Aae, an autotransporter involved in adhesion of Actinobacillus actinomycetemcomitans to epithelial cells.

The periodontal pathogen Actinobacillus actinomycetemcomitans possesses myriad virulence factors, among them the ability to adhere to and invade epithelial cells. Recent advances in the molecular manipulation of this pathogen and the sequencing of strain HK 1651 (http://www.genome.ou.edu/act.html) have facilitated examination of the genetics of its interaction with epithelial cells. The related gram-negative organism, Haemophilus influenzae, possesses autotransporter adhesins. A search of the sequence database of strain HK 1651 revealed a homologue with similarity in the pore-forming domain to that of the H. influenzae autotransporter, Hap. A. actinomycetemcomitans mutants deficient in the homologue, Aae, showed reduced binding to epithelial cells. A method for making A. actinomycetemcomitans SUNY 465 transiently resistant to spectinomycin was used with conjugation to generate an isogenic aae mutant. An allelic replacement mutant was created in the naturally transformable A. actinomycetemcomitans strain ATCC 29523. Lactoferrin, an important part of the innate host defense system, protects against bacterial infection by bactericidal and antiadhesion mechanisms. Lactoferrin in human milk removes or cleaves Hap and another autotransporter, an immunoglobulin A1 protease, from the surface of H. influenzae, thereby reducing their binding to epithelial cells. Human milk whey had similar effects on Aae from A. actinomycetemcomitans ATCC 29523 and its binding to epithelial cells; however, there was little effect on the binding of SUNY 465. A difference in the genetic structure of aae in the two strains, apparently due to the copy number of a 135-base repeated sequence, may be the cause of the differential action of lactoferrin. aae is the first A. actinomycetemcomitans gene involved in adhesion to epithelial cells to be identified.

Effect of Incubation Temperature on Repair of Heat-Injured Listeria in Milk.

Heat-injured Listeria species were examined for their ability to repair in pasteurized whole and 2% (fat) bovine milk. Listeria monocytogenes F5069 (serotype 4B) and F5027 (serotype 1/2a) and Listeria innocua CWD139 were heated at 55°C. After 20 min, 99% of the surviving population was injured as determined by their inability to grow in the presence of 4% NaCl. Bacterial cells were immediately suspended in sterile milk at a concentration of 102 to 103 per ml and incubated at 4, 10, 26 and 37°C. For all of the Listeria tested, repair at 4°C was initiated between days 8 and 10 and was complete between days 16 and 19; at 10°C, repair began immediately and was complete in 4 d; at 26 and 37°C, repair was complete by 13 and 9 h, respectively. The kinetics of repair were similar in whole and 2% (fat) milk. The relationship between the time required for repair and increasing temperature was nonlinear and indicated that repair of heat-injured Listeria in milk is highly sensitive to minor increases in temperature. Current Listeria detection techniques are not adequate for the detection of injured organisms. The public health consequences associated with failure to detect injured L. monocytogenes which subsequently repair in milk may be significant.