DETECTION AND CHARACTERIZATION OF ORNITHINE DECARBOXYLASE
ACTIVITY IN RAT PNEUMOCYSTIS CARINII: IMPLICATIONS FOR ANTI-PNEUMOCYSTIS
Thesis (PH.D.)--NEW YORK UNIVERSITY. 1992. 178p.
Dissertation Abstracts International. Volume: 53-08, Section: B, page: 4090.
Pneumocystis carinii, a microorganism most likely of fungal origin, causes a
severe pneumonia in immunocompromised individuals. Although eflornithine,
a specific ornithine decarboxylase (ODC) inhibitor, has been
successfully used as an anti-Pneumocystis agent, the activity of the enzyme
target has been reported to be absent from the parasite. Pneumocystis carinii
was re-examined and ODC was found to be present. The therapeutic effect of eflornithine
thus results from a direct anti-Pneumocystis action and not from interference
with host metabolism as it had been presupposed.
Rats immunosuppressed with dexamethasone and maintained under near-aseptic
conditions were inoculated intratracheally with lung homogenates from
previously infected rats. Pneumocystis carinii cells were separated from lungs
according to a novel isolation procedure consisting of tissue homogenization,
host-cell lysis, differential centrifugation and enzymatic digestion. The
isolated parasite cells were lyzed and ODC activity measured by capturing and
counting 14CO2 released from carboxyl carbon-labeled ornithine.
Significant and reproducible ODC activity was detectable only after removing
low-molecular weight compounds. Unlabeled ornithine, found to be
present in high concentrations in Pneumocystis carinii lysates, is most likely
the interfering compound which blocked ODC detection by previous workers. The
measured activity was linear with respect to protein concentration and was
inhibitable by eflornithine.
The possibility that the measured ODC activity was of host origin was
considered and rejected for the following four reasons. Mammalian ODC is a
cytosolic enzyme and the isolated Pneumocystis carinii contained neither host
cells nor intact organelles as judged by electron microscopy. The activity in
homogenates of Pneumocystis carinii-infected lungs correlated with the degree
of Pneumocystis carinii enrichment. Although pH optima curves for both
Pneumocystis carinii and host ODC peaked at 7.5, the two curves were clearly
distinct. The eflornithine IC50 values for isolated Pneumocystis
carinii of 3.5 μM was 2.7 times that of host ODC.
This demonstration of polyamine biosynthesis, which after folate biosynthesis
is only the second biochemical pathway clearly characterized in Pneumocystis
carinii, identifies a drug target for Pneumocystis carinii pneumonia.
Clarkson, Allen B, Jr.
9402. Revised: 940113.