Bordetella pertussis invasive adenylate cyclase. Partial resolution and properties of its cellular penetration.

E Hanski,Z Farfel

doi:10.1016/s0021-9258(18)89054-8

Abstract

The existence of an invasive adenylate cyclase in dialyzed urea extracts of the bacterium Bordetella pertussis has been suggested recently. Gel filtration of B. pertussis dialyzed urea extract shows that the invasive enzyme constitutes only a small portion of the total adenylate cyclase activity found in the extract. Its size is different than the size of the two peaks of adenylate cyclase activity identified in the extract. Ca2+ is absolutely required for the penetration of the invasive enzyme, it also controls the rate of intracellular cAMP accumulation in human lymphocytes exposed to dialyzed extract. These characteristics may be attributed to the increase in the size of the invasive enzyme as found by gel filtration chromatography of the extract in the absence of Ca2+. Removal of nonpenetrating adenylate cyclase that adheres to lymphocytes permits a direct assay of the intracellular enzyme. The time course of intracellular accumulation of adenylate cyclase activity is similar to the time course of intracellular accumulation of cAMP, suggesting that the invasive enzyme is rapidly deactivated, but not degraded, since it can be detected upon cell disruption. No appreciable amount of the enzyme is introduced when cells are incubated with extract at 4 degrees C for 120 min, then washed and incubated further at 37 degrees C. Concanavalin A inhibits cAMP accumulation irrespective of the time of its addition, and EGTA prevents penetration of the invasive enzyme even if added 20 min after addition of extract. These findings are different from those observed in other bacterial toxins thought to be internalized by receptor-mediated endocytosis. However, the cellular penetration of B. pertussis adenylate cyclase is cell-selective. It does not occur in human erythrocytes. In addition to human lymphocytes, S49 cyc- murine lymphoma, turkey erythrocytes, and rat oocytes accumulate cAMP in response to B. pertussis extract.

Highlights

The existence of an invasive adenylate cyclase in B. pertussis products by human neutrophils leads to extensive dialyzed urea extractosf the bacteriumBordetellaper- production of intracellular adenosine 3‘5‘-monophosphate, tussis has been suggested recently
Dose D e ~ and~Time~Cour~se of eCAMPA c ~ u r n ~ ~ ~ ~ n in ~ u ~ y ~~ ~ n~ c~xypot seedsto B. p e r t ~ Us r~ea Extract-As depicted from the experiments shown in Fig. 1, a and b, intracellular cAMP accumulation in human lymphocytes subjected to urea extract of B. pertussis is dose- and time-dependent
B. pertussis invasive adenylate cyclasewhich causes an increase in intracellular cAMP constitutes only a small por

Summary

EXPERIMENTAL PROCEDURES

The cells were collected, washed twice by centrifugation at 1400 rpm (IEC centrifuge) for 10 min, and resuspended in phosphate-buffered saline (PBS’), pH 7.4,containing 1 mM CaC12. The abbreviations used are: PBS, phosphate-buffered saline; IBMX, 3-isobutyl-1-methylxanthine;PMSF, phenylmethylsulfonyl fluoride; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; dansyl, 5-dimetbylaminoaphthalene-1-sulfonylC;onA, concanavalin A EGTA, ethylene glycol bid@-aminoethylether)-N,N,N’,N’tetraacetic acid. S49 cyc- Murine Lymphoma”S49 cyc- cells were grown in suspension as described previously [9].The cells were washed twice by centrifugation as above and resuspended in PBS buffer containing 1 mM CaC1,.

RESULTS

Successive washes

20 Adenylate

DISCUSSION