Phylogenetic Analysis of Filifactor alocis Strains Isolated from Several Oral Infections Identified a Novel RTX Toxin, FtxA

Abstract

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod of the phylum Firmicutes, and is considered an emerging pathogen in various oral infections, including periodontitis. We here aimed to perform phylogenetic analysis of a genome-sequenced F. alocis type strain (ATCC 35896; CCUG 47790), as well as nine clinical oral strains that we have independently isolated and sequenced, for identification and deeper characterization of novel genomic elements of virulence in this species. We identified that 60% of the strains carried a gene encoding a hitherto unrecognized member of the large repeats-in-toxins (RTX) family, which we have designated as FtxA. The clinical infection origin of the ftxA-positive isolates largely varied. However, according to MLST, a clear monophylogeny was reveled for all ftxA-positive strains, along with a high co-occurrence of lactate dehydrogenase (ldh)-positivity. Cloning and expression of ftxA in E. coli, and purification of soluble FtxA yielded a protein of the predicted molecular size of approximately 250 kDa. Additional functional and proteomics analyses using both the recombinant protein and the ftxA-positive, and -negative isolates may reveal a possible role and mechanism(s) of FtxA in the virulence properties of F.alocis, and whether the gene might be a candidate diagnostic marker for more virulent strains.

Keywords: Filifactor alocis; FtxA; multilocus sequence typing (MLST); oral infections; phylogenetic tree.

Figure 1

(A) Overlapping conserved domains in the FtxA protein sequence, deduced using InterPro. The C-terminal portion displays homology to the Hemolysin-type calcium binding-related domain (IPR010566), and the serralysin-like metalloprotease superfamily (IPR011049), and contains repeats-in-toxins (RTX) calcium-binding nonapeptide repeats (IPR001343), and Hemolysin-type calcium-binding conserved sites (IPR018511). (B) One archetypal RTX gene cluster (hlyCABD) encodes the E. coli alpha-hemolysin, HlyA. HlyA is synthesized as an inactive protoxin, ProHlyA, which is post-translationally activated in two steps, first via HlyC-directed acylation in the cytoplasm, and then by binding Ca²⁺ in the extracellular medium [13]. The cyaE gene of B. pertussis encodes a homologue to E. coli tolC and plays a critical role in the secretion of CyaA [11]. We identified no equivalent to an HlyC-homologue in the F. alocis genomes. The hypothetical protein encoded upstream of ftxA exhibits no apparent homology to hlyC or to other known proteins.

Figure 2

Phylogenetic relationships among the ten F. alocis strains, based on multilocus sequence typing (MLST) analysis as described in Materials and Methods. The tree with the highest log likelihood is shown. The percentages of trees in which the associated strains clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The ftxA and ldh genotypes, respectively, are indicated.

Figure 3

Coomassie-staining reveals purified recombinant FtxA as a high molecular weight band near 250 kDa (lane 2). A molecular weight marker was loaded in lane 1, and the molecular sizes (kDa) of the individual protein bands are indicated.