Outer membrane proteins of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus studied by SDS‐PAGE and immunoblotting

Abstract

This investigation characterized and compared outer membrane proteins (OMP) of the closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by means of SDS-PAGE patterns and reactions on immunoblots with rabbit antiserum against A. actinomycetemcomitans FDC Y4. Reactions with serum from a patient with Papillon Lefévre syndrome (PLS), from whom periodontal wild strains of A. actinomycetemcomitans had been isolated, were also studied. OMP were purified with selective solubilization from lyophilized cells of 10 wild and 4 reference strains of A. actinomycetemcomitans and 4 reference strains of H. aphrophilus. OMP profiles from wild and reference strains of A. actinomycetemcomitans were similar while those from A. actinomycetemcomitans and H. aphrophilus differed. The most prominent difference was absence of a heat modifiable protein in H. aphrophilus strains. Immunoblotting revealed strong common antigens in most strains, including a heat modifiable protein with mol wt 34 kDa, as well as a 29 kDa and a 16.5 kDa protein. Treatment with pronase and sodium periodate confirmed the protein nature of the major OMP antigens.

Extracellular 37-kDa Antigenic Protein from Actinobacillus actinomycetemcomitans Induces TNF-α, IL-1β, and IL-6 in Murine Macrophages

“…These are mainly cell-surface antigens, such as LPS (Page et al, 1991), capsular PS (Califano et al, 1989), and outer membrane proteins. The outer membrane proteins include the 29-kDa and 16.5-kDa heat-modifiable proteins (Bolstad et al, 1990;Wilson, 1991;Spinola et al, 1993), and the 39-kDa protein (Kokeguchi et al, 1991). These outer membrane proteins were isolated from cells by detergent, whereas the 37-kDa protein was not.…”

“…To identify the heat-modifiable protein, we incubated our samples in SDS-lysis buffer at 37°C for 30 min or 100°C for 5 min. The heat-modifiable outer membrane protein migrates at -1--lf 9q bn If tCor cral1-h-li7:vtion :l 37°C, and to around 35 kDa at 100°C (Bolstad et al, 1990;Wilson, 1991 (Bolstad et al, 1990;Spinola et al, 1993) and 5 periodontopathic bacteria (P. gingivalis 381, P. intermedia ATCC 25611, C. ochracea 25, E. corrodens ATCC 23834, and F. ni/cleatiom ATCC 10953) were ultrasonically disrupted and centrifuged at 10,000 x g for 20 min. We used ELISA to investigate the reactions of these 10 bacteria against anti-37-kDa protein mouse serum.…”

Microbial dysbiosis and colon carcinogenesis: could colon cancer be considered a bacteria-related disease?

“…It is a key component of periodontal plaque due to its abundance and its ability to coaggregate with other species in the oral cavity. This bacterium shows the ability to associate with viruses, which adhere to host tissue cells and modulate the host's immune response [Bolstad et al 1996]. However, a causative role of Fusobacterium has not been demonstrated and so this bacterium could be a cofactor of an as yet unidentified cause.…”

Variation in outer-membrane protein and lipopolysaccharide profiles of Pasteurella haemolytica isolates of serotypes A1 and A2 obtained from pneumonic and healthy cattle

“…Typing schemes based on OMP patterns in SDSpolyacrylamide gels have been developed in both human (Loeb & Smith, 1980;Mocca & Frasch, 1982;Achtman e t al., 1983;Blaser e t al., 1983;Odumeru e t al., 1983) and veterinary (Lugtenberg e t al., 1984;Rapp e t al., 1986;Davies, 1991) pathogens, while LPS analysis has led to the identification of different SDS-PAGE profiles in Haemophilzis injuenxae and proven useful in epidemiological studies (Inzana, 1983 ;Inzana & Pichichero, 1984). Western blotting has been used successfully for strain differentiation and in epidemiological studies of both Gram-negative (Bolstad e t al., 1990;Hansman & Lawrence, 1993) and Gram-positive (Mulligan e t al., 1988) pathogens. However, the combined analysis of both OMP and LPS profiles by SDS-PAGE has been carried out with less frequency, although such studies have been performed on Escherichia coli (Achtman et al, 1986) and Pasteurella multocida (Lugtenberg et al , 1984).…”