Phenotypic variation in Actinobacillus actionmycetemcomitans during laboratory growth: implications for virulence

Abstract

This study examined alteration of specific virulence traits associated with phenotypic changes seen when a low-passage disease-associated and well maintained parent strain of Actinobacillus actinomycetemcomitans was compared to a laboratory-grown spontaneous variantlmutant. Clinical isolates of A. actinomycetemcomitans recovered from periodontitis patients typically grow as rough, adherent colonies on primary culture but undergo transformation to smooth, non-adherent colonies following repeated passage in vitro. The relationship of these phenotypic changes to the virulence of the organism or to the processes that underlie this transformation are not understood. A fresh clinical isolate, designated strain CUlOOO, was obtained from the first molar site of a patient with classical signs of localized juvenile periodontitis and used as the parent strain to study virulence-related phenotypes. Following several passages of CUlOOO on selective agar, a spontaneous variant that demonstrated smooth, opaque, non-adherent colonies was isolated and designated strain CU1060. This study compared the properties of these two strains with respect to colony morphology, autoaggregation, surface appendages, adherence to saliva-coated hydroxyapatite (SHA), LPS chemotype and activity, induction of fibroblast proteinase activity and antigenic properties. CUlOOO demonstrated rough, raised, star-positive colonies which upon electron microscopic examination revealed the presence of large, flexible, bundled fibrils. In addition, CUlOOO showed adherence to SHA, several unique protein antigens and elevated endotoxin and fibroblast proteinase activity. CU1060, on the other hand, showed minimal adherence to SHA and fewer reactive proteins compared to the fresh clinical isolates. This strain formed smooth, opaque colonies on agar, showed minimal fibril formation and limited endotoxin and fibroblastproteinase-inducing activity. These findings demonstrate that clinical isolates of A. actinomycetemcomitans undergo significant virulence-reducing phenotypic alterations during in vitro passage and support the need to study this organism in its clinical form.

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

“…JP2 produces a wild type LtxA (LtxA wt ), CS001 lacks the ltxC gene and therefore produces an unacylated LtxA (LtxA CS001 ), and SM003 has been complemented with the ltxC gene in trans and produces an acylated LtxA (LtxA SM003 ). The A. actinomycetemcomitans strains were grown overnight in AAGM broth (19) supplemented with antibiotics (JP2: 12.5 g/ml vancomycin and 75 g/ml bacitracin, CS001: 40 g/ml kanamycin, SM003: 40 g/ml kanamycin and 2 g/ml chloramphenicol). LtxA was purified as described previously (20).…”

Tight-adherence genes of Actinobacillus actinomycetemcomitans are required for virulence in a rat model

“…Cells adhere avidly to solid surfaces, including plastic, glass, and hydroxyapatite (14)(15)(16). Clinical isolates also autoaggregate and produce bundled pili that mediate nonspecific adherence (16)(17)(18)(19). The tight-adherence phenotype is thought to be important for colonization of host surfaces, and recently we reported that an adherent clinical isolate was able to colonize, survive, and persist in the oral cavity of rats (2).…”

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

“…Clinical isolates autoaggregate and secrete long, bundled pili (Flp fibrils) (Holt et al, 1980;Scannapieco et al, 1987;Rosan et al, 1988;Inoue et al, 1998;Kachlany et al, 2001b;Henderson et al, 2003;Fine et al, 2006). In vitro A. actinomycetemcomitans can produce rare, stable, nonadherent, and nonaggregative genetic variants that form smooth, large colonies and fail to produce Flp fibrils (Wyss, 1989;Inouye et al, 1990;Fine et al, 1999Fine et al, , 2006Kachlany et al, 2000Kachlany et al, , 2001aHenderson et al, 2003).…”