3D rendered models of malaria merozoites. DNA is shown in blue, the rhoptry protein RAP1 in red and the rhoptry protein Cerli1 in green. Acquired with ZEISS confocal microscope with Airyscan detector.

Exploring Malaria Parasite Entry into Red Blood Cells

Dr. Sonja Frölich caught our attention with her tweet demonstrating her use of ZEISS Airyscan super-resolution technology to study an organelle which the malaria parasite uses to invade red blood cells by analyzing thousands of samples in a non-biased, semi-automated fashion.

Dr. Sonja Frölich is a postdoctoral researcher at the Malaria Biology (Wilson) Laboratory located in the Research Centre of Infectious Diseases, the University of Adelaide, Australia. The focus of her work is in understanding the biophysical interactions between the malaria parasite and the human red blood cell (RBC).

Dr. Frölich is developing new approaches that dramatically improve visibility into the parasite proteins and underlying forces that control an essential organelle needed for RBC-entry, called the rhoptry, and thus replication of disease causing blood stage parasites. 

Dr. Frölich, along with former postgraduate student, Benjamin Liffner, recently published a paper which describes how functional knock-down of a newly identified rhoptry-associated protein termed PfCERLI1 results in failure of the parasite to infect a red blood cell. This loss of infectivity could be targeted for the development of antimalarial compounds that reduce disease pathology in patients infected with malaria. We asked her to tell us more about her research.   

Tell us about the Malaria Biology Laboratory.

Tear drop shaped malaria merozoites infecting red blood cells. Video acquired with the ZEISS Axio Observer inverted microscope using transmitted light brightfield microscopy.

The Wilson Lab applies multi-disciplinary approaches to understand the unique biology that allows malaria parasites to infect human red blood cells and cause disease.

By identifying and characterizing the key proteins that enable malaria parasites to infect red blood cells, we hope to identify new drug targets that can be developed to reduce the debilitating and costly burden of malaria.

In our recent publication, our approach was to develop a robust and quantitative super-resolution microscopy-based image analysis pipeline to characterize what happens when we removed PfCERLI1s function through gene-editing. 

What findings did you recently publish?

Rhoptries, an essential organelle needed for RBC-entry, are ubiquitous throughout the phylum Apicomplexa, which includes some of the most important single-celled parasites of humans and animals. This specialized secretory organelle is located at the anterior pole of the parasite where it appears as a set of large club-shaped organelles. Upon contact with the host cell, the rhoptry organelles secrete proteins involved in early parasite attachment to the RBCs, mechanical entry and formation of a vacuole within which the parasite grows and replicates.

In the present study, we used electron microscopy, super-resolution (Airyscan) microscopy and computational image analysis to determine the subcellular localization of PfCERLI1 and the effect on rhoptry structure with PfCERLI1 depletion. Furthermore, RBC-entry and secretion assays in PfCERLI1 depleted parasites showed aberrations in proteolytic processing and blocked secretion of key rhoptry antigens which prevented the parasite from infecting red blood cells.

Super-resolved and 3D reconstructed malaria merozoite rhoptries stained with antibodies to RAP1 and PfCERLI1 protein and their respective location/size within the rhoptry organelle. Image acquired with ZEISS LSM 800 with Airyscan. Courtesy of S. Froelich, Malaria Biology (Wilson) Laboratory, Research Centre of Infectious Diseases, the University of Adelaide, Australia

Super-resolved and 3D reconstructed malaria merozoite rhoptries stained with antibodies to RAP1 and PfCERLI1 protein and their respective location/size within the rhoptry organelle. Image acquired with ZEISS confocal microscope with Airyscan.

Our work provides important mechanistic insights supporting a role for PfCERLI1 in normal rhoptry function, which is essential for the establishment of red blood cell infection and multiplication of disease causing blood stage parasites. 

How did you use Airyscan microscopy in this work?

To complete the project, we combined genetic engineering to inducibly ablate PfCERLI1 function and used a ZEISS confocal microscope equipped with the Airyscan detector to collect z-stacks from hundreds of wild type and knockdown parasites. Using this super-resolution technology, we could pass the physical limits of light and see spatial positioning of multiple proteins simultaneously within the rhoptry in three-dimensions.

It took two years to complete the imaging side of the project. It has been an enormous effort that would not have been possible to achieve with a conventional confocal microscope.  

Workflow used to validate image segmentation for quantitative analysis of malaria invasion organelles based on super-resolved 3D Airyscan images:

Workflow used to validate image segmentation for quantitative analysis of malaria invasion organelles based on super-resolved 3D Airyscan images. Courtesy: S. Froelich, Malaria Biology (Wilson) Laboratory, Research Centre of Infectious Diseases, the University of Adelaide, Australia
Workflow used to validate image segmentation for quantitative analysis of malaria invasion organelles based on super-resolved 3D Airyscan images. Courtesy: S. Froelich, Malaria Biology (Wilson) Laboratory, Research Centre of Infectious Diseases, the University of Adelaide, Australia

What next projects are you working on?

Being able to acquire thousands of images using automated high content imaging and then automated data analysis using ZEISS ZEN Intellesis with minimum user input will speed up our research and provide further insights into the biophysical interactions between the malaria parasite and the human red blood cell.

We are currently exploring the application of the ZEISS Axioscan automated slide scanning platform to probe phenotypical changes in Giemsa stained thin blood smears of PfCERLI1 knockdown parasites.

Giemsa stain of malaria parasites rupturing and invading red blood cells. Schz: Schizont - young malaria parasite. Yellow arrow - ruptured cells. Red arrows - liberated daughter parasites, merozoites (Mz). Image acquired with ZEISS Axioscan. Courtesy: S. Froelich, Malaria Biology (Wilson) Laboratory, Research Centre of Infectious Diseases, the University of Adelaide, Australia

Giemsa stain of malaria parasites rupturing and invading red blood cells. Schz: Schizont – young malaria parasite. Yellow arrow – ruptured cells. Red arrows – liberated daughter parasites, merozoites (Mz). Image acquired with ZEISS Axioscan.

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