Effects of environment conditions on the production of leukotoxin by Actinobacillus actinomycetemcomitans

伴放线放线杆菌（Actinobacillusactinomycetemcomitans）是一种牙周病革兰氏阴性细菌，产生的白细胞毒素是RTX细胞毒素家族的成员。虽然基因可能在毒素分泌中起作用，但白细胞毒素不分泌到细胞外，而是与细菌细胞表面结合。我们在这里报告说，这种毒素细胞表面协会是由核酸介导的，并直接表明，细胞外分泌的毒素发生在不断增长的文化与介质的离子强度增加。所有检查均使用从厌氧果糖限制恒化器培养物中新鲜收获的伴放线菌301-B进行。通过用限制性内切酶EcoRI或HindIII直接消化整个细胞，产生许多DNA片段，显示了细胞表面定位的DNA的发生。细胞表面DNA约占细胞总DNA的20%，经DNA酶Ⅰ和限制性内切酶消化后，白细胞毒素从整个细胞中释放出来。因为白细胞毒素离子结合DNA，它依赖于缓冲液或介质的离子强度。相应地，在存在增加量的MgCl_2（0至10 mM）或NaCl（0至50 mM）的情况下，毒素从悬浮在pH 7.5的盐水中的细胞中释放。此外，当将琥珀酸钠溶液作为额外的盐（30 mM，然后50 mM）泵入稳态时，在果糖限制恒化器培养物的培养上清液中检测到相当数量的白细胞毒素。当这些菌株在恒化器培养物中生长时，在充分表征的菌株中也发现了这种毒素-DNA缔合，所述菌株不仅包括产生白细胞毒素的ATCC 29522，而且包括毒素产生可变的ATCC 29523和不产生白细胞毒素的ATCC 33384。

Actinobacillus actinomycetemcomitans, a periodontopathic gram-negative bacterium, produces a leukotoxin that is a member of the RTX cytotoxin family. Although genes may function in toxin secretion, the leukotoxin is not secreted extracellularly but remains associated with the bacterial cell surface. We report here that this toxin-cell surface association is mediated by nucleic acids and directly demonstrate that the extracellular secretion of toxin occurs in growing cultures with increased ionic strength of medium. All examinations were performed with freshly harvested A.actinomycetemcomitans 301-b from anaerobic fructose-limited chemostat cultures. The occurrence of cell surface-localized DNA was shown by directly digesting whole cells with the restriction endonuclease EcoRI or HindIII, which yielded many DNA fragments. The cell surface DNA constituted about 20% of the total cellular DNA.The leukotoxin was released from the whole cells by digestion with DNase I as well as restriction endonucleases. Because the leukotoxin binds ionically to DNA, it is dependent on the ionic strength of buffers or media. Accordingly, the toxin was released from cells suspended in saline at pH 7.5 in the presence of increasing amounts of MgCl_2 (0 to 10 mM) or NaCl (0 to 50 mM). Moreover, a considerable quantity of leukotoxin was detected in the culture supernatant of fructose-limited chemostat cultures when sodium succinate solution was pumped into the steady state as an additional salt (30 and then 50 mM). This toxin-DNA association was also found in well-characterized strains including not only the leukotoxin-producing ATCC 29522 but also the toxin production-variable ATCC 29523 and the non-leukotoxin-producing ATCC 33384 when these strains were grown in the chemostat culture.