Plasmodium falciparum: New Developments

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Electron micrograph of Plasmodium falciparum. 2006.

By Charley Myers

Background

Plasmodium falciparum is a protozoan of the eukaryotic domain. It is widely known in today's world as one of the most common malarial parasites. This particular species causes malignant malaria, which leads to the most complications and mortality rates of all malaria-causing agents. It is estimated that between 300 million and 500 million people are afflicted with malaria annually (WHO). The majority of these incidences of malaria occur in sub-Saharan Africa and affect children under 5. According to the CDC, there are 156 species of Plasmodium, four of which are considered parasitic to humans. These include P. falciparum, P. vivax, P. ovale and P. malaria. Plasmodium falciparum has the highest rate of malarial infection among the four species . We know for a fact that P. falciparum is a malarial parasite that targets humans. What is relatively unknown, however, is the exact mechanism by which the protozoa are able to enter the cell and cause disease.

The Physical Microbe

Taxonomy

Kingdom: Protista Subkingdom: Protozoa Phylum: Apicomplexa Class: Sporozoasida Order: Eucoccidiorida Family: Plasmodiidae Genus: Plasmodium Species: Falciparum

Metabolism

Plasmodium falciparum is a highly proliferating organism whose job is very energetically costly. The protozoa must maintain a high metabolic rate in order to infect its various hosts. It is clear that the metabolism of P. falciparum is closely intertwined with that of its host, as the two share a very intimate relationship. The metabolism of the microbe must adapt to its changing environments. For example, when in the blood stage of its lifecycle, P. falciparum acquires most of its energy from oxidizing glucose into lactate through the process of glycolysis. This parasite utilizes up to 75% times more glucose than its uninfected erythrocyte counterparts. Glycolysis without the TCA cycle is not very productive in the gathering of energy, as it generally only produces 2 ATP per molecule of glucose. A reasonable way to generate more ATP would be to carry out the TCA cycle. However, as the mammalian blood is teeming with glucose, it is efficient enough a process to perform glycolysis alone.

There is evidence that proposes hemoglobin degradation as an additional form of generating energy in P. falciparum, on top of glycolysis. During the intraerythrotic stage of the P. falciparum life cycle, in which the protozoa is parasitizing a vertebrate, the cytoplasm of the host cell is consumed and between 60% and 80% of the hemoglobin in the cell is degraded. After the degradation the heme moiety is stored as a polymer, known as the malaria pigment hemozoin, instead of being recycled. P. falciparum may then utilize this polymer in order to produce amino acids, which are then integrated into parasite proteins and may also be used in metabolism. This is a good example of parasitism because the parasite, P. falciparum is pooling the resources of the host (the hemoglobin) and turning them into an integral part of its own survival (amino acids) (Figure 2).

Figure 2. Hemoglobin degradation pathway in P. falciparum. Francis et al 1997.

Hemoglobin metabolism is not fully sufficient for the metabolism of P. falciparum. But it can be combined with exogenous amino acid synthesis in order to allow for P. falciparum survival. Knowledge about hemoglobin degradation has proven very useful to researchers targeting malaria outbreaks worldwide. This is because the enzymes that are involved in hemoglobin breakdown, or proteolysis, may be potential targets for antimalarial drug therapy (see section on Malarial Drug Therapy).

Life Cycle and Transmission

Plasmodium falciparum have a complicated life cycle. They are able to produce both sexually and asexually, although may only do so at different times during the cycle. To carry out the asexual cycle, the parasite must have a vertebrate host, whereas a female Anopheles mosquito is needed for the completion of the sexual cycle. Transmission of P. falciparum, and therefore malaria, begins when the parasite is in the sexual stage of its life cycle. A female Anopheles mosquito injects P. falciparum sporozoites into the vertebrate bloodstream during a blood meal. Human infection begins in the liver, as these sporozoites travel to the liver and invade the hepatocytes. This stage is known as pre-erythrocytic development. Once this is complete, the asexual stage of P. falciparum’s life cycle can begin via the release of merozoites into the host bloodstream (Figure 3).

Figure 3. Nature Reviews. Life cycle of P. falciparum in both human host and mosquito vector.

Some of these merozoites mature into sexual gametocytes, which is the only vector through which transmission from humans to mosquitos may occur. The remaining gametocytes, those of stages I-IV are sequestered into the bone marrow while stage V gametocytes may circulate freely in the peripheral blood. Once these mature (Stage V) gametocytes are ingested by a female mosquito, gamete fusion occurs in the midgut of the mosquito and produces a mobile ookinete which can form oocysts in the midgut wall. As time goes on, these oocysts grow and mature and eventually burst, releasing sporozoites that travel to the salivary glands of the mosquito. These sporozoites then go on to infect humans during a blood meal, starting the cycle again.

Transmission of malaria can vary by environment. For example, areas in which the vector (Anopheles) has ample water habitats and therefore longer life spans, giving the parasite more time to mature, see higher intensity of transmission. Climactic conditions, such as rainfall, temperature and humidity, also affect transmission of P. falciparum to humans. Transmission is often of a seasonal nature, with peaks in intensity during and immediately after the rainy season. Anopheles, especially those found in Africa, prefer to lay their eggs in shallow freshwater, such as puddles. These reservoirs of standing freshwater are obviously more abundant in the rainy season, leading to the increased rates of transmission seen.

Historical Significance

Malaria is not a 21st century problem; Plasmodium falciparum has been problematic for thousands of years. Additionally, it was not always concentrated in Africa, as it might be easy to assume today. In fact, the bustling metropolis of ancient Rome had some of the highest transmission rates of malaria. Because Rome is not a tropical place and because the infection of P. falciparum was concentrated in young children, there must have been extenuating circumstances which allowed for such prevalence of this disease. Transmission rates and therefore the overall incidence of malaria were exceptionally high in ancient Rome, a condition which is known as hyperendemicity. It is estimated that somewhere between 50 and 75% of inhabitants of ancient Rome carried the malaria parasite P. falciparum. Those who were infected in early life but survived were able to develop a gradual immunity to their local strains of P. falciparum. As a result, instances of infection and illness due to P. falciparum became less and less frequent with age. The above situation seems beneficial for the human hosts of P. falciparum and victims of malaria; a lot of built up resistance should be a good thing. However, imperial Rome saw throngs of adult immigrants moving to the crowded areas of the city, most of whom did not have the necessary resistance to the local strains of P. falciparum. Many accounts from ancient writers emphasize the susceptibility of adult immigrants to malaria. The infections themselves manifested in quite different ways among hosts, with children experiencing different symptoms than adults. This difference even lead to different terminologies for describing the same disease; children experience “quotidian fever” whereas adults suffered from “semitertian fever”. Additionally, certain demographics seemed to be hit harder than others. In particular, small children and adult men, as mentioned above, were frequent sufferers of a “falciparian infection”.

How do we know for sure that P. falciparum was the parasite causing these deadly infections in ancient Rome? Researchers were able to answer just this question when, in the 1990s, a large infant cemetery was discovered on the outskirts of Rome. This find was unusual, as babies rarely received grand official burials in ancient Rome. Plasmodium falciparum was extracted from the bones of one of the inhabitants of the cemetery, a 2 to 3-year-old girl (Figure 4).

Figure 4. Salleres et al 2004. “Infant burial no. 36, an inhumation burial, at the cemetery at Lugnano, in Taverna.”

Scientists were successfully able to amplify a section of ribosomal DNA of P. falciparum via a Polymerase Chain Reaction. This evidence proves that malaria was indeed the cause of death of many in the ancient world.

The Disease Today

Malaria

According to the latest estimates from the World Health Organization, there were 212 million cases of malaria worldwide, resulting in 429,000 deaths in 2015. The majority (90%) of the cases occurred in sub-Saharan Africa. More strikingly, over two thirds (70%) of malaria related deaths are in children under 5; every two minutes a child dies from malaria. This is obviously a pressing issue that should fall on all of humanity.

Symptoms

Malaria, caused by Plasmodium falciparum has a range of symptoms. Symptoms may be virtually absent or very severe and may result in death. The disease itself may even be classified as uncomplicated, or asymptomatic, or severe/complicated, exhibiting serious symptoms. The symptoms displayed by a malaria patient are all caused by the asexual life stage of Plasmodium falciparum. When the protozoa mature in the blood of the human host they release many waste products, many of which are unknown to us, into the bloodstream. The toxins that are released into the body stimulate an immune response within the host. This response manifests in fever and rigors, as a result of cytokine release. In the case of the “uncomplicated” or milder form of malaria, patients may present with the following symptoms: fever, chills, general feeling of malaise, headaches, sweating, nausea and vomiting, enlarged liver or spleen, weakness, mile jaundice, increased respiratory rate, and body aches. In countries where malaria is not very prevalent these symptoms may go unnoticed as malaria and may instead be attributed to something less dangerous, like the common cold or the flu. However, in areas familiar with malaria, such as sub-Saharan Africa, the sufferer may more easily recognize the symptoms as those of deadly malaria and may even treat themselves without a diagnosis. Severe malaria obviously has more serious, life threatening symptoms compared to its uncomplicated counterpart. These can include a combination of: severe anemia, low blood pressure, acute kidney failure, hyperparasitemia, metabolic acidosis, hypoglycemia, abnormal blood coagulation, acute respiratory distress syndrome or ARDS, cerebral malaria accompanied with abnormal behavior, seizures, impairment of consciousness, coma, or hemoglobinuria. The patient may also present these symptoms in conjunction with those of uncomplicated malaria. In order to officially diagnose malaria one must present the parasite, P. falciparum, in the blood. This can be tested with the help of a microscope. Malarial deaths are most often caused by related complications including: cerebral malaria, a swelling of the brain due to infected blood vessels, breathing problems caused by a pulmonary edema, or gathered fluid in the lungs, organ failure, including the liver or the kidneys, anemia, due to malarial damage to the red blood cells, or low blood sugar, caused by a medication used to treat malaria, or by the disease itself. Symptoms generally present themselves anywhere between 7 to 30 days after the Anopheles bite. The incubation period of P. falciparum is usually shorter than other malaria causing strains of Plasmodium, like Plasmodium malaria.

Immunity and Resistance

Researchers argue that falciparian disease is one of the biggest selective pressure on humans in today’s world. Interestingly, there are some humans are naturally resistant to malaria. A classic example of this is the heterozygote advantage of sickle cell anemia. The sickle cell trait has proven to be advantageous against malaria, especially when caused by Plasmodium falciparum. The abnormal shape of the hemoglobin in sickle cell patients confers resistance to P. falciparum in its erythrocyte stage compared to those with normal hemoglobin. There are also other genes in the human genome that, when mutated, can bring about resistance to malaria. These include the ABO gene, G6PD, HLA, -thalassaemia and -thalassaemia. Malaria resistance genes frequently extremely variable in nature. The aforementioned genes are some of the most variable of the whole human genome. The variation seen may be due to the fact that these genes are rapidly producing new adaptive alleles at a high mutation rate.

Politics and Prevention

As mentioned above, the highest instances of malaria appear in Africa and other tropical and sub-tropical regions. However, this does not mean that the inhabitants of those areas are the only ones affected by the disease. Malaria is a worldwide issue and we need to work to eradicate it. In 2005, President George W. Bush began the President’s Malaria Initiative in order to eliminate malaria. President Barack Obama expanded it further under his leadership. At its conception the program was endowed with $30 million; in 2016 it had $670 million. This governmental program works alongside private programs, like the Bill and Melinda Gates Foundation. Most of the funding that programs like these goes toward research and prevention. The most effective way to prevent malaria in high-prevalence areas is to stop the mosquito bite before it happens. International travelers to destinations like these are encouraged to take a prescription medicine before, during, and after the trip in order to prevent malaria. This drug acts via a mechanism that is not completely known, however it has proven relatively effective at preventing malaria in travelers. Travelers are also advised to cover as much skin as possible and use the recommended amount of insect repellant. Bed nets are also highly recommended in the prevention of malaria transmission. Jessica Cohen, professor of global health at Harvard’s Chan School, argues that bed nets should be much more widely used among the local populations affected by the disease. They are an inexpensive yet effective prevention mechanism and are more accessible than the expensive repellant, pills and clothing. However, the most effective solution yet would be a vaccine. A vaccine will greatly speed up the process of the total eradication of P. falciparum malaria. With all the research being done to target Plasmodium falciparum and its insect vector, the microbes and the mosquitos are developing resistance to insecticides and medicines. A vaccine is the solution to these problems.

Drug Therapies

Even though prevention proves to be the most effective way of eradicating malaria worldwide, drug therapy also proves very successful in treating patients who have already contracted the disease. The existing drugs used to treat malaria today include a whole slew of anti-infectives. Quinolines are some of the most common anti-malarial drugs. They work by targeting the DNA synthesis of a potential parasite or pathogen. Synthetic quinones, such as chloroquine and amodiaquine, are inexpensive and have only a three-day course of treatment, making them relatively accessible to those most affected by this epidemic. However, use of this drug has decreased over time due to its ability to confer resistance in the target strain of P. falciparum. To combat this resistance, quinones are often used alongside other antimalarial drugs, such as Artemisinins. Artemisinins are a class of drug that act quickly against the sexual stages of the P. falciparum life cycle. Artemisinin derivatives have the ability to rapidly eliminate the parasite itself which makes it a good partner of quinone drugs. New drugs are constantly being researched and tested. A possible new target for antimalarial drugs is hemoglobin degradation (Francis et al). Specifically, researchers are trying to target the enzymes involved in the proteolysis of hemoglobin, as were mentioned above in the Metabolism section.

New Developments in the Transmission of Plasmodium falciparum

A huge question in the field of malarial research and drug therapy remains. How do the malaria sporozoites migrate through host cells in order to spread infection? Until now, not much was known about human cell traversal by Plasmodium falciparum organisms. Antibodies have been known to inhibit traversal within the human liver. These include antibodies against circumsporozoite protein (CSP), a protein involved in hepatocyte binding in human hosts, and those induced by chloroquine prophylaxis. Additionally, a receptor for hepatocyte invasion in humans was identified in 2015, although its functions are not yet well understood.

In 2017, a research team led by Annie Yang set out to discover how Plasmodium falciparum is able to traverse the cell membrane of host cells and cause infection. In order to do so, they performed a knock-out experiment in which they generated traversal-deficient sporozoites lacking sporozoite microneme protein essential for cell traversal (SPECT) and perforin-like protein 1 (PLP1). They performed this experiment on humanized mice with liver infections. The researchers hypothesized that the sporozoites required both aforementioned proteins for successful cell traversal; their data supports their hypothesis.

Two proteins, sporozoite microneme protein essential for cell traversal (SPECT) and perforin-like protein (PLP) were found to be localized on the surface of the P. falciparum cell, indicating their ability to successfully interact with host cells (Figure 5).

Figure 5. Yang et al 2017. Immunofluorescence microscopy labeling SPECT and PLP1 proteins in Plasmodium falciparum (Pf).

PLP is very likely to be involved with cell entry, as it has a highly-conserved perforin/MAC domain. Domains like these are generally associated with lysis of host cell membrane due to pore formation and membrane destabilization. Strains of P. falciparum were mutated via double cross-over homologous recombination in order to knock out either the SPECT or PLP gene. These mutated strains of P. falciparum, missing either the SPECT gene or the PLP gene were not able to traverse through human cells. For an in vivo experiment, the PLP knockout strain infected humanized mice livers. No P. falciparum was detected in the livers of infected mice (Figure 6). Therefore, PLP is an integral part of liver infection in the vertebrate host.

Figure 6. Yang et al 2017. Measurements of P. falciparum presence in human liver cells. NF54 are wild type while PfPLP1 D2 are the PLP knockout strains of P. falciparum.

It was previously thought that the PLP protein was necessary for egress of merozoites from erythrocytes. However, it was proven that PLP is not present in the asexual stage of the parasitic cycle and, additionally, a PLP knockout successfully carries out blood-stage growth and gametocyte development. New developments like these should prove extremely useful in future anti-malarial endeavors. The more we find out about the parasite itself and its mechanisms of entry and infection of humans, the more ways we will have to target with drug therapies.

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2017, Kenyon College.