# Plasmodium actin-like proteins are essential for DNA segregation during male gametogenesis and malaria transmission

## TL;DR

This study shows that two actin-like proteins are essential for DNA segregation during male gametogenesis in malaria parasites, which is crucial for malaria transmission.

## Contribution

The study identifies Alp5a and Alp5b as essential for DNA segregation during male gametogenesis in Plasmodium, revealing a potential target for transmission-blocking interventions.

## Key findings

- Alp5a and Alp5b are essential for DNA segregation during male gametogenesis in Plasmodium.
- Deletion of Alp5a or Alp5b leads to impaired oocyst development and male gamete integrity.
- Alp5a and Alp5b are structurally similar to human Arp2 and Arp3 and localize to the nucleus.

## Abstract

Protozoan parasites of the genus Plasmodium cause malaria and involve infection of multiple hosts and cell types during the life cycle. Producing sexually fit gametocytes is essential for transmitting the Plasmodium parasite into an anopheline mosquito vector. After the uptake of malaria parasites, male gametocytes undergo three rounds of DNA replication to produce eight nucleated flagellar gametes. Here, we report that the actin-like proteins Alp5a and Alp5b are involved in DNA segregation during male gametogenesis. The Plasmodium-specific Alp5a and Alp5b can be superimposed on human Arp2 and Arp3, localize to the nucleus, and interact with each other. Alp5a and Alp5b are individually dispensable for the development of P. berghei blood stages, but are simultaneously indispensable for parasite viability. Consistent with genetic studies, the inhibitory activity of the Arp2/3 complex inhibitor in Plasmodium supports an essential role for this complex during the blood stage. Deletion of Alp5a or Alp5b had no impact on actin nucleation, parasite growth, or gametocytemia during the blood stage. The knockout parasites were able to invade the mosquito midgut and form oocysts; however, these oocysts were significantly smaller in size and failed to mature, ultimately leading to their death. Genetic crosses revealed defects in male gamete integrity. We found that the reduced oocyst development was due to impaired DNA segregation during male gametogenesis. Our study provides molecular insights into the fundamental requirements of the Alps in Plasmodium, which are essential for malaria transmission.

The Arp2/3 complex, comprising seven subunits (ARPC1–5, Arp2, and Arp3), orchestrates actin polymerization by nucleating branched actin networks. This complex is pivotal for spindle actin assembly during mitosis and meiosis, thereby ensuring accurate chromosome segregation. Transmission of the malaria parasite relies on a highly synchronized and rapid process known as male gametogony. Gametocytes formed during the asexual blood stage are activated in the mosquito midgut, where male gametogenesis involves three rapid rounds of DNA replication and mitosis, resulting in an octoploid genome. However, the mechanisms ensuring equal DNA partitioning during this process remain poorly understood. In this study, we studied two actin-like proteins, ALP5a and ALP5b, which interact with each other and show structural similarity to human Arp3 and Arp2. We demonstrate that the Alp5 subunit of the Arp2/3 complex localizes to the nucleus and is essential for proper DNA segregation during male gametogenesis. These findings reveal a critical role for the Alp5 proteins in nuclear division during Plasmodium male gametogenesis and identify them as a potential target for transmission-blocking interventions against malaria.