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Antimicrobial Agents and Chemotherapy, March 1998, p. 715-716, Vol. 42, No. 3
Growth and Development Section, Laboratory of
Parasitic Diseases, National Institutes of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland 20892-0425
Received 28 October 1997/Returned for modification 1 December
1997/Accepted 22 December 1997
The antibiotic micrococcin is a potent growth inhibitor of the
human malaria parasite Plasmodium falciparum, with a 50%
inhibitory concentration of 35 nM. This is comparable to or less than
the corresponding levels of commonly used antimalarial drugs.
Micrococcin, like thiostrepton, putatively targets protein synthesis in
the plastid-like organelle of the parasite.
Antibiotics of the
thiocillin-thiazolyl class, which are collectively known as
thiopeptides (16), are highly modified peptides whose site
of action lies within eubacterial large subunit (LSU) rRNA
(1). The most familiar member of this class of antibiotics is thiostrepton, which is produced by Streptomyces azureus.
It binds tightly to a small region of eubacterial LSU rRNA
(18) and inhibits the GTPase reaction catalyzed by ribosomes
in the presence of EF-G (10). Although eukaryotic ribosomes
are insensitive to thiostrepton (19), we found that
thiostrepton inhibits growth of blood-stage cultures of the malaria
parasite Plasmodium falciparum, probably by inhibiting
protein synthesis in the unusual plastid-like organelle of the parasite
(7). Thiostrepton also interacts directly with an RNA
fragment derived from the plastid-encoded rRNA, and not with
nucleus-encoded rRNA fragments (13). Since in
Escherichia coli thiostrepton binding is correlated with
inhibition of protein synthesis (18), the target for
thiostrepton in P. falciparum is presumably plastid-encoded
protein synthesis. The plastid genome was recently characterized in
detail (20) and is, perhaps, a general feature of the phylum
Apicomplexa (6, 8). Antibiotics that target the
plastid may, therefore, be of considerable interest as novel
chemotherapeutic agents against these parasites which are responsible
for important human and animal diseases. In particular, the spread of
chloroquine and antifolate resistance in P. falciparum
suggests that modification of existing drugs may not circumvent the
problem of multidrug-resistant parasites (12).
Like thiostrepton, micrococcin is a thiopeptide, although it is
produced by Bacillus and Micrococcus spp.
(2). Both compounds inhibit binding of aminoacyl-tRNA to the
ribosomal A site as well as other functions linked to eubacterial
ribosomal GTP hydrolysis (summarized in reference
1). However, subtle differences in the modes of
action of the two drugs, as described below, prompted us to test the
effects of micrococcin on parasite growth and protein synthesis, which
were correlated with the uptake of [3H]hypoxanthine and
[3H]leucine, respectively, in blood stage cultures
(5). The results were calculated as the means of
triplicate determinations (Fig. 1) and
revealed effects of micrococcin on both growth (incorporation of
[3H]hypoxanthine) and protein synthesis (incorporation of
[3H]leucine) that are statistically significant. While
the effects of thiostrepton were similar to those seen previously
(7), comparable inhibition with micrococcin occurred at
about 100-fold lower drug concentrations. For micrococcin, the
estimated 50% inhibitory concentration (IC50) for growth
was 35 ± 7.9 nM, while that of thiostrepton was 3.2 ± 0.9 M. Inhibition of protein synthesis also required higher drug
concentrations; the IC50 for micrococcin was 90 ± 22 nM and that for thiostrepton 15 ± 4 M. The greater sensitivity of
growth as opposed to protein synthesis was consistent with selective
inhibition of a minor component of total protein synthesis, as expected
if these drugs targeted plastid-encoded rRNA.
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Copyright © 1998, American Society for Microbiology. All rights reserved.
The Antibiotic Micrococcin Is a Potent Inhibitor of
Growth and Protein Synthesis in the Malaria Parasite
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FIG. 1.
Concentration-response curves describing the in vitro
inhibition of growth and protein synthesis in P. falciparum
by micrococcin and thiostrepton. Data are the percent reduction values
from no drug controls and are means of three determinations. Dashed
lines, micrococcin; solid lines, thiostrepton. Incorporation of
[3H]hypoxanthine ( 
) and [3H]leucine
(
) was measured at the concentrations of drug shown. For the assays,
an in vitro culture of P. falciparum (strains 3D7 and LF4)
was diluted in microtiter plates as described previously
(7). Micrococcin P (Mr, 1,120 [a
kind gift from J. Walker]) and thiostrepton
(Mr, 1,660 [Calbiochem]) were dissolved at 100 mM in sterile dimethyl sulfoxide (silylation grade [Pierce]); at the
highest concentrations used, the drugs precipitated in the media.
Parasites were grown for 48 h in the presence of serial dilutions
of thiostrepton or micrococcin, together with drug-free controls.
Labeling with [3H]hypoxanthine or
[3H]leucine in medium lacking hypoxanthine or leucine,
respectively, was as described elsewhere (7). Cells were
harvested and lysed, and incorporated radioactivity was estimated. Data
shown are for P. falciparum 3D7, but these were similar to
data for LF4 (IC50 for growth, 39 nM).
It is intriguing why micrococcin is much more potent than thiostrepton in inhibition of the growth of P. falciparum, since both antibiotics interact in similar ways with eubacterial LSU rRNA (14). However, there are subtle differences in the ways in which the two drugs act. For example, micrococcin stimulates ribosome-dependent GTP hydrolysis in the presence of EF-G, whereas thiostrepton completely inhibits this process (2). Whether the mechanistic difference between the drugs is related to the 100-fold discrepancy in their potencies against P. falciparum is unclear, since the effectiveness of micrococcin might result from fortuitously concentrating in the plastid-like organelle. This is similar to accumulation of chloroquine in the food vacuole of the parasite (17), with resistance genetically linked to active transport of the drug (15). Since development of the erythrocytic stages of Plasmodium takes place in a cell with no organelles and little metabolic activity, the parasite forms Golgi-like intraerythrocytic tubovesicular structures (4), which are presumably contiguous with the parasite membrane, to traffic proteins and exchange nutrients. Either mechanistic differences or active transport would provide a framework for new drug development, targeting plastid protein synthesis.
The growth-inhibitory effects of micrococcin compare favorably with those of proven antimalarial drugs tested in vitro against sensitive strains of the parasite (Table 1). Antimalarials such as pyrimethamine, chloroquine, and mefloquine act rapidly and have in vitro IC50s that are in the nanomolar range for sensitive strains and can be as high as 100 nM for resistant strains. Antibiotics are generally poorly effective as antimalarial agents, since they are slow acting, although they are used in part because of their nontoxic effects (11). Since the IC50 of tetracycline derivatives for in vitro cultures of P. falciparum is 26 to 39 M (3), the 1,000-fold greater effectiveness of micrococcin prompts investigation of this perhaps forgotten antibiotic as a potential chemotherapeutic against Apicomplexa in general. Also, the potencies of these antibiotics against the mouse malaria parasite Plasmodium berghei, including chloroquine-resistant strains, are worth investigating.
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ACKNOWLEDGMENTS |
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This work was supported by the NIH Intramural Research Program and by LamBed (E.C.).
We are grateful to Normal Heatley and James Walker (deceased) for supplies of micrococcin and micrococcin P, respectively.
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FOOTNOTES |
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* Corresponding author. Mailing address: Growth and Development Section, Laboratory of Parasitic Diseases, Building 4, Room B1-28, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425. Phone: (301) 496-6149. Fax: (301) 402-0079. E-mail: mcutchan{at}helix.nih.gov.
Present address: Antimicrobial Group, Du Pont Merck Pharmaceutical
Company, Wilmington, DE 19880-0400.
Present address: Department of Biochemistry, University of
Leicester, Leicester LE1 7RH, United Kingdom.
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Antimicrobial Agents and Chemotherapy, March 1998, p. 715-716, Vol. 42, No. 3
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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