Phenotypic variation in Actinobacillus actinomycetemcomitans during laboratory growth: implications for virulence.
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 variant/mutant. 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 CU1000, 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 CU1000 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. CU1000 demonstrated rough, raised, star-positive colonies which upon electron microscopic examination revealed the presence of large, flexible, bundled fibrils. In addition, CU1000 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 fibroblast-proteinase-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. (+info)
Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f.
The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of L-rhamnose and 2-acetamido-2-deoxy-D-galactose (molar ratio, 2:1) with the structure -->2)-alpha-L-Rhap-(1-3)-2-O-(beta-D-GalpNAc)-alpha-L-Rhap-(1-->* O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903phikan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted. (+info)
Tight-adherence genes of Actinobacillus actinomycetemcomitans are required for virulence in a rat model.
Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that has been associated with localized aggressive periodontitis and infections of the heart, brain, and urinary tract. Wild-type clinical isolates have the remarkable ability to adhere tenaciously and nonspecifically to solid surfaces such as glass, plastic, and hydroxyapatite. Adherence by A. actinomycetemcomitans is mediated by the tight-adherence (tad) gene locus, which consists of 14 genes (flp-1-flp-2-tadV-rcpCAB-tadZABCDEFG). All but 2 of the genes have been shown to be required for the secretion and assembly of long, bundled Flp1 fibrils. To test whether the tad locus is required for colonization and disease, we developed a rat model for periodontal disease. To mimic the natural route of infection, Sprague-Dawley rats were inoculated orally by adding bacteria directly to their food for 8 days. After inoculation with wild-type or mutant strains defective in adherence (flp-1 and tadA), the rats were assessed for colonization of the oral cavity and pathogenesis. Wild-type A. actinomycetemcomitans was able to colonize and persist for at least 12 weeks in the oral cavity, elicit a humoral immune response, and cause significant bone loss in rats. In contrast, rats fed flp-1 or tadA mutant strains showed no bone loss and their immune responses were indistinguishable from those of the uninoculated controls. These results demonstrate the critical importance of the tad locus in the colonization and pathogenesis of A. actinomycetemcomitans. (+info)
Novel surface structures are associated with the adhesion of Actinobacillus actinomycetemcomitans to collagen.
Actinobacillus actinomycetemcomitans is a gram-negative, facultative, anaerobic bacterium that colonizes the human oral cavity and the upper respiratory tract. This bacterium is strongly associated with localized aggressive periodontitis and adult periodontitis and is the causative agent for other serious systemic infections. Recently, we have identified a protein, EmaA (extracellular matrix protein adhesin A), that mediates the adhesion of A. actinomycetemcomitans to collagen. The conserved sequence and predicted secondary structure suggest that EmaA is an orthologue of the Yersinia enterocolitica adhesin YadA. Electron microscopy examinations of A. actinomycetemcomitans have identified antenna-like protrusions associated with the surface of the bacterium. These structures are absent on emaA mutant strains and can be restored by transformation of the mutant strain with emaA in trans. The loss of these structures is associated with a decrease in the binding of this bacterium to collagen. The antenna-like structures are composed of a long rod that terminates in an ellipsoidal head region. The analysis of these structures using image processing techniques has provided an initial estimate of the overall dimensions, which suggests that the appendages are oligomeric structures formed by either three or four subunits. Together, the data suggest that emaA is required for the expression of novel appendages on the surface of A. actinomycetemcomitans that mediate the adhesion of the bacterium to collagen. (+info)
HACEK infective endocarditis: characteristics and outcomes from a large, multi-national cohort.
Epithelial cell invasion by Actinobacillus actinomycetemcomitans strains from restriction fragment-length polymorphism groups associated with juvenile periodontitis or carrier status.
The epithelial cell invasiveness of Actinobacillus actinomycetemcomitans strains of different restriction fragment-length polymorphism (RFLP) groups associated with disease conversion and asymptomatic carrier status in localized juvenile periodontitis was examined. Twenty clinical isolates were studied for their ability to invade KB monolayers, using the quantitative gentamicin killing assay. Five isolates were found to be invasive, five were not invasive; and the other 10 did not invade better than an invasion negative control Haemophilus aphrophilus strain ATCC 19415. Using probe-specific DNA fingerprinting. 11 strains were assigned to RFLP group II (disease-associated); 4 to RFLP type XIII (carrier status associated); and the other to groups III, IV, V and VII. Eight isolates, all RFLP group II, were leukotoxin producers as determined by PCR amplification of the lkt promoter region. No correlation was found between invasiveness and RFLP group. Leukotoxin production was more associated with noninvasive than invasive strains. (+info)