TUBERCULOSIS: secrets of a polymer-building enzyme

Laura Cassiday : July 15, 2011
Solving The Mystery Of Sugar Chain Growth : Assay based on mass spectrometry reveals mechanistic secrets of a polymer-building enzyme

 CDC/ Dr. Ray Butler
IMPENETRABLE WALL Carbohydrate polymers strengthen the cell wall of Mycobacterium tuberculosis to protect the pathogen.Email this article to a friend

Solving The Mystery Of Sugar Chain Growth Assay based on mass spectrometry reveals mechanistic secrets of a polymer-building enzyme.

Mycobacterium tuberculosis, the microbe responsible for tuberculosis, uses an unusually strong cell wall, fortified with a carbohydrate called galactan, to protect itself from harsh environments and its host's immune system. Sugar polymers like galactan are built by enzymes called glycosyltransferases, which add successive sugar molecules to a chain, like beads on a string. Now researchers have developed an assay based on mass spectrometry to answer a long-standing question about the mechanism of these enzymes (J. Am. Chem. Soc., DOI: 10.1021/ja204448t).

Understanding how M. tuberculosis builds galactan could allow researchers to design drugs that interfere with the process, allowing infected people to mount an effective immune response, says Laura Kiessling, a chemical biologist at the University of Wisconsin, Madison.
Kiessling and her colleagues wanted to know whether GlfT2, one of the glycosyltransferases that synthesize galactan, uses what biochemists call a processive or distributive mechanism. Processive enzymes remain bound to the carbohydrate chain as they add monomers, while distributive enzymes fall off the sugar chain after each addition and then bind another chain. Drug designers would like to exploit such a mechanistic understanding in developing enzyme inhibitors, Kiessling says.
To distinguish between the two mechanisms, the researchers designed an assay that detects how often GlfT2 swaps polymer partners during elongation of the sugar chain. Initially, the team exposed GlfT2 to a lipid-linked oligosaccharide that mimics the base of the growing galactan chain. After allowing the enzyme to bind this molecule and start adding sugar monomers, the scientists introduced a deuterium-labeled version of the lipid-linked molecule. After the enzyme continued working to add monomers, the researchers analyzed the reaction products with matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry to measure the length of the sugar chains and to determine whether or not they were labeled with deuterium.
Kiessling expected that if the enzyme was processive, the longest carbohydrate chains would be unlabeled, because the enzyme would tightly latch onto the initial, unlabeled base molecule and wouldn't get distracted when the labeled one appeared. In contrast, a distributive mechanism would produce labeled and unlabeled carbohydrate chains of roughly equal lengths because the two lipid-linked molecules would have equal access to the enzyme each time it fell off a chain.
When Kiessling and her colleagues examined the mass spectrometry data, they found that none of the longest chains were labeled, suggesting to them that GlfT2 used a processive mechanism. Moreover, the researchers quantified the degree of processivity, or the probability that an enzyme bound to the chain would add another sugar before it fell off the chain. They determined that GlfT2's processivity increased as the sugar chain grew, suggesting that the enzyme, like some other polymer-building enzymes, binds more tightly to longer chains.
"This elegant, straightforward approach will be widely used by researchers studying carbohydrate polymerization, and could be applied to other polymers as well," says Todd Lowary, a chemist at the University of Alberta. The knowledge that GlfT2 shows greater affinity for longer sugar chains could be used to design inhibitors for use as antibiotics, he adds, although a crystal structure of the enzyme would better aid drug design.

http://pubs.acs.org/cen/news/89/i29/8929scene3.html

MALARIA: Rain forest reduction enhances spread of malaria

A small reduction in tropical rainforest cover can increase malaria incidence by nearly 50 per cent, a study in the Brazilian Amazon has found.
Open spaces and partially sunlit pools of water, typical conditions of deforested landscapes, provide an ideal habitat in which the Anopheles darlingi mosquito— the main vector of the malaria parasite in the Amazon — can live and lay its eggs, according to the study, published online early in Emerging Infectious Diseases.
The authors, from the University of Wisconsin, Madison in the United States, and Santo Antonio Energia in Brazil, an energy consortium, studied high-resolution satellite data of land cover from 54 Brazilian health districts bordering Peru from 1996 to 2006. They also examined health data collected in the same areas in 2006.
They found that a four per cent change in forest cover was associated with a 48 per cent increase in malaria incidence.
Areas with less deforestation had a lower malaria risk, suggesting a link between conservation practices and health.
"We believe that the small change in deforestation is greatly amplifying the number of mosquito vectors, and thereby increasing malaria risk in humans," said Sarah Olson, lead author of the study.
"Local land management and development policies should weigh this human health risk along with the economic benefits of deforestation."
Kevin Lafferty, an ecologist at the University of California Santa Barbara, who questioned the links between climate change and malaria last year, said:
"Certainly, deforestation can create conditions that favour mosquitoes. Also, human populations tend to move into areas that are recently deforested. If these are migrants that bring malaria with them, they can set off epidemics.
"But it is important to formally quantify the link between deforestation and malaria, whatever the causal chain.
However, he added that "economics trumps climate change [and deforestation] when it comes to determining the future of malaria.
"[Economics] is an overlooked aspect of malaria given the current emphasis on climate change, but there is good evidence that endemic malaria is much more likely in poor countries and that malaria makes poor countries even poorer. It is a vicious cycle."
http://www.scidev.net/en/news/small-change-in-forest-cover-can-double-malaria-rate.html

MALARIA: Amazon tree cutting increases malaria incidence

MADISON — Establishing a firm link between environmental change and human disease has always been an iffy proposition.
Now, however, a team of scientists from the University of Wisconsin-Madison, writing in the current (June 16, 2010) online issue of the CDC journal Emerging Infectious Diseases, presents the most enumerated case to date linking increased incidence of malaria to land-use practices in the Amazon.
The report, which combines detailed information on the incidence of malaria in 54 Brazilian health districts and high-resolution satellite imagery of the extent of logging in the Amazon forest, shows that clearing tropical forest landscapes boosts the incidence of malaria by nearly 50 percent.
"It appears that deforestation is one of the initial ecological factors that can trigger a malaria epidemic," says Sarah Olson, the lead author of the new report and a postdoctoral fellow at the Nelson Institute, Center for Sustainability and the Global Environment.
The clearing of tropical forests, say Olson and senior author Jonathan Patz of the University of Wisconsin School of Medicine and Public Health, creates conditions that favor malaria's primary carrier in the Amazon, the mosquito Anopheles darlingi, which transmits the malaria parasite if it draws its blood meals from infected humans.
"The deforested landscape, with more open spaces and partially sunlit pools of water, appears to provide ideal habitat for this mosquito," Olson says, noting that Anopheles darlingi has been shown to displace other types of mosquitoes that prefer forest and that are far less prone to transmit malaria.
"This study of human malaria cases complements our previous work that focused more on the abundance of the malaria-carrying mosquito," Patz adds. "In those studies from the Peruvian Amazon, we showed a correlation between this mosquito's larvae and aquatic breeding sites in disturbed habitats following land clearance."
The new Wisconsin study focused on 54 Brazilian health districts in a corner of the Brazilian Amazon near Peru and where detailed health and population data were collected in 2006 by Brazilian researchers. Combined with high-resolution satellite data of changes in land cover, the health data reveals the large human-health impact of relatively small changes to the forest landscape.
"A 4 percent change in forest cover was associated with a 48 percent increase in malaria incidence in these 54 health districts," notes Olson. "The health data used in the study is of the highest quality and spatial resolution. Unlike previous studies, our data allowed us to zoom in on areas where people are being exposed to malaria and to exclude areas where they are not being exposed."
The health districts reflected in the Wisconsin study are typical of many of the thousands of such districts spread across Brazil and its Amazon region. Since 2001, the Brazilian Ministry of Health has similarly monitored and treated malaria in more than 7,000 districts. Deforestation in the districts, says Olson, is occurring as it typically does in Amazonia, and occurs mostly near rivers, the backbone of the region's transportation system, and spreads outward.
The new work, argues Patz, an authority on environmental change and human health, shows how deforestation and land clearing contribute to malaria's dynamic at the frontier of settlement. "In 2006, the county that encompasses these health districts was in the top five of all Brazilian counties with malaria," Patz says. "Even after we adjusted for human populations, access to healthcare and other factors, malaria hotspots paralleled locations with the most destruction of rainforests."
The message from the study, say Olson and Patz, is that tropical forest conservation may benefit human health more than we realized.
"Land-management practices show promise as useful interventions to reduce malaria risk factors," says Olson.
Patz and Olson believe the new work provides a template that could be used to spatially track environmental risk factors and the incidence of malaria, which infected an estimated 500,000 Brazilians annually across the Amazon basin from 1997 to 2006. "The technology is there. The health data is there. Health officers with cell phones could gather data for the whole Amazon region," Olson says.
http://www.sciencecodex.com/incidence_of_malaria_jumps_when_amazon_forests_are_cut