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Updated: Jul 18, 2026
High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity
Published on: November 16, 2016
In vitro efficacy, resistance selection, and structural modeling studies implicate the malarial parasite apicoplast
Amar Bir Singh Sidhu1, Qingan Sun, Louis J Nkrumah
1Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Abstract:
Azithromycin (AZ), a broad-spectrum antibacterial macrolide that inhibits protein synthesis, also manifests reasonable efficacy as an antimalarial. Its mode of action against malarial parasites, however, has remained undefined. Our in vitro investigations with the human malarial parasite Plasmodium falciparum document a remarkable increase in AZ potency when exposure is prolonged from one to two generations of intraerythrocytic growth, with AZ producing 50% inhibition of parasite growth at concentrations in the mid to low nanomolar range. In our culture-adapted lines, AZ displayed no synergy with chloroquine (CQ), amodiaquine, or artesunate. AZ activity was also unaffected by mutations in the pfcrt (P. falciparum chloroquine resistance transporter) or pfmdr1 (P. falciparum multidrug resistance-1) drug resistance loci, as determined using transgenic lines. We have selected mutant, AZ-resistant 7G8 and Dd2 parasite lines. In the AZ-resistant 7G8 line, the bacterial-like apicoplast large subunit ribosomal RNA harbored a U438C mutation in domain I. Both AZ-resistant lines revealed a G76V mutation in a conserved region of the apicoplast-encoded P. falciparum ribosomal protein L4 (PfRpl4). This protein is predicted to associate with the nuclear genome-encoded P. falciparum ribosomal protein L22 (PfRpl22) and the large subunit rRNA to form the 50 S ribosome polypeptide exit tunnel that can be occupied by AZ. The PfRpl22 sequence remained unchanged. Molecular modeling of mutant PfRpl4 with AZ suggests an altered orientation of the L75 side chain that could preclude AZ binding. These data imply that AZ acts on the apicoplast bacterial-like translation machinery and identify Pfrpl4 as a potential marker of resistance.
Insights
Azithromycin (AZ) shows increased antimalarial potency against Plasmodium falciparum with prolonged exposure. Resistance arises from mutations in ribosomal protein L4 (PfRpl4) within the apicoplast, suggesting AZ targets this bacterial-like machinery.
Area of Science:
- Microbiology
- Parasitology
- Molecular Biology
Background:
- Azithromycin (AZ), a macrolide antibiotic, exhibits antimalarial activity against Plasmodium falciparum.
- The precise mechanism of AZ's action against malaria parasites remains unclear.
- Understanding AZ's antimalarial mechanism is crucial for developing new therapeutic strategies.
Purpose of the Study:
- To investigate the in vitro efficacy and mechanism of action of Azithromycin (AZ) against Plasmodium falciparum.
- To identify genetic factors conferring resistance to AZ in malaria parasites.
- To explore potential drug resistance markers for AZ.
Main Methods:
- In vitro culture of Plasmodium falciparum with varying Azithromycin exposure durations.
- Drug synergy testing with standard antimalarials (chloroquine, amodiaquine, artesunate).
- Selection and genetic analysis of Azithromycin-resistant parasite lines, including sequencing of apicoplast ribosomal RNA and ribosomal protein L4 (PfRpl4).
- Molecular modeling to predict the impact of mutations on AZ binding.
Main Results:
- Prolonged AZ exposure (1-2 generations) significantly enhanced potency, achieving 50% inhibition at nanomolar concentrations.
- AZ showed no synergy with chloroquine, amodiaquine, or artesunate and was unaffected by pfcrt or pfmdr1 mutations.
- Azithromycin resistance was associated with a G76V mutation in PfRpl4 and a U438C mutation in apicoplast rRNA.
- Molecular modeling suggested the PfRpl4 G76V mutation impedes AZ binding to the ribosome's polypeptide exit tunnel.
Conclusions:
- Azithromycin exerts its antimalarial effect by targeting the bacterial-like translation machinery in the apicoplast.
- The PfRpl4 gene, specifically the G76V mutation, is identified as a key determinant of AZ resistance in Plasmodium falciparum.
- PfRpl4 mutations could serve as potential biomarkers for predicting AZ resistance in malaria treatment.
