Fusobacterium nucleatum apoptosis-inducing outer membrane protein

Abstract

The periodontal pathogen Fusobacterium nucleatum induces apoptosis in lymphocytes. We previously identified the autotransporter protein Fap2 in F. nucleatum strain PK1594 that induced apoptosis in lymphocytes when expressed in Escherichia coli. In this study, we identified protein homologs of Fap2 in the transformable F. nucleatum strain ATCC 23726, to determine their role in the induction of apoptosis in lymphocytes. We used a new gene-inactivation vector conferring thiamphenicol resistance (pHS31) to construct a mutant deficient in one of the homologs, aim1. Transcriptional analyses demonstrated disruption of aim1 expression, and phenotypic analyses revealed a 41% decrease in the ability of the mutant to induce apoptosis in Jurkat cells, as compared with the parental strain. These studies demonstrate, in the native host cell background, the contribution of aim1 to F. nucleatum induction of apoptosis and, to the best of our knowledge, represent the first report of a genetically defined and phenotypically characterized mutation in F. nucleatum.

Figure 1

Structural and transcriptional analyses of wild-type and aimless1 mutant strains. (A) Schematic illustration of mutagenesis: organization of aim1 and flanking genes on the F. nucleatum ATCC 23726 wild-type and aimless1 mutant chromosome. Arrows indicate PCR primers used for construction and analysis of the aim1 mutant; arrowheads indicate real-time PCR primers; black shaded area indicates intergenic region; bracket indicates location of autotransporter domain. Full-length aim1 (5625 bases) was submitted to GenBank and has been assigned accession number DQ020251. The Fig. is not to scale. (B) PCR analysis confirming insertion into aim1. PCR analysis of chromosomal DNA from ATCC 23726 and the aimless1 mutant. Plasmid pIP-aim1, the construct used to create the inactivation in aim1, was used as the control DNA template. (C) Transcriptional analysis of aim1 and the downstream gene Fn2057. The relative RNA abundance of F. nucleatum wild-type and aimless1 was determined by real-time PCR. Results are expressed as relative means (mutant/wt) ± SEMs (error bars) of triplicate experiments.

Figure 2

Induction of apoptosis in Jurkat cells after incubation with F. nucleatum wild-type or with aimless1. Jurkat cells were incubated with wild-type or aimless1 for 18 hrs before assay (n = 3). Uninfected Jurkat cells were incubated alone as a control. Values shown are relative to wild-type apoptosis levels after background subtraction. Error bars represent SD. **p < 0.0001.