# BORDETELLA CYCLASE:  STRUCTURE AND BIOLOGICAL ACTIVITY

> **NIH NIH R01** · UNIVERSITY OF VIRGINIA · 1993 · $253,475

## Abstract

Infection with Bordetella pertussis remains a cause of pediatric morbidity
and mortality and adult morbidity worldwide.  Adenylate cyclase (AC) toxin
from B. pertussis is a major virulence factor hypothesized to be involved
in evasion of host defenses and development of local pathology.  In this
project, AC toxin structure and function will be studied for three
purposes:  1) understanding its mechanism of action; 2) considering it as
a vaccine antigen in acellular pertussis vaccine; and 3) contributing to
the overall knowledge of bacterial toxins which enter target cells
(traverse intact cell membranes).  AC toxin is a calmodulin-regulate
protein which catalyzes the production of cyclic AMP.  Its novel features
include its ability to enter intact eukaryotic cells to produce
supraphysiologic levels of cAMP and its ability to hemolyze erythrocytes.
AC toxin is synthesized in an inactive form which requires modification by
a separate protein (product of the cyaC gene) for expression of full toxin
and hemolytic activities.  The c-gene product (CyaC) will be expressed,
isolated and characterized.  Monoclonal antibodies will be prepared against
CyaC to facilitate study of its site and mechanism of action.  The
activation of AC toxin by the product of CyaC will be studied in vitro, in
order to determine site of protein modification and structural
consequences.

Activated AC toxin binds calcium and undergoes a conformational change
which allows it to enter eukaryotic target cells.  This conformational
changes does not occur in toxin from mutants missing CyaC, despite the fact
that the toxin from such strains binds calcium.  The role of CyaC-dependent
activation in the structural response to calcium will be explored.  Using
purified, activated AC toxin the binding and entry processes will be
dissected using functional techniques as well as electron microscopy.  The
study of toxin structure and translocation across target cell membranes
will be used to formulate a model of toxin entry which will be specific to
AC toxin as well as generally applicable to toxins which cross host-cell
membranes.  The apparent production of a transmembrane pore by the toxin
molecule will be investigated by electro-physiologic methods in intact
cells and artificial membranes.

Serum from children immunized with whole cell pertussis vaccines or
convalescent from pertussis will be evaluated for antibody response to AC
toxin.  In addition, purified AC toxin will be tested for its capacity to
protect mice from B. pertussis infection and death in the intracerebral and
aerosol challenge models.  Inactive mutants of AC toxin will be evaluated
as genetically toxoided antigens if AC toxin protective activity is
demonstrated.

In summary, these structure/function studies will facilitate better
understanding of the mechanism of AC toxin as well as bacterial toxin
action in general and will provide an antigen potentially useful in the
prevention of clinical pertussis.

## Key facts

- **NIH application ID:** 3127629
- **Project number:** 5R01AI018000-12
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **SAM.gov UEI:** JJG6HU8PA4S5
- **Principal Investigator:** ERIK L HEWLETT
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1993
- **Award amount:** $253,475
- **Award type:** 5
- **Project period:** 1980-09-01 → 1995-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3127629

## Citation

> US National Institutes of Health, RePORTER application 3127629, BORDETELLA CYCLASE:  STRUCTURE AND BIOLOGICAL ACTIVITY (5R01AI018000-12). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3127629. Licensed CC0.

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