Malaria parasite resistance to azithromycin is not readily transmitted by mosquitoes

Reviewer #1 (Public Review):

In this manuscript by Buchanan and colleagues, the authors set out to determine if mutations associated with resistance to the Plasmodium apicoplast inhibitor azithromycin (AZ) had a measurable impact on the fitness of Plasmodium berghei and P. falciparum parasites as they traverse both the mosquito host and vertebrate liver.

The Plasmodium endosymbiotic organelles - the mitochondrion and apicoplast - are attractive drug targets as they (1) possess essential functions across the multi-host multi-compartment life cycle of these parasites, and (2) are of bacterial origin and thus are vulnerable to inhibition both to extant antibiotics, and novel drugs with high parasite specificity.

Historically however the high resistance propensity of drug targets encoded in the organellar genomes (most notably atovaquone and doxycycline) has precluded the use of these drugs in an endemic setting, limiting these potent compounds to use in prophylaxis for travelers from non-endemic countries. Several studies in the last decade now fairly definitively show that mutations conferring resistance to atovaquone in the mitochondrial gene cytochrome b are, in a mutation-dependent manner, totally or near-totally compromised in their ability to infect, grow, and escape the mosquito host, leading to a reexamination of the potential utility of this extraordinarily potent drug in endemic settings. Symmetries exist between the Plasmodium mitochondrion and apicoplast, which both appear to have highly fexpanded roles in the mosquito and liver relative to the blood stages. Thus, the authors set out to explore whether mutations in essential apicoplast genes were, in a similar manner to mutations in cytochrome B, associated with fitness effects in the mosquito and/or liver.

Towards this, the authors selected for several AZ-resistant parasite populations, all of which acquired mutations in the apicoplast genome-encoded ribosomal protein Rpl4. Interestingly, the authors observed contrasting fitness effects caused by these mutations, both between mutants within Plasmodium species, and between species. In P. berghei, AZ mutants were compromised in their ability to form oocysts and sporozoites, and a large proportion of sporozoites lacked an intact apicoplast and displayed aberrant gliding behaviour. Similarly, in the liver, Rpl4 mutant P. berghei liver schizonts were smaller, had fewer nuclei, and appeared extremely limited in their ability to cause a patent infection - crucially in particular via mosquito bites. Surprisingly, a P. falciparum Rpl4 mutant (notably in a different position of the protein) had no impact on sporogony but appeared to have a strong impact on liver schizont development in a liver-humanized mouse model, suggesting that establishment of blood stage infection in a subsequent human host would be less likely for mutant parasites.

This is a well-executed study, that presents novel and noteworthy findings. The impact of drug-resistance-conferring mutations in Plasmodium outside of the blood stage is woefully understudied, primarily due to significant challenges associated with studying Plasmodium, especially P. falciparum, in both the mosquito and liver which the authors navigate commendably. The results presented in this manuscript leverage state-of-the-art techniques and clearly support the authors' conclusion that AZ-conferring resistance mutations have a strong negative effect on the ability of Plasmodium parasites to both reinfect and cause symptomatic infection in a subsequent vertebrate host. This could indicate that apicoplast-targeted inhibitors are more attractive as co-drugs for malaria treatment than previously thought, due to the reduced probability of the spread of resistance, which has been a perennial issue in malaria therapeutic care.

Reviewer #2 (Public Review):

Summary:

This manuscript answers an important question about the transmission of Plasmodium parasites resistant to apicoplast inhibitors, specifically azithromycin. This study builds on previous work showing the inability to transmit parasites resistant to mitochondrial inhibitor, atovaquone, based on fitness defects in transmission stages in the mosquito. The transmissibility of drug-resistant parasites is grounded in the basic biology of the Plasmodium lifecycle and has implications for the selection of drug regimens for clinical treatment, so these questions are highly significant. The authors clearly demonstrate severe defects in mosquito stages of azithromycin-resistant (AZR) P. berghei (rodent species) inhibiting transmission of AZR parasites. However, surprisingly AZR P. falciparum (human species) is unaffected in mosquito stages, rather defects are observed in liver-stage development suggesting AZR P. falciparum can transmit but may not mount a productive blood infection. The differences in the observed defects in the 2 species are important and well demonstrated in the results but are obscured in the title/summary of the manuscript. The results demonstrate that AZR parasites are unlikely to spread.

Strengths:

The authors performed experiments with both P. berghei (rodent species) and P. falciparum (human species). P. falciparum is the more relevant species from a clinical standpoint, however, there are limitations to studying the full lifecycle of P. falciparum which only infects humans and some primates, for example requiring humanized mice without intact immune systems. Pberghei is commonly used in lifecycle analyses as a proxy for experimental tractability, however, there are cases where the biology of P. berghei does not reflect that in P. falciparum. So the use of both species is complementary and most informative. Specific modification of the apicoplast genome, where AZR mutations are located, is not currently possible so matched genotypes could not be produced but multiple AZR mutants were analyzed for each species. Acknowledging these limitations in the experimental systems available, the authors perform a thorough set of experiments to pinpoint the specific defects in AZR Pb vs Pf during mosquito and liver developmental stages. The results show phenotypic differences between AZR Pb and Pf in mosquito stages which was not expected but in line with differences in apicoplast biology of Pb vs Pf that are important to document and be aware of when using P. berghei as a model for P. falciparum development.

Weaknesses:

The claim that human AZR malaria parasites (P. falciparum) is not readily transmitted to mosquitoes is incorrect, as stated in the title and abstract. Strictly speaking, transmission refers to the infection of a human host by another via mosquitos. The evidence that AZR Pf is unaffected in mosquito development indicates that transmission is not reduced compared to WT Pf. Rather transmitted AZR Pf has disrupted liver stage development and may not mount a productive blood infection. This distinction between the phenotypes of AZR Pb vs Pf is surprising, significant (suggesting differences between Pb and Pf and/or specific mutations in Rpl4), and should be more accurately represented in the title/abstract. To their credit, the authors performed thorough experiments to pinpoint the specific defects in AZR Pb vs Pf, but the current claim about AZR Pf is misleading.

Additional context:

Clinical trials (MORDOR studies) in several African countries have shown that biannual administration of azithromycin reduces childhood mortality (PMID: 31167050). The mechanism of this survival benefit is unknown and may be multifactorial. The findings in this manuscript can also be considered in the context that azithromycin is a commonly used antibiotic and may be administered for purposes other than malaria treatment. In theory, AZR-resistant parasites could be selected in asymptomatic patients not receiving malaria treatment but receiving azithromycin. This study indicates that mass distribution of azithromycin for other clinical applications would not result in adverse effects on malaria transmissions in the same population.

Reviewer #3 (Public Review):

Summary:

Drug resistance is a perennial problem for malaria control and strategies to prevent the acquisition and spread of drug resistance mutations are desperately needed. One strategy is to identify drug resistance mutations that arise in blood-stage parasites, but cannot be readily spread to a new human host. Since malaria parasites must survive and replicate in mosquitoes in order to be transmitted, mutations with elevated mosquito-stage fitness defects will not spread efficiently. Buchanan and coworkers focus on the drug azithromycin and its known role of inhibiting the ribosomes found in parasite apicoplast organelles. Apicoplast organelles are known to have elevated metabolic activity in mosquito stage parasites and azithromycin resistance mutations could interfere with mosquito stage parasite development and parasite transmission.

To address this hypothesis, azithromycin-resistant P. berghei and P. falciparum parasites were generated and analyzed for transmission defects. All lines had mutations in the apicoplast ribosomal protein Rpl4 consistent with the known role of azithromycin inhibiting the 50S ribosomal subunit. Overall, the three lines (3 berghei and one falciparum) had phenotypes that should limit parasite transmission, however, detailed characterization showed that there were surprising differences between the two parasite species and even between the P. berghei lines. The P. berghei lines produced fewer oocysts and sporozoites with aberrant apicoplast morphology compared to wild-type controls. Sporozoites from azithromycin-resistant lines appeared to have motility defects and typically were not able to infect mice (one strain produced infections when 10,000 sporozoites were injected, but not when 1,000 were).

By contrast, the azithromycin-resistant P. falciparum strain did not display any mosquito-stage phenotypes and produced motile sporozoites with intact apicoplast organelles. These sporozoites, however, developed abnormally in a humanized mouse model with reduced liver-stage nuclear division and abnormal apicoplast morphology. These defects combined with a five-fold lower prevalence suggest that azithromycin-resistant P. falciparum parasites experience significant fitness costs during liver stage development (at least those harboring the G76V mutation).

Strengths:

This work was carefully conducted and transparently presented. It provides a comprehensive view of how parasite development is impacted by azithromycin resistance mutations during the mosquito and liver stages in P. berghei and P. falciparum. It adds a new dimension to the growing literature on the transmissibility of drug-resistant parasites, by showing that mutations in the apicoplast genome can impact transmission.

Weaknesses:

Whether these liver-stage defects in P. falciparum are severe enough to completely block subsequent blood-stage infection remains to be seen and would require experiments with humanized mice continuously grafted with human red blood cells - a difficult and expensive model system.

Author Response

We would like to thank the reviewers for their positive comments and valuable suggestions for improvements to the manuscript. We intend to revisit the discussion to clarify our interpretation of how azithromycin resistance mutations impact the transmission potential of P. falciparum and expand on the differences between mouse and human malaria. Additionally, we intend to adjust the title to better align with the revised interpretation of the main findings. These changes will be reflected in the revised manuscript to be submitted as the eLife Version of Record.