Invasion On The Sly

A new microscopy shows how malaria 
menace wreaks havoc, reports Biplab Das 

Every year the malaria parasite claims numerous lives. The parasite is so powerful that it easily evades even smart drugs. Shortly after entering the host's body, it takes refuge inside the red blood cells (RBCs), where it wreaks havoc. 

Till now, it is unclear how the parasite gets inside an RBC. But a research team led by Dr. Tapan Guha of University Science Instrumentation Centre (USIC), Calcutta University, and Prof. R. L. Brahmachary, formerly with the embryology unit of the Indian Statistical Institute (ISI) and now with USIC, has detected tiny pits on the outer membrane of the RBCs which directly open into the cytoplasm of the blood cells. This knowledge might guide experts in future to devise a defence mechanism against the parasite. 

"The malaria parasite might prefer those pits to launch an invasion," write Guha and Brahmachary in the latest issue of Current Science. The research team comprising Kajol Bhattacharya, an honorary research student at USIC, and R. Bhar, a research scholar from USIC, Jadavpur University, used a technique called Atomic Force Microscopy (AFM) to make images of an RBC's outer surface. The team was supported by Dr. A. Sen, head of USIC, and Dr. V. Ganesan, a physicist from the University of Indore. 

In AFM, the sample to be studied is not prepared in any solution. After the blood sample is drawn, it is smeared on a glass slide and air dried. The tip of an extremely small probe is mounted on the end of a small flexible cantilever. When the surface of an RBC comes in contact with the tip, a weak attraction called Van der Waals' force comes into play between them. This force deflects the cantilever. The deflection is detected by a laser beam which is then reflected onto a photo-detector. "The sensitive photo-detector generates a measurable signal voltage, producing the images of the RBC's surface, a tiny landscape interspersed with pits and depressions," explains Guha. 

The images they took revealed a lot about the RBC's outer surface. It was found that the tiny pits or holes are surrounded by depressions. Surprisingly, the pits are much smaller than the pores found on the membrane of a cell's nucleus. "It has been revealed that the depressions consist of blebs," Guha says. "Blebs are thought to be sculpted by nanometre-size particles made of either protein or lipid molecules. One nanometre (nm) equals one billionth of a metre." 

On the outer surface of an RBC, at high magnification, at least nine depressions or holes have been found between closely packed nanometre-size particles. The size of the particles is mostly in the 75-100 nm range. 

Guha and his teammates have designed an experiment to study the blebs. "If a fat-digesting enzyme dissolves the blebs then they are made of lipids," he says. "On the other hand, blebs dissolved by a protein-digesting enzyme are made of proteins." 

Now the team has employed a special type of microscope called Lateral Force Microscope (LFM). With LFM, the varying roughness of an RBC's surface can be imaged. In the image, white portions show the roughest parts of the outer membrane, and the darker parts show the smoother region. It reveals that the varying structure of molecules contributes to the varying roughness of the RBC's surface. "It has been found that holes appear due to the network of proteins like spectrin and actin," says Guha. "Those protein molecules, being plastic, contribute to the changing shape of RBCs." This comes in handy when RBCs pass through the extremely thin passage of capillaries. 

Apart from the insight into the malarial parasite, the study of RBCs shows other promising aspects as well. Abnormal RBCs of leukaemia and thalassaemia have shown the distinctive structural changes caused by the diseases. This finding might have implications in selecting and delivering drugs. "Studying abnormal RBCs could reveal why their shape has been distorted, thereby uncovering the protein responsible," says Dr. Tapan Kumar Bhattacharya, a cardiologist formerly from R. G. Kar Medical College who collaborated in this research. 

Before the discovery of holes, it was known that opening and closing of protein channels on the cell membrane control the exit and entry of ions and other particles. Drugs act by binding with receptor molecules on the membrane. "But finding permanent holes or pits has challenged the existing theories," says Bhattacharya. "Future study could show whether ions and drugs can pass through the holes." The surface holes might also have implications in allergic and immunological reactions. 

Guha's team is now planning to study the deformed RBCs of sickle cell anaemia. People with sickle cell anaemia are resistant to malaria. The team wants to find out why this is so. According to Guha, AFM can be used to study other types of human cells and their vulnerability to diseases. "A cell's metabolic functions could be studied while it is kept alive in a nutrient medium," he says. There is also the prospect of studying the effects of drugs on living cells using AFM. 

Although AFM can magnify an object up to one million times, its full scope was not exploited in this study. "Using the full potential of AFM, many other unknown facets of the RBC's outer surface will be unravelled," says Guha.

    September 30, 2002.