Exploring Malaria Parasite Entry into Red Blood Cells
Introduction

Exploring Malaria Parasite Entry into Red Blood Cells

Hunting new drug targets to reduce the debilitating and costly burden of malaria

The rhoptry is an organelle found in malaria parasites that is essential to their ability to invade red blood cells. A recent paper from Dr. Danny Wilson's group at the Research Centre of Infectious Diseases, University of Adelaide, Australia, describes how functional knock-down of a newly identified rhoptry-associated protein, 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. Confocal super-resolution microscopy was used to help characterize PfCERLI1 in this work.

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

Research at the Wilson Lab

The Wilson Lab - also known as the Malaria Biology Laboratory - is located at the Research Centre for Infectious Diseases. They apply 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, they hope to identify new drug targets that can be developed to reduce the debilitating and costly burden of malaria.

The Malaria Rhoptry

An Organelle Essential for Malaria Parasite Red Blood Cell Invasion Visualized using Confocal Super-resolution Microscopy

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.
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.

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.

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.

Understanding Malaria Parasite Invasion with Super-resolution Microscopy of the Rhoptry

The rhoptry is an essential organelle needed for the malaria parasite to enter red blood cells. 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 red blood cell, mechanical entry and formation of a vacuole where the invaded parasite will then grow and replicate.

Dr. Sonja Frölich is a postdoctoral researcher in the Wilson lab and is working to develop new approaches that dramatically improve visibility into the rhoptry, an essential organelle in the parasite needed for red blood cell entry and establishment of blood stage parasites.

Dr. Sonja Frölich, University of Adelaide, Australia

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 PfCERLI1 - a newly identified rhoptry protein - function through gene-editing.

Dr. Sonja Frölich

Wilson Laboratory, Research Centre of Infectious Diseases, University of Adelaide, Australia

Image segmentation validation for quantitative analysis of malaria invasion organelles
Image segmentation validation for quantitative analysis of malaria invasion organelles

Image Segmentation Validation for Quantitative Analysis of Malaria Invasion Organelles

A) Merozoite organelles imaged by fluorescence microscopy often appear as irregularly shaped objects, and image pre-processing and segmentation methods must be implemented to subtract background from genuine signal. In this example, PfCERLI1 immuno-labelled with anti-HA antibodies (green) have been segmented at threshold values to separate signal from noise. Signals are then converted into objects, based on minimal/maximal size, shape and signal intensity. Data is then extracted from each of these objects, including shape (sphericity), intensity, volume and area.

B) Representative super-resolution micrographs of immune-labelled rhoptries in control (untreated) and glucosamine PfCERLIHAGlmS schizonts. Data obtained from object analysis can then be compared between two different treatment to assess the influence of the treatment on the fluorescent marker of interest (Nature Communications, 2020).

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.

Dr. Sonja Frölich

Wilson Laboratory, Research Centre of Infectious Diseases, University of Adelaide, Australia

Giemsa stain of malaria parasites rupturing and invading red blood cells. Schz: Schizont – young malaria parasite.
Giemsa stain of malaria parasites rupturing and invading red blood cells. Schz: Schizont – young malaria parasite.

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.

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.

Increasing Efficiency with Automation

The Wilson team is currently testing the capabilities of the ZEISS Axioscan automated slide scanner to acquire thousands of images using automated high content imaging and then automated data analysis using ZEISS ZEN Intellesis. With minimum user input required, this will speed up their research and provide further insights into the biophysical interactions between the malaria parasite and the human red blood cell.

They are currently exploring using the ZEISS Axioscan to capture phenotypical changes in Giemsa stained thin blood smears of PfCERLI1 knockdown parasites.


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