Actinobacillus actinomycetemcomitans, the etiologic agent for localized juvenile periodontitis and certain other human infections, such as endocarditis, expresses a leukotoxin that acts on polymorphonuclear leukocytes and macrophages. Leukotoxin is a member of the highly conserved repeat toxin (RTX) family of bacterial toxins expressed by a variety of pathogenic bacteria. While the RTX toxins of other bacterial species are secreted, the leukotoxin of A. actinomycetemcomitans is thought to remain associated with the bacterial cell. We have examined leukotoxin production and localization in rough (adherent) and smooth (nonadherent) strains of A. actinomycetemcomitans. We found that leukotoxin expressed by the rough, adherent, clinical isolate CU1000N is indeed cell associated, as expected. However, we were surprised to find that smooth, nonadherent strains of A. actinomycetemcomitans, including Y4, JP2 (a strain expressing a high level of toxin), and CU1060N (an isogenic smooth variant of CU1000N), secrete an abundance of leukotoxin into the culture supernatants during early stages of growth. After longer times of incubation, leukotoxin disappears from the supernatants, and its loss is accompanied by the appearance of a number of low-molecular-weight polypeptides. The secreted leukotoxin is active, as evidenced by its ability to kill HL-60 cells in vitro. We found that the growth phase and initial pH of the growth medium significantly affect the abundance of secreted leukotoxin, and we have developed a rapid (<2 h) method to partially purify large amounts of leukotoxin. Remarkably, mutations in the tad genes, which are required for tight nonspecific adherence of A. actinomycetemcomitans to surfaces, cause leukotoxin to be released from the bacterial cell. These studies show that A. actinomycetemcomitans has the potential to secrete abundant leukotoxin. It is therefore appropriate to consider a possible role for leukotoxin secretion in the pathogenesis of A. actinomycetemcomitans.

译文

放线杆菌放线杆菌(Actinobacillus actinomycetemcomitans)是局部性青少年牙周炎和某些其他人类感染(如心内膜炎)的病原体,其表达的白细胞毒素作用于多形核白细胞和巨噬细胞。白细胞毒素是多种致病细菌表达的细菌毒素的高度保守重复毒素(RTX)家族的成员。虽然其他细菌物种的RTX毒素被分泌出来,但认为放线放线杆菌的白细胞毒素仍与细菌细胞结合。我们已经检查了粗毒素(粘附)和光滑(非粘附)菌株放线放线杆菌中白细胞毒素的产生和定位。我们发现,如预期的那样,由粗糙的,粘附的临床分离株CU1000N表达的白细胞毒素确实与细胞相关。但是,我们惊讶地发现光滑的,非粘附性的放线放线杆菌菌株,包括Y4,JP2(表达高水平毒素的菌株)和CU1060N(CU1000N的同基因光滑变体),向其中分泌了大量白细胞毒素。在生长的早期培养上清液。经过较长时间的培养,白细胞毒素从上清液中消失,其丢失伴随着许多低分子量多肽的出现。分泌的白细胞毒素具有活性,其体外杀伤HL-60细胞的能力证明了这一点。我们发现,生长培养基的生长期和初始pH值会显着影响分泌的白细胞毒素的丰度,因此,我们开发了一种快速(<2小时)方法来部分纯化大量白细胞毒素。值得注意的是,tad基因中的突变是放线放线杆菌紧密粘附于表面所需的突变,导致白细胞毒素从细菌细胞中释放出来。这些研究表明,放线放线杆菌有可能分泌丰富的白细胞毒素。因此,有必要考虑白细胞毒素分泌在放线放线杆菌的发病机理中的可能作用。

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