Gonococci of the colonial types that are associated with virulence, types 1 and 2, have pili that enable the bacteria both to attach in vitro to human epithelial cells and to resist phagocytosis by polymorphonuclear leukocytes. These piliated gonococci also agglutinate various mammalian and chicken erythrocytes. Gonococci of an avirulent colonial type, i.e., type 4, have no pili and neither attach to epithelial cells or erythrocytes nor resist phagocytosis. Like the type 4 bacteria, mechanically or enzymatically (trypsin) depiliated type 1 gonococci failed to attach to epithelial cells and erythrocytes and were susceptible to phagocytosis. Pili of types 1 and 2 gonococci were antigenically similar. Both type 1 gonococci and pili isolated from them induced in rabbits antibody that (i) precipitated gonococcal pili in immunodiffusion, (ii) reacted with piliated gonococci as tested by indirect immunofluorescent analysis, (iii) inhibited attachment of piliated gonococci to both human epithelial cells and erythrocytes, and (iv) opsonized piliated gonococci.

Analysis of mature spores or their integuments by extraction with sodium dodecyl sulphate/dithioerythritol followed by electrophoresis shows that the coat contains four major proteins and about ten others. Nine offie 14 proteins begin to be synthesized in stage I1 or stage I11 and their synthesis must be controlled by stage I1 or stage I11 operons. Some of these proteins are incorporated into the spore structure from about t, onwards (i.e. 4 h after induction of sporulation). Their deposition in the coat between t, and t , is not stopped by chloramphenicol, with the exception of one protein (mol. wt 36000) which begins to be synthesized only at t,. Spores were isolated at various stages from about t, onwards, and the surface proteins were labelled with 1251. The labelling patterns show that proteins which are exposed on the surface at t5.3 are successively overlaid as the spores mature. It appears that the coat of the mature spore contains at least three layers. The outermost layer is mainly composed of an alkali-soluble protein (mol. wt 12000), which is synthesized early (t2), and the 36000 mol. wt protein, which is synthesized very late. Deposition of the former seems to require processing by proteolytic action, and both proteins are apparently necessary for the acquisition of resistance to lysozyme, though not for resistance to heat or organic solvents. The results are discussed in relation to the classification of sporulation events and the nomenclature of the genetic loci controlling sporulation.? Present address: is described. The results suggest that the times of synthesis of the coat proteins are determined by an order of gene expression beginning at stage I1 but that their incorporation into the structure of the spore does not necessarily follow the same order. M E T H O D SChemicals. Acrylamide (purified) and N,N'-methylenebisacrylamide were obtained from BDH. Radioactively labelled compounds were purchased from The Radiochemical Centre, Amersham. All other chemicals were from Sigma unless otherwise specified.Bacteria. Bacillus subtilis 168 trpC2, which requires indole or tryptophan and sporulates normally, was used. Stocks were kept at 4 "C as dilute spore suspensions in distilled water.induction of sporulation. Cells in the exponential phase of growth in a hydrolysed casein medium at 37 OC were harvested by centrifugation when the culture contained about 0.25 mg dry wt bacteria m1-I. The pellet was resuspended to the same density in warm resuspension medium containing glutamate, inorganic salts and 20 pg tryptophan ml-I (Sterlini & Mandelstam. 1969) and incubated with shaking. About 80% of the cells contained refractile spores at 8 h after resuspension. The time of resuspension is denoted to and subsequent times (h) as t , , t,, etc.Determination of spore incidence. Spores were counted by phase-contrast microscopy and expressed as a percentage of the total number of cells. The term 'phase-bright' describes all gradations from dull white to bright spores; earlier visible sporal inclusions are termed da...

Colonial varieties of Neisseria gonorrhoeae that are associated with virulence, types 1 and 2, were more resistant to phagocytosis by rabbit exudative polymorphonuclear leukocytes than colonial, types of lesser virulence, types 3 and 4. Type 1 bacteria were resistant and type 4 gonococci were susceptible to phagocytosis by human polymorphonuclear leukocytes. Recent local type 1 isolates were similar in resistance to type 1 organisms of a standard laboratory strain (F62). Living and Formalin-treated, heat-killed, type 1 gonococci were equally resistant to phagocytosis. The antiphagocytic property of virulent colonial types was independent of leukotoxic action. Phagocytosis of both type 1 and type 4 gonococci by rabbit and human leukocytes was bactericidal. Rabbit leukocytes were superior to human leukocytes in killing gonococci. The results suggest that N. gonorrhoeae has virulence properties similar to those of extracellular bacterial pathogens, i.e., virulence is associated with antiphagocytic properties.on August 1, 2020 by guest http://iai.asm.org/ Downloaded from

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