virulence factor i n B, bacilliformis. The presence of a secretory signal sequence and amino a cid sequence similarity to two known outer membrane proteins involved in vi rulence suggested that IalB was an outer membrane protein, To develop an an tiserum for protein localization, the ialB gene was cloned in frame into an expression vector with a six-histidine tag and under control of the lacZ p romoter. The IalB fusion protein was purified by nickel affinity chromatogr aphy and used to raise polyclonal antibodies. IalB was initially localized to the bacterial membrane fraction. To further localize IalB, B, bacillifor mis inner and outer membranes were fractionated by sucrose density gradient centrifugation and identified by appearance, buoyant density (rho), and cy tochrome b content. Inner and outer membrane proteins were analyzed by sodi um dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and IalB was positively identified by Western blot. Contrary to expectations, IalB w as localized to the inner membrane of the pathogen, To directly demonstrate a role for IalB in erythrocyte parasitism, the B. bacilliformis ialB gene was disrupted by insertional mutagenesis, The resulting ialB mutant strain was complemented in irans with a replicative plasmid encoding the full-leng th ialB gene. PCR and high-stringency DNA hybridization confirmed mutagenes is and transcomplementation events. Abrogation and restoration of ialB expr ession was verified by SDS-PAGE and immunoblotting. In vitro virulence assa ys showed that mutagenesis of ialB decreased bacterial association and inva sion of human erythrocytes by 47 to 53% relative to controls. Transcompleme ntation of ialB restored erythrocyte association and invasion rates to leve ls observed in the parental strain. These data provide direct evidence for IalB's role in erythrocyte parasitism and represent the first demonstration of molecular Koch's postulates for a Bartonella species.