New research highlights survival skills of disease-causing E. coli

January 30, 2012

Escherichia coli bacteria thrive in the lower intestine of humans and other animals, including birds. Most are vital constituents of the healthy gut flora, but certain forms of E. coli cause a range of diseases in both humans and poultry. 

In this month’s issue of the journal PLoS ONE, a team of researchers at ASU’s Biodesign Institute, investigates disease-causing E. coli strains known as APEC (for Avian Pathogenic E. coli). By studying circular segments of bacterial DNA known as plasmids, the group uncovered some of the tricks used by these highly adaptive organisms to survive, even in the face of daunting environmental challenges.   Download Full Image

According to Melha Mellata, assistant research professor and lead author of the current study, the research is an important step toward a more thorough understanding of the genetic underpinnings of pathogenic E. coli: “E. coli bacteria that are able to persist and cause diseases have developed multiple strategies to achieve this,” she says. “It is important to elucidate the genetic mechanisms used by these bacteria so that we can turn their own weapons against them.”

Birds, including chickens can become infected with APEC, causing colibacillosis – an acute and often fatal disease, resulting in significant economic loss for the poultry industry. Further, because APEC bacteria bear a genetic blueprint similar to that of other members of the group of Extra-intestinal pathogenic E. coli or ExPEC, to which they belong, the danger exists for such avian bacterial strains to cross the genetic barrier to infect humans, causing so-called zoonotic diseases. 

Retail chicken products are also believed to act as reservoirs for existing E. coli strains responsible for human ExPEC infections. As their name implies, these extra-intestinal bacterial pathogens cause infections outside their customary habitat in the gut. They are responsible for illnesses including septicemia, newborn meningitis and urinary tract infections. ExPEC infections result in significant loss of life and cost the U.S. health care industry billions of dollars. 

While the genetic kinship of human and avian pathogenic E. coli strains is cause for concern, it may also provide an opportunity for the development of a vaccine capable of cross-protecting humans and birds, if a group of genes common to all extra-intestinal E. coli can be identified and targeted. Roy Curtiss, director of Biodesign’s Center for Infectious Diseases and Vaccinology, oversees a project aimed at achieving this goal. 

In the current study, the team – including undergraduate researchers, Jacob Maddux and Timothy Nam – investigated the genetic sequence of several large plasmids in a strain of APEC commonly used for research purposes. The presence of multiple large plasmids is characteristic of ExPEC bacteria, particularly APEC. Previously, the first of three large plasmids had been sequenced and analyzed by the group and found to code for virulence factors, which help the bacterium infect its host. The two other large plasmids were sequenced for the first time in the present study, as well as a smaller APEC plasmid, whose significance remains obscure. 

Unlike the first of the three large plasmids examined, the second and third do not encode for common virulence factors and appear to play only a minor role in the actual infection process of APEC bacteria. The team hypothesized that these plasmids instead conferred heightened survival potential during stressful environmental situations, including bacterial subsistence soils, poultry litter or under acidic conditions.  

In order to test the hypothesis, the group began by fully sequencing these two large plasmids as well as a smaller plasmid. They next examined the contribution of all four plasmids, both individually and in combination, as the APEC bacteria colonized human intestinal epithelial cells. The APEC strains, with their complement of plasmids, were studied under varying environmental conditions to assess their resistance to acid and bile in the human GI tract; growth under iron-poor conditions and varying carbon sources; and ability to clump together to form biofilms—a critical component of the infection process. 

In order to study the role of plasmids on APEC interaction with enteric cells, the group used a 3-D cell culture model of human intestinal epithelium, which has been shown to more accurately mimic the structure-function of the in vivo tissue than traditional monolayer cultures. Cheryl Nickerson’s research group at the Biodesign Institute – participants in the current study – have worked extensively in the development and application of 3-D cell cultures as human surrogate infection models. The application of these advanced enteric models to dissect the molecular mechanisms of APEC pathogenesis was a logical choice for these studies. 

The large plasmids under investigation did not appear to have a significant effect on the ability of APEC-derived strains to associate with and invade human intestinal epithelial cells. Further experiments however implicated large plasmids – for the first time – in APEC’s ability to resist acid and bile, two critically important tools for E. coli survival, particularly under the low pH conditions found in certain foods and in the stomach. 

Many pathogenic bacteria, APEC included, form aggregates of material known as biofilms. Biofilms are implicated in 65-80 percent of human infections. Their mechanisms of formation are therefore a matter of considerable medical concern. ExPEC cells form biofilm concentrations in both the gastrointestinal and urinary tracts. By examining the function of three large plasmids in biofilm formation (separately and in combination) at varying temperatures, the group was able to tease out some of the key features of ExPEC biofilm formation. They found that 4 distinct kinds of biofilm formed under the influence of the large plasmids, depending on temperature conditions. 

Plasmid-driven biofilm formation may play an essential role in the virulence of APEC and other ExPEC forms, by conveying survival advantages in various environmental niches found in the host. Likewise, the means by which ExPEC bacteria are able to modify their metabolism to make use of available nutrients is an important factor in their pathogenesis. A gene cluster located on one of the large plasmids was found to code for an alternate sugar pathway, again improving the pathogen’s prospects for survival under changing nutrient conditions. (Intriguingly, the gene cluster does not occur in other forms of E. coli, though it is present in another important pathogen – Salmonella.)

The combined results are a significant advance toward a comprehensive understanding of extra-intestinal E. coli pathogens and their mechanisms of survival. In earlier work, assistant research professor Mellata generated vaccine candidates specifically targeting APEC infection. The current research improves the prospects for a new range of vaccine candidates conveying cross protection from ExPEC infections in both human and avian populations. “We are very confident that our strategy in designing a much broader vaccine targeting multiple subgroups of pathogenic E. coli will result in positive health and economic impacts,” Melha says. 

In addition to their appointments at the Biodesign Institute, Roy Curtiss and Cheryl Nickerson are professors in the College of Liberal Arts and Sciences, School of Life Sciences.

Richard Harth

Science writer, Biodesign Institute at ASU


Weightless Wonder: ASU students selected to fly in zero gravity plane

January 30, 2012

Made up of ASU undergraduate students, the Dust Devils Microgravity Team has been selected to fly in NASA’s 2012 Reduced Gravity Education Flight Program.

The team is one of 14 teams from across the nation selected for the highly competitive program. The students will fly in June on the Weightless Wonder, an airplane run by NASA that provides zero gravity. dust devils team Download Full Image

The NASA program provides a unique academic experience for undergraduates to successfully propose, design, fabricate, fly and assess a reduced gravity experiment of their choice over the course of six months. The experience includes scientific research, hands-on experimental design, test operations, and educational and public outreach activities.

The Dust Devils Microgravity Team is composed of five student flyers and two faculty advisers from ASU’s School of Earth and Space Exploration (SESE) and the Ira A. Fulton Schools of Engineering. The team is headed by Pye Pye Zaw (SESE) and includes Jacob Higgins (SESE), Dani Hoots (SESE, history, anthropology), Emily McBryan (aerospace engineering, astronautics) and Amy Kaczmarowski (SESE and aerospace engineering).

The Dust Devils propose to utilize the Reduced Gravity Education Flight Program to observe the coagulation of dust particles in microgravity environments, as a function of factors such as size and composition.

Steve Desch, associate professor, advised the students on how to conduct the science experiment – providing them background literature and the implications for understanding solar system formation (coagulation is how planets start). Chris Groppi, assistant professor, advised them on the design of the instrument, the format of the proposal, and the preparation of a project budget, among other things.

“We are looking at the electrostatic properties of different variations of dust particles to better understand what causes the attraction that allows for these sets of particles to coagulate in the absence of gravity,” said Zaw. “Understanding this is important because it answers some questions about the interstellar medium, and in addition NASA is interested because this relationship may be harmful or beneficial to rover missions to places such as Mars where there are dust devils displaying some similar effects.”

Microgravity is absolutely necessary to study this because gravity will overwhelm the weaker forces in dust coagulation.

In addition, this experiment will include a simulation of a protoplanetary disk environment by testing coagulation of meteorite powders. The powders are being donated by the Center for Meteorite Studies at ASU, courtesy of its director, Meenakshi Wadhwa, also a professor in the School of Earth and Space Exploration. Better understanding of the coagulation mechanism of small particles in zero gravity will provide insight into the formation of planets from proto-planetary disks as well as the charging effects of particles on planetary surfaces.

The team will use the next six months to collect funds, build the experiment test structure, and prepare the experiment for flight week.

“We have a lot of outreach, fundraising and experiment safety preps to do between now and June, in addition to physically building our experiments,” Zaw said.

Although the cost of the microgravity flight is covered by NASA, the students will have to raise money to pay for their trip, hotel and supplies.

“We’ve raised about 54 percent of the $13,000 we need,” Zaw added.

The ASU/NASA Space Grant has supported the Reduced Gravity Flight Program in years past and will be supporting more than 50 percent of the cost for the Dust Devils Microgravity Team for this year’s competition. Three of the team members – Zaw, McBryan and Kaczmarowski – are current and past Space Grant interns. 

The Dust Devils also will be participating in various outreach events throughout the state to promote interest in science, technology, engineering and math (STEM) for the program. In addition to hands-on outreach activities, the team has created a website that includes a resource page for educators with “How To’s” on performing simple gravity simulations, models and experiments in their classrooms.

In June, the students will report to Johnson Space Center (JSC) in Houston to test their hypothesis aboard NASA’s Weightless Wonder – a modified McDonnell Douglas DC-9 jetliner that takes 45-degree nosedives to simulate zero gravity. The reduced gravity aircraft generally flies 30 parabolic maneuvers over the Gulf of Mexico. This parabolic pattern provides about 30 seconds of hypergravity (about 1.8-2g’s) as the plane climbs to the top of the parabola. Once the plane starts to “nose over” the top of the parabola to descend toward Earth, the plane experiences about 18 seconds of microgravity (0g).

The students will spend nine days on-site at JSC learning about NASA, undergoing physical training and performing their experiments aboard the Weightless Wonder. The program tries to put students through the same procedures as those followed by full-time professional research scientists.

“This was a motivated, hard-working group of students,” said Desch. “We are very proud of this success.”

The ASU team was among 14 teams selected from 60 that applied. Other teams selected include those from the Massachusetts Institute of Technology and Yale University.

The Dust Devils Microgravity Team is the fifth such team ever selected from ASU. Other ASU teams participated in the Reduced Gravity Education Flight Program in 1999, 2001, 2002 and 2003 and were supported by the ASU/NASA Space Grant Program.

Nikki Cassis

marketing and communications director, School of Earth and Space Exploration