Actinomyces naeslundii displays variant fimP and fimA fimbrial subunit genes corresponding to different types of acidic proline-rich protein and beta-linked galactosamine binding specificity

Abstract

Actinomyces naeslundii genospecies 1 and 2 bind to acidic proline-rich proteins (APRPs) and statherin via type 1 fimbriae and to beta-linked galactosamine (GalNAcbeta) structures via type 2 fimbriae. In addition, A. naeslundii displays two types of binding specificity for both APRPs-statherin and GalNAcbeta, while Actinomyces odontolyticus binds to unknown structures. To study the molecular basis for these binding specificities, DNA fragments spanning the entire or central portions of fimP (type 1) and fimA (type 2) fimbrial subunit genes were amplified by PCR from strains of genospecies 1 and 2 and hybridized with DNA from two independent collections of oral Actinomyces isolates. Isolates of genospecies 1 and 2 and A. odontolyticus, but no other Actinomyces species, were positive for hybridization with fimP and fimA full-length probes irrespective of binding to APRPs and statherin, GalNAcbeta, or unknown structures. Isolates of genospecies 1 and 2, with deviating patterns of GalNAcbeta1-3Galalpha-O-ethyl-inhibitable coaggregation with Streptococcus oralis Ss34 and MPB1, were distinguished by a fimA central probe from genospecies 1 and 2, respectively. Furthermore, isolates of genospecies 1 and 2 displaying preferential binding to APRPs over statherin were positive with a fimP central probe, while a genospecies 2 strain with the opposite binding preference was not. The sequences of fimP and fimA central gene segments were highly conserved among isolates with the same, but diversified between those with a variant, binding specificity. In conclusion, A. naeslundii exhibits variant fimP and fimA genes corresponding to diverse APRP and GalNAcbeta specificities, respectively, while A. odontolyticus has a genetically related but distinct adhesin binding specificity.

FIG. 1

Schematic illustration of the fimP and fimA fimbrial subunit genes from A. naeslundii. DNA probes representing full-length and central (gray area) gene segments specific for different adhesin specificities were PCR amplified by using synthetic oligonucleotide primers specific for fimP from A. naeslundii T14V (genospecies 2), fimA from ATCC 12104^T (genospecies 1), and fimA from P-1-N (genospecies 2, strain CCUG 33910). The nucleotide positions of all primers are indicated by arrowheads and labeled p1 through p12 (see Materials and Methods). Primers set as superscripts to position numbers were used to amplify full-length gene segments; primers set as subscripts to position numbers were used to amplify central gene segments. The central DNA probes were generated from a region of the gene sequences having comparably low homology and being devoid of highly conserved proline-containing segments. The overall nucleotide sequence identities between the three fimbrial subunit genes are indicated on the right. An asterisk marks the position of the BamHI cleavage site in the type 2:1 fimbrial subunit gene.

FIG. 2

The type 1 and type 2 fimbrial subunits are encoded by single gene copies in Actinomyces isolates. Southern blot hybridization of chromosomal DNA from A. naeslundii isolates with the fimP type 1 central DNA probe (left panel: lane 1, strain B-1-K; lane 2, P-1-K; lane 3, T14V; and lane 4, LY7), the fimA type 2:1 central DNA probe (middle panel: lane 1, strain Pn-22-E; lane 2, Pn-6-GA; lane 3, P-5-N; and lane 4, ATCC 12104^T), and the fimA type 2:2 central DNA probe (right panel: lane 1, strain P-1-K; lane 2, P-1-N; and lane 3, LY7). Chromosomal DNA was digested with BamHI prior to hybridization with either type 1 or type 2:1 DNA probes and with PstI prior to hybridization with the type 2:2 DNA probe. The sizes (in kilobases) of HindIII-digested bacteriophage lambda DNA standards (lanes kb) are indicated on the right.

FIG. 3

Alignment of the deduced amino acid sequences (in single-letter code) of central segments of the fimP and fimA type 2:1 and type 2:2 fimbrial subunit genes (see Fig. 1). Identical amino acids are indicated by gray shading, and those conserved in structure are indicated by a colon (:). Amino acids in boldface type are variable in fimbrial subunit sequences corresponding to the same binding specificity. The type 1 sequence data were collected from five isolates of genospecies 2 (strains B-10-K, B-1-K, P-1-K, P-3-K, and LY7), the type 2:1 sequence data are from six strains of genospecies 1 (strains P-3-N, P-5-N, Pn-1-GA, Pn-6-N, Pn-20-E, and Pn-22-E), and the type 2:2 sequence data are from a comparison of three strains of genospecies 2 (strains P-1-N, P-1-K, and LY7).