Aggregatibacter菌種水平轉移基因分析

Abstract

Horizontal gene transfer (HGT) is a process by which bacteria acquire genes from organisms of related or distant taxa. HGT is now recognized as a major driving force in the evolution of bacterial pathogens. Through this process, bacteria may accumulate blocks of DNA such as genomic islands (GEIs) that encode fitness or virulence factors. Aggregatibacter actinomycetemcomitans and Aggregatibacter aphrophilus are Gram-negative facultative oral bacteria. These two species share a high degree of genetic relatedness and identical oral ecological niches. However, the former is considered a major periodontal pathogen, while the latter is a commensal bacterium found frequently in healthy individuals. Moreover, A. actinomycetemcomitans has also been known to exhibit variable virulence potentials among strains. It was postulated that variation in virulence potentials between these Aggregatibacter spp., and among A. actinomycetemcomitans strains could be explained by different genomic contents due to HGT of individual virulence genes or GEI that carry virulence genes. Presumably HGT acquired genes will function in closely related bacterial host species such as A. actinomycetemcomitans and A. aphrophilus. Based on the hypothesis, we made the following predictions: (1) there are genomic islands unique to some strains of A. actinomycetemcomitans. The deletion of some of the islands may lead to reduced virulence potentials of the strains. (2) There are virulence genes presenting in A. actinomycetemcomitans but not in A. aphrophilus. The A. actinomycetemcomitans-specific virulence genes could be transferred and function in A. aphrophilus. Two aims were set to examine the hypothesis. First, two known virulence determinants and seven strain-specific GEIs were deleted by the markless Cre/loxP system in natural transformable A. actinomycetemcomitans strain D7S-1. Subsequently the wildtype and mutants were tested for their growth rates and biofilm formation. The results indicated the deletion of the strain-specific 285-island (encoding proteins related to cross-membrane translocations and the multidrug efflux pump), significantly increased the growth rate of D7S-1. No other phenotypes were detected in other mutants. Also, two identified strain-specific genomic islands AAI-1 and AAI-2 in A. actinomycetemcomitans strain HK 1651 were deleted by the markless Cre/loxP system via conjugation. The functional annotations of AAI-1 are hypothetical proteins, putative adhesion and putative transposase, and AAI-2 encodes hypothetical proteins and Rhs family protein. The insertional inactivation of comM byAAI-2 may relate to the loss of natural competence of strain HK1651. While it was recognized that AAI-1 and AAI-2 were acquired by HGT, the growth curve study of the knockout mutants of AAI-1and AAI-2 did not reveal a notable variation in their phenotype of growth rate. In the second aim, the simulation of the acquisition of specific virulence determinants between Aggregatibacter species was developed by transferring the kat gene, an A. actinomycetemcomitans specific virulence factor, to A. aphrophilus strain NJ8700. The protective function against the bactericidal effect of hydrogen peroxide was carried out in the wild type A. actinomycetemcomitans D7S-1, rescued D7S-1 kat-deleted mutant and mutant A. aphrophilus expressing kat, while wild type NJ8700 and kat-deleted mutants of D7S-1 were sensitive to hydrogen peroxide treatment. The results supported that the lack of species barrier in the transfer and expression of genes between these two species. In conclusion, the strain specific GEI may play a role in the phenotype variations of A. actinomycetemcomitans. The natural transformation characteristic of A. actinomycetemcomitans and Cre/loxP recombination system proved to be an efficient and reliable approach in the construction of A. actinomycetemcomitans mutants with gene(s) deletion. In addition, Kat gene is able to be transferred form A. actinomycetemcomitans to A. aphrophilus, suggested that differential acquisition of genes for virulence determinants could occur during the evolution of the Aggregatibacter species.