A closer look at a deadly bacterium sets the stage for new vaccines

May 21, 2015

Tularemia, or rabbit fever, is a severely debilitating and sometimes fatal disease, and the pathogen involved has potential as a biological weapon. To better understand the disease – and to better design potential vaccines – an ASU team looked at a key cellular protein in unprecedented detail.

The bacterium Francisella tularensis is responsible for tularemia, which can spread from rabbits to people. In addition to being endemic in North America, Europe and Asia, the highly infectious pathogen is easily aerosolized, leading to its designation as a Class A bioterrorism agent. This figure shows how scientists progress from tackling a disease to vaccine Download Full Image

In new research, Petra Fromme of Arizona State University’s Biodesign Institute and her ASU colleagues examine a key component responsible for the tularemia’s profoundly infectious character, using nuclear magnetic resonance spectroscopy (NMR) to explore features of the bacteria’s surface protein in unprecedented detail.

The study marks the first structural determination at atomic scale for any lipoprotein of the genus Francisella, and it holds the promise of improved therapies and potential vaccines.

According to James Zook of ASU’s Department of Chemistry and Biochemistry and lead author of the new study:

“Observing this virulence determinant at an atomic scale has shown us that this protein is a possible drug target. We now have an opportunity to develope a new, specialized pharmaceutical that specifically affects Francisella infections. This pathogen is a potential bioweapon; we want as many countermeasures as we can get.”

The group’s research findings appear in today’s issue of the journal Structure.

A natural (and man-made) threat

Tularemia is a spectrum of illnesses caused by F. tularensis, a bacterium existing in different subtypes bearing varying degrees of infectiousness. Rabbits, hares and pikas carry the most common strain of the bacteria, known as type A. The resulting disease is often referred to as rabbit fever.

The number of cases of tularemia in endemic regions is generally fairly low, with prevalence linked to areas of poor sanitation, lacking modern health care. Conflict zones are also vulnerable areas, with one of the largest modern outbreaks occurring in postwar Kosovo between October 1999 and May 2000, with 327 confirmed cases.

Vectors for the disease-causing bacterium include ticks, deer flies and other arthropods. After initial infection, tularemia undergoes an incubation period of one to 14 days, with most infections appearing after three to five days.

Symptoms of tularemia include loss of appetite, fever, lethargy and signs of sepsis – in severe cases leading to death. Additional symptoms include reddening and inflammation of the eyes and lymph node inflammation, which may be accompanied by suppuration (pus) and high fever, mimicking symptoms of plague.

The bacterium can enter the body through damaged skin and mucous membranes or through bacterial inhalation. Because F. tularensis in an intracellular pathogen, it can persist as a parasite within the cells of its host.

Typically, the bacterium infects white blood cells known as macrophages, using these as a refuge to evade recognition by the immune system. During the course of disease, the pathogen can spread to multiple organ systems, including the lungs, liver, spleen and lymphatic system. 

F. tularensis has also been identified as a potential biological warfare agent by the Centers for Disease Control and Prevention. It has existed as a component in biowarfare projects in the United States, Russia and Japan at various times.

Several factors contribute to the bacterium’s attractiveness as a biological weapon, including the ease with which it can be aerosolized, its extremely infectious nature and the high degree of incapacitation produced in infected persons.

Vaccines needed

Treatment for tularemia is intensive and often unavailable in endemic regions, making vaccines the most attractive means of addressing the disease. Presently, there is a live attenuated vaccine available, though several factors limit its applicability. If improperly administered, the vaccine can induce infection when insufficient immunity is acquired following injection. This insufficiency leaves some 30-40 percent of a vaccinated population subject to infection.

For these reasons, the live vaccine strain, or LVS, vaccine for tularemia is not approved for use in the U.S. The authors of the current study therefore stress that an improved vaccine alternative is vitally needed.

The F. tularensis bacterium has a profound capacity for infection. Fewer than 10 cells are required and in some cases, a single bacillus is sufficient to cause disease. What accounts for this high propensity to cause disease or virulence? One answer, and the focus of the current study, is known as Flpp3, an outer membrane protein linked with aggressive infectiousness and implicated as a possible target of future vaccine efforts against tularemia.

Flpp3 is structurally related to a family of proteins known as Bet v1, which are responsible for many allergic reactions, including peanut and birch allergies. The fact that allergies caused by this protein family induce strong immune responses, suggests the related Flpp3 protein may be a promising target for vaccine and drug delivery efforts.

In earlier studies, a significant decrease in tularemia virulence was observed when a mutation was introduced into the DNA region coding for Flpp3, rendering the protein inoperative.

Probing with resonance

In the current study, nuclear magnetic resonance spectroscopy (NMR) was used to more closely home in on the membrane Flpp3. The protein examined with NMR was derived from a particularly virulent strain of tularensis, known as Type A SCHU S4.

The study marks the first atomic-scale structural investigation of this critical membrane protein. The precise function of Flpp3 remains obscure, and the authors suggest detailed structural analysis is the first step to unlocking this mystery.

Nuclear magnetic resonance for protein research (sometimes called protein NMR) is a powerful technique for revealing protein structure. It is similar in basic outline to magnetic resonance imaging (MRI) commonly used to examine human tissue in hospital settings, though protein NMR provides much higher sensitivity in order to capture structures more than a million times smaller than those studied by MRI radiologists. Here, at the scale of nanometers, molecular structure can be observed and characterized.

Protein NMR involves placing the sample inside a powerful magnet, passing radio-frequency signals through the sample, and measuring the level of absorption of these signals. Nuclei in individual atoms will absorb different radio frequencies, allowing researchers to determine distances between nuclei and compose detailed models of overall protein structure.

The study successfully determined the structure for Flpp3. The protein is largely spherical, containing a six-strand beta-sheet, two alpha-helices and three flexible loop regions. The NMR analysis revealed an internal binding pocket in the protein – a plausible target for vaccine and drug development.

In addition to uncovering structural details, the NMR study also provided new insights into the atomic-level dynamics of the protein, at rapid time scales. The authors suggest the loop structures of the protein may act like a gateway, blocking or enabling access to the protein’s internal binding cavity, depending on the variable conformation of these loops.

Ongoing research will further explore protein functionality, with particular focus on experimental and computational studies of drug activity.

Joe Caspermeyer

Manager (natural sciences), Media Relations & Strategic Communications


Experts discuss need for cross-border collaborations in higher education, research

May 21, 2015

University presidents and faculty, international leaders and experts in the field convened at Arizona State University to discuss the role that higher education and research institutions play in advancing quality of life, education, sustainability and economic development in the Arizona-Sonora region.

The Arizona-Sonora Colloquium at ASU's McCord Hall on May 20-21 kicked off with a keynote address by ASU President Michael M. Crow, who noted that the university’s brand-new charter establishes the importance of the institution’s local impact and social embeddedness. Arizona-Sonora Colloquium ASU Download Full Image

“ASU is an institution built around inclusion versus exclusion and the success of the students that we include. And what that means with the level of socioeconomic diversity that we have in this part of the U.S. and the level of ethnic diversity we have in this part of the U.S. is that the institution is not successful unless it is in its entirety representative of the entire socioeconomic diversity of this region,” Crow said.

One way of ensuring that is through the development of collaborations among the many universities and centers for research and innovation within the Arizona-Sonora region.

Attendee and panel speaker Pablo Wong Gonzalez, general director of public research institution Centro de Investigacion en Alimentacion y Desarrollo, noted the importance of maintaining awareness of existing collaborations while creating interest in developing new ones.

“We need to complement each other, to help each other. I think we need to have a global vision with a local focus,” he said.

Speakers at the colloquium’s closing roundtable discussion pointed out how such issues as water scarcity and immigration affect the entire region, regardless of where the “real border” between the U.S. and Mexico is, which only highlights the importance of working together.

“This all has to do with the well-being of the region overall,” said Heriberto Grijalva Monteverde, rector of Universidad de Sonora.

Jim O’Brien, senior vice president of University Affairs and chief of staff to President Crow at ASU, laid out some simple steps to achieve that overall well-being: identify project areas, create an agenda of issues important to the region, and hold each other accountable for working toward those goals together.

“We can do more when we work with others than when we try do it alone,” O’Brien said. “It’s too big a task to take on water issues alone, to take on education alone, to take on social issues of the region alone. We need partners, and we’re committed to taking advantage of this opportunity to build new partnerships.”

After the closing roundtable discussion, Francisco Lara, associate professor in ASU’s School of Transborder Studies, fielded questions from attendees on topics such as health and involving the private sector in collaborations.

“We have to try new things,” Lara told the crowd. “We should not be afraid of failure. We have to work together. We have to think of the region.”

The Arizona-Sonora colloquium is coordinated by the ASU Program for Transborder Communities (PTC) of the School of Transborder Studies and the College of Liberal Arts and Sciences, in collaboration with the Universidad de Sonora (UNISON), the Centro de Investigación en Alimentación y Desarrollo (CIAD) and El Colegio de Sonora (COLSON). The colloquium is also supported by ASU’s Mexico and Latin America Initiatives at the Office of University Affairs.

Emma Greguska

Editor, ASU News

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