MicrobiologyBytes

There’s a great collection of freely available resources On the Nature website under Nature Collections – Malaria (maybe NPG is finally getting the message about open access – hey Nature, it’s good to share :-)

The world is on the verge of making major inroads against malaria – a deadly disease that still claims the lives of more than 1 million people annually, mostly children less than 5 years of age. Over the past decade, scientists, large pharmaceutical companies and small biotechnology firms, governments and philanthropic organizations have come together to mount a full frontal attack on malaria, and there is now even talk of the ‘E word’ – that is, eradication. This collection highlights advances in the deployment of existing tools, and in the basic science of malaria – particularly those flowing from sequencing of the malaria parasite genomes – that will underpin the next generation of malaria-control tools, which will be needed if the scourge of malaria is to be eradicated.

Contents:

• Malaria: The end of the beginning – After decades of work, a pioneering malaria vaccine may soon reach the final phase of clinical trials. A vaccine that is far from perfect – but which may provide new direction and save thousands of lives.
• Malaria vaccine gets shot in the arm from tests – Promising results pave the way for a vaccine candidate to undergo full-blown trials across Africa.
• Malaria: The big push – Zambia, with help from partners around the world, is stepping up its battle against malaria.
• The billion-dollar malaria moment – For years the global malaria effort has been asking for more resources. Now the field needs to figure out a systematic strategy for spending the money effectively.
• Review: Malaria research in the post-genomic era

Articles:

• Comparative genomics of the neglected human malaria parasite Plasmodium vivax
• Genome sequence of the human malaria parasite Plasmodium falciparum
• Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii
• The genome of the simian and human malaria parasite Plasmodium knowlesi

Related:

• MicrobiologyBytes: Malaria

Chagas disease is considered the largest parasitic disease burden in Latin America with a cost of the loss of 667,000 Disability Adjusted Life Years in 2002. Trypanosoma cruzi, the parasite that causes Chagas disease, infects approximately 9.8 million people in the Americas with 200,000 new Chagas cases annually. Most transmission occurs by contamination with the parasite-containing faeces of triatomine insect vectors (“kissing bugs”). There is no vaccine available and treatment shows limited effectiveness, comes with troublesome side effects, and is out of reach of most people in endemic countries. Therefore, as with most parasitic infections, control of transmission by the vectors is the control strategy of choice.

Pesticide spraying has effectively halted transmission in most of southern South America, especially where the bugs live exclusively inside houses. In Mesoamerica, bugs living in the forest readily reinfest treated houses. In addition, one of the main species of insect that transmits Chagas in Mesoamerica, Triatoma dimidiata, although it looks similar in different localities, may consist of genetically distinct populations, even different species, which differ in how efficiently they transmit the parasite: characteristics which confound control efforts. Nuclear and mitochondrial DNA were analyzed to characterize different populations of T. dimidiata from Mexico and Central America. Both the nuclear and mitochondrial DNA show that there is a very distinct population of T. dimidiata, perhaps even a different species, that lives in very close proximity with other T. dimidiata in Mexico and Guatemala. The nuclear DNA divides the remaining T. dimidiata into three additional genetically distinct groups. However, the mitochondrial DNA does not distinguish these additional groups. This study helps inform control efforts by showing where genetically distinct populations of T. dimidiata occur.

Since 1997, the Central America Initiative for the Control of Chagas disease has shown dramatically different results following insecticide spraying in houses, e.g. in Nicaragua, the bugs did not return; in stark contrast to rapid reinfestation in Jutiapa, Guatemala. It is important to understand how much of the differences in epidemiology and control outcomes are due to distinct taxa of T. dimidiata. The area of Peten, Guatemala has not been included in the control program since most are forest populations. Deforestation and increasing encroachment of human populations in the area means that T. dimidiata could become domesticated in this region. It is critical to realize that there are at least two distinct T. dimidiata populations in this area (and in Mexico and Belize) as control measures are designed. For effective control it will be imperative to understand the mechanisms maintaining this reproductive isolation and the epidemiological importance of distinct taxa.

Two Distinct Triatoma dimidiata (Latreille, 1811) Taxa Are Found in Sympatry in Guatemala and Mexico. PLoS Negl Trop Dis 3(3): e393
Approximately 10 million people are infected with Trypanosoma cruzi, the causative agent of Chagas disease, which remains the most serious parasitic disease in the Americas. Most people are infected via triatomine vectors. Transmission has been largely halted in South America in areas with predominantly domestic vectors. However, one of the main Chagas vectors in Mesoamerica, Triatoma dimidiata, poses special challenges to control due to its diversity across its large geographic range (from Mexico into northern South America), and peridomestic and sylvatic populations that repopulate houses following pesticide treatment. Recent evidence suggests T. dimidiata may be a complex of species, perhaps including cryptic species; taxonomic ambiguity which confounds control. The nuclear sequence of the internal transcribed spacer 2 (ITS2) of the ribosomal DNA and the mitochondrial cytochrome b (mt cyt b) gene were used to analyze the taxonomy of T. dimidiata from southern Mexico throughout Central America. ITS2 sequence divides T. dimidiata into four taxa. The first three are found mostly localized to specific geographic regions with some overlap: (1) southern Mexico and Guatemala (Group 2); (2) Guatemala, Honduras, El Salvador, Nicaragua, and Costa Rica (Group 1A); (3) and Panama (Group 1B). We extend ITS2 Group 1A south into Costa Rica, Group 2 into southern Guatemala and show the first information on isolates in Belize, identifying Groups 2 and 3 in that country. The fourth group (Group 3), a potential cryptic species, is dispersed across parts of Mexico, Guatemala, and Belize. We show it exists in sympatry with other groups in Peten, Guatemala, and Yucatan, Mexico. Mitochondrial cyt b data supports this putative cryptic species in sympatry with others. However, unlike the clear distinction of the remaining groups by ITS2, the remaining groups are not separated by mt cyt b. This work contributes to an understanding of the taxonomy and population subdivision of T. dimidiata, essential for designing effective control strategies.

Related:

• Neglected Tropical Diseases in Latin America and the Caribbean

Unlike the marine environment, bioluminescence in terrestrial bacteria is not yet understood. In this article in Microbiology Today, Susan Joyce and David Clarke describe the complex association between Photorhabdus, its nematode host and their insect prey:

Next time that you are on the beach, walk into the dunes and take a sample of the sandy soil within the area where the dune grass is growing. Place your soil sample in a flask, add a few insect larvae (readily available from your local bait or pet shop) and the chances are good that the insects will be dead within 2–3 days. Take these dead insects into the darkest room in your house and within 5–10 minutes you should see that some, if not all, of the insect cadavers will glow in the dark. This bioluminescence is due to the presence of the bacterium Photorhabdus, a highly virulent insect pathogen (entomopathogen) that you have isolated in a nematode vector from the soil. Together, Photorhabdus and the nematode vector (Heterorhabditis) form a deadly complex that is naturally lethal to insect larvae.

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Microsporidia are single-celled parasites that are capable of causing infections in humans – primarily in people with compromised immune systems, such as those infected with HIV or who have undergone organ transplants. A new article documents a recently discovered species of microsporidian which infects Caenorhabditis elegans, the harmless worm used as a model system by developmental biologists. The work is a breakthrough for public health researchers who had been looking for a suitable laboratory model in which to study microsporidia. Microsporidia are classified as priority pathogens by the U.S. National Institutes of Health, since they have been found in water supplies and because no drugs are available for treating the most common infections they cause in humans. The new species infects C. elegans, a small roundworm that is very easy to study in the lab, and so provides a powerful system in which to study these mysterious microbes, learn how animals respond to infection and develop new drugs to fight infections of microsporidia.

The discovery is particularly exciting because it potentially offers a unique opportunity to identify new drugs for treating human microsporidian infections. The researchers named the new species of microsporidian Nematocida parisii, or “nematode killer from Paris”, because it was discovered in the intestines of roundworms found in Parisian compost pits. Since then, scientists have found closely related naturally-occurring pathogens in Portugal and India, leading them to conclude that they are widespread natural parasites of C. elegans, which has intestinal cells that look almost exactly like human intestinal cells, so researchers are able to tell what the parasites are doing to intestinal cells as they invading and exit the cells. Because the worms are completely transparent, they can take intact worms, put them on slides and see what’s happening. They have already seen some interesting changes in the structure of intestinal cells during different time periods of infection.

Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans. 2008 PLoS Biol 6(12): e309
For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wildcaught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.

Cholera is an infectious form of gastroenteritis caused by the enterotoxin-producing strains of the curved Gram-negative bacillus Vibrio cholerae. Transmission to humans occurs through ingesting food or water that is contaminated with faecal matter from infected people. Classically the disease occurs where there is a lack or failure of sanitation, e.g. in crowded urban conditions in developing countries or following natural disasters. In its most severe forms, cholera is one of the most rapidly fatal illnesses known – in some circumstances infected patients may die within hours if medical treatment is not provided. Usually the disease progresses from the first liquid stool to shock (due to dehydration) in 4 to 12 hours, with death following in 18 hours to several days, unless oral rehydration therapy is provided.

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How does a tiny bacterium cause such rapid deaths? Cholera is a toxin-mediated disease. Vibrio cholerae produces cholera toxin, an enterotoxin which acts on the mucosal epithelium lining the small intestine, causing cell death and massive loss of body fluids into the gut, resulting in the the characteristic massive diarrhoea associated with the disease. Death is caused by hypovolemic shock due to the loss of body fluids, so first aid for cholera involves oral rehydration with isotonic liquids to combat these symptoms. Antibiotics are then given to speed up resolution of the infection.

Cholera seems to have originated in the Indian subcontinent and the disease spread by trade routes to Russia, then to Western Europe, and from Europe to North America during the nineteenth century. The history of cholera has been marked by a series of pandemics:

• 1816-1826 – First cholera pandemic
• 1829-1851 – Second cholera pandemic
• 1852-1860 – Third cholera pandemic. During this pandemic in 1854 John Snow identified contaminated water as the source of the disease by removal of the handle of the Broad Street pump in London.
• 1863-1875 – Fourth cholera pandemic
• 1881-1896 – Fifth cholera pandemic
• 1899-1923 – Sixth cholera pandemic
• 1961-1970s – Seventh cholera pandemic

Currently the disease is following a more endemic pattern, cropping up in poor countries and after natural disasters.

Vaccines against cholera are available but are not currently recommended for routine use. New oral vaccines against cholera are being developed, including a live-attenuated vaccine containing genetically manipulated V. cholerae, and an alternative vaccine containing killed whole-cell V. cholerae in combination with purified recombinant B subunit of the cholera toxin. Although the ultimate answer to cholera lies in public health and sanitation, at present the only feasible answer to cholera in poor countries is in vaccine development.

Cholera: Latest News

Related:

• Cracking the cholera code
• Bacterial toxins

One of the main problems in fighting malaria is the speed with which Plasmodium falciparum, the causative agent of human malaria, is able to vary its genetic makeup. This allows antigenic variation, which makes the creation of effective vaccines very difficult. Antigenic variability also gives P. falciparum the ability to persist in the face of an immune reaction and to reinfect people who have been previously exposed to the disease. Effective immunity to malaria requires repeated infections and is slow to develop, so children under ten years of age are most susceptible to illness. The entry of malaria parasites into red blood cells during the replication cycle creates two opportunities to evade host immunity. First, infected red blood cells do not induce a CTL response due to their lack of MHC I expression. Second, malaria antigens exposed on the surface of the cell are highly variable. The P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a key virulence factor which is expressed on the surface of infected erythrocytes and causes the blood cells to stick to the walls of small blood vessels, preventing infected cells from going through the general circulation and to the spleen (see: Giving malaria the slip).

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Red blood cells infected with Plasmodium display immunodominant parasite antigens on their surface. The reason for this is not clear, but it may be to modify the physical properties of the host cells so that they are not trapped and destroyed in the spleen. The expression of the immunodominant surface protein PfEMP1 is also linked to suppression of host interferon-gamma in the early immune response to the parasite, and low interferon-gamma levels may improve parasite survival.

PfEMP1 is in fact a family of cell surface molecules, encoded by approximately 60 var genes. Antigenic variation is controlled by epigenetic factors including monoallelic var transcription in separate domains at the nuclear periphery, differential histones on otherwise identical var genes, and var gene silencing mediated by telomeres (Antigenic variation in Plasmodium falciparum. Annu Rev Microbiol. 2008 62: 445-470).

Targeting the mechanisms responsible for antigen switching could be a promising approach to tackle the malaria parasite without having to deal with phenotypic variation of the surface molecule. The development of specific biological assays that target antigenic variation could uncover crucial mechanisms required for export to the cell surface, repression of the var gene family, or switching to new variants and would allow the screening of drugs which block these essential processes. Plasmodium’s trump card could yet prove to be its undoing.

Related:

• Malaria, mosquitoes and the legacy of Ronald Ross
• Malaria: now you see me, now you don’t
• Researchers characterize potential protein targets for malaria vaccine
• Blood group O protects against severe malaria
• Tough Choices – DDT or Malaria?

Wolbachia are members of the Rickettsia, a diverse group of intracellular bacteria that comprises bacteria with parasitic, mutualistic and commensal relationships with their hosts. Typical Rickettsia have life cycles that include an invertebrate vector and mammalian host. However, unlike its close relatives, Wolbachia does not routinely infect vertebrates. Wolbachia species have attracted considerable interest in the past decade primarily because of their abundance, fascinating effects on their hosts and their potential in pest and disease vector control (Wolbachia: master manipulators of invertebrate biology. 2008 Nature Reviews Microbiology 6, 741-751).

Wolbachia have small genomes (1-2 Mb) that are within the size range of the other Rickettsia. Until the early 1990s, Wolbachia were considered to be rare, but with the advent of PCR, Wolbachia were found to be widespread and are in fact common in insects and other arthropods, as well as in nematodes. Recent work estimates that over 65% of insect species harbour Wolbachia, making it among the most abundant intracellular bacterial genus so far discovered, infecting over a million insect species alone.

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Together with their widespread distribution, another interesting feature of Wolbachia is the various host manipulations they induce. The effects of Wolbachia infection include: feminization of genetic males; parthenogenetic induction, resulting in the development of unfertilized eggs; the killing of male progeny from infected females; and sperm–egg incompatibility. Each of these reproductive alterations helps the bacterium by enhancing the production of infected female hosts, and this is referred to as reproductive parasitism. Whether Wolbachia have a role in accelerating the evolution of their hosts is a controversial question, but there is good evidence that parthenogenesis-inducing bacteria have led to the evolution of parthenogenetic insect species.

Considerable progress in understanding the biology of Wolbachia has been made in the past ten years. However, important questions still remain, including: how do Wolbachia manipulate host reproduction; how is the abundance and distribution of Wolbachia maintained globally; can Wolbachia be effectively used in disease control; do Wolbachia have important roles in the evolution of their hosts; and do Wolbachia accelerate the rates of speciation in invertebrates and contribute to novel gene acquisition.

Related:

• Parasite genomes
• Malaria Researchers Identify New Mosquito Virus
• Jumping Genes, Wolbachia Style
• PubMed: Wolbachia

Every day 2000 children die from malaria in Africa alone. The infection is transmitted from human to human by biting mosquitoes. Despite many years of effort, a vaccine is still not available to fight the deadly disease. Once injected by a mosquito, parasites migrate to the liver where they mature and then their sporozoites (infective cells) are released into the blood, causing disease and fatal complications. Human malaria is caused by Plasmodium falciparum, a unicellular protozoan parasite that is transmitted by Anopheles mosquitoes. An infectious mosquito injects saliva containing sporozoite forms of the parasite and these then migrate from the skin to the liver, where they establish an infection. Many intervention strategies are currently focused on preventing the establishment of infection by sporozoites. Clearly, an understanding of the biology of the sporozoite is essential for developing new intervention strategies. Sporozoites are produced within the oocyst, located on the outside wall of the mosquito midgut, and migrate after release from the oocysts to the salivary glands where they are stored as mature infectious forms. Comparison of the proteomes of sporozoites derived from either the oocyst or from the salivary gland reveals remarkable differences in the protein content of these stages despite their similar morphology. The changes in protein content reflect the very specific preparations the sporozoites make in order to establish an infection of the liver. Analysis of the function of several previously uncharacterized, conserved proteins revealed proteins essential for sporozoite development at distinct points of their maturation.

Researchers characterized a large number of parasite proteins that may prove useful in the development of a human malaria vaccine. A promising method for vaccination is to sufficiently weaken these parasites such that they invade liver cells and stimulate an immune response, but don’t develop further. This can be achieved by genetically inactivating individual parasite genes that are active during the parasite’s growth in the liver. The researchers achieved this by modifying the proteins essential for sporozoite development, which their study identified. Collaborators had previously shown how to successfully vaccinate mice using a rodent malaria which had one of these liver stage genes removed, specifically p36p.

A related article shows the first transition of such a vaccination from the rodent system to humans, by inactivating the equivalent gene (p52) in the major human malaria parasite, P. falciparum. Similar to the results with the rodent parasite, these human parasites are unable to develop in liver cells. This is the first time that genetic modification of a human parasite results in its growth arrest in a liver cell, opening up promising possibilities for its use as a human vaccine. These studies show how results obtained in rodent models of malaria can be pipelined to form the basis for clinical development of anti-malaria vaccines in humans.

Proteomic Profiling of Plasmodium Sporozoite Maturation Identifies New Proteins Essential for Parasite Development and Infectivity. PLoS Pathog 4(10): e1000195
Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito – early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans.

Related:

• Gametogenesis in malaria parasites
• Malaria, mosquitoes and the legacy of Ronald Ross
• New target for anti-malaria drugs
• Blood group O protects against severe malaria

Researchers have reported positive results from a safety and efficacy study pertaining to tribendimidine, a broad-based treatment for intestinal worm infections. The group’s results demonstrate the success of a new drug from China versus that of the standard albendazole for the treatment of hookworm, large roundworm, whipworm, and, for the first time, threadworm and tapeworm. Globally, more than one billion people are infected with intestinal worms. These chronic infections negatively impact on child and maternal health, nutritional status, physical performance, and cognitive development. The current control strategy relies on drugs to reduce morbidity, ideally complemented by the provision of safe water and sanitation to curb transmission. Only four drugs are currently recommended by the World Health Organization for treating soil-transmitted helminth infections, making the potential development of drug resistance a concern. Tribendimidine belongs to a different chemical class than current worm treatments. The new trial involved 123 individuals who were screened for intestinal helminth infections, and randomly allocated to tribendimidine or the widely used albendazole treatment. The researchers’ administration of a single oral dose of tribendimidine cured up to 92% of the common soil-transmitted helminth infections in humans in a highly endemic setting in China. Encouraging results were also found against threadworm and tapeworm infections. After treatment, these two parasites were absent in 55% and 67% of those initially infected, respectively. The infection intensity of large roundworms and hookworms was significantly reduced by both drugs, and no adverse treatment-related events were noted among the final study cohort. The results obtained need to be validated in larger patient cohorts and different epidemiological settings, and repeated dosing should be tested to further improve treatment outcomes.

Tribendimidine and Albendazole for Treating Soil-Transmitted Helminths, Strongyloides stercoralis and Taenia spp: Open-Label Randomized Trial. 2008 PLoS Negl Trop Dis 2(10): e322
Tribendimidine is an anthelminthic drug with a broad spectrum of activity. In 2004 the drug was approved by Chinese authorities for human use. The efficacy of tribendimidine against soil-transmitted helminths (Ascaris lumbricoides, hookworm, and Trichuris trichiura) has been established, and new laboratory investigations point to activity against cestodes and Strongyloides ratti. In an open-label randomized trial, the safety and efficacy of a single oral dose of albendazole or tribendimidine against soil-transmitted helminths, Strongyloides stercoralis, and Taenia spp. were assessed in a village in Yunnan province, People’s Republic of China. The analysis was on a per-protocol basis and the trial is registered with controlled-trials.com (number ISRCTN01779485). Both albendazole and tribendimidine were highly efficacious against A. lumbricoides and, moderately, against hookworm. The efficacy against T. trichiura was low. Among 57 individuals who received tribendimidine, the prevalence of S. stercoralis was reduced from 19.3% to 8.8% and that of Taenia spp. from 26.3% to 8.8%. Similar prevalence reductions were noted among the 66 albendazole recipients. Taking into account ‘‘new’’ infections discovered at treatment evaluation, which were most likely missed pre-treatment due to the lack of sensitivity of available diagnostic approaches, the difference between the drug-specific net Taenia spp. cure rates was highly significant in favor of tribendimidine. No significant adverse events of either drug were observed. Our results suggest that single-dose oral tribendimidine can be employed in settings with extensive intestinal polyparasitism, and its efficacy against A. lumbricoides and hookworm was confirmed. The promising results obtained with tribendimidine against S. stercoralis and Taenia spp. warrant further investigations. In a next step, multiple-dose schedules should be evaluated.

Related:

• Multiple parasite infections synergize to increase the risk of anemia
• New Drugs for Nasty Nematodes