Genetic Control of Serological Specificity in Bacteriophage

Abstract

Discussion and Summary The cross-absorption technic has served to demonstrate the antigenic complexity of the phages in the T5 serological group and the corresponding complexity of their antisera. For instance, anti-T5 serum contains an antibody which will neutralize phage PB and which is readily absorbed from solution by phage PB. It also contains an antibody which will neutralize phage T5 but not phage PB, but which can nevertheless be absorbed from solution by large amounts of phage PB. Also present is a smaller amount of T5-neutralizing antibody which is not removed by absorption with even large amounts of PB phage. There is also present an antibody which can fix complement with phage PB and agglutinate phage PB and which may or may not be identical with one of the previously mentioned antibodies since it has not been separated from them. This anti-T5 serum also contains an antibody which will agglutinate hybrid 12 and fix complement with hybrid 12 and which differs from the previously-described antibodies in not being absorbed from solution by large amounts of phage PB. Thus anti-T5 serum contains at least three distinguishable antibodies and possibly more. Phage T5 itself contains at least two functionally-distinct antigens—one which is involved in neutralization and which is presumably situated at the adsorption site on the tail, and a second which is not involved in neutralization, but can be detected by complement fixation and by agglutination and which is presumably situated on the phage head. It is probable that phage PB and anti-PB serum are equally complex. The situation with these phages is comparable to that with phage T2 where it has been conclusively demonstrated that the phage head is antigenically distinct from the phage tail. Antibodies against the phage head agglutinate and fix complement but do not neutralize, whereas antibodies against the tail do all three (4, 5). The use of cross-absorbed sera has also served to demonstrate that some of the T5-PB hybrids are antigenically more complex than either parent. It was possible to prepare anti-T5 sera which would neutralize and fix complement with phage T5 but not with phage PB; and also anti-PB sera which were equally specific for phage PB. Some of the hybrids, of which hybrid 12 is a good example, were neutralized by both absorbed sera and fixed complement with both absorbed sera. These hybrid phages contain antigenic substances for which the controlling genetic material must have been derived from both parents. These experiments indicate that it is feasible to use antigenic specificity as a genetic marker in phage. An interesting by-product of these studies was the observation that in certain combinations of antiserum with distantly-related phages the rate of phage inactivation increased when the phage concentration was raised from 106 to 108 per ml. In one case of heavily-absorbed antiserum there was no detectable inactivation at a phage concentration of 106 per ml, but quite rapid inactivation at 108 per ml. This breakdown of the classical percentage law (16) is due to the fact that the sera contain a large amount of agglutinating antibody and little or no neutralizing antibody. At a phage concentration of 106 per ml there are so few collisions between phage particles that agglutination does not contribute appreciably to a decrease in the number of plaque-forming particles and any inactivation observed must be due to neutralization. At concentrations of 108 particles per ml, collisions between phage particles are very rapid and in the presence of agglutinating antibody lead to aggregation with a decrease in the number of discrete plaque-forming particles. However, if the serum contains large amounts of neutralizing antibody, these antibody molecules will react more rapidly with the phage tails than the phage particles can collide with each other and so the effect of any agglutinating antibody present will not be noticed even at phage concentrations of 108 per ml. In the case of phage T2 it was possible to demonstrate that the non-neutralizing antibody which agglutinated phage particles at concentrations of 108 per ml was directed against antigens located in the phage head (4, 5). It seems reasonable to make a similar assumption in the case of the T5 group of phages and to conclude that the non-neutralizing antibody which agglutinates and fixes complement is directed against the phage head, whereas the neutralizing antibody is directed against one or another antigen in the phage tail.