Novel Plague Virulence Factor Identified
DURHAM, N.C. -- Researchers at Duke University Medical Center have identified a previously unknown family of virulence factors that make the bacterium responsible for the plague especially efficient at killing its host.
In the process, the team not only demonstrated that the use of the common roundworm is a valid model for studying the virulence of Yersinia pestis, the bacterium that causes plague. They also showed that the interaction between Y. pestis and the worm is quite similar to what occurs in mammals, including humans. The work indicates that the pathogen may use similar virulence mechanisms to infect evolutionary disperse organisms.
These findings are important, the researchers continued, since the pathogenesis system using the Caenorhabditis elegans worm can accelerate the process of better understanding Y. pestis pathogenesis. The shorter time and increased ease of experimentation can be especially important, the researchers continued, given potential use of Y. pestis as a biological weapon, they said.
The results of the Duke research will appear Aug. 26, 2005, in the on-line edition of European Molecular Biology Organization (EMBO) Reports to be published in print in October. The research was supported by the National Institutes of Health's National Institute of General Medical Science, the Southeast Regional Center for Emerging Infections and Biodefense (SERCEB) and the Duke Center for Translational Research.
"Our experiments have demonstrated how closely the Y. pestis-C. elegans model we developed mimics what happens when Y. pestis infects mammals," said Duke microbiologist Alejandro Aballay, Ph.D., lead researcher of the team. "This system should help speed the characterization of both pathogen and host functions that potentially can be targeted for intervention."
The Y. pestis bacterium primarily infects wild rodents, such as mice, rats and squirrels. It is usually transmitted by fleas, which spread the infection as they feed on the blood of mammals. There are different forms of plague in humans -- bubonic, pneumonic and septicemic -- depending on site of infection; and infections in humans are highly lethal if not immediately treated.
"Dr. Aballay has developed a new model for dissecting the ways pathogens such as the plague can infect and kill their hosts," said Pamela Marino, Ph.D., scientist at the National Institute of General Medical Science. "This creative approach should improve our ability to develop new medicines to treat such diseases."
Aballay has used the C. elegans, a worm commonly found in the soil, as a model to study the virulence mechanisms of other bacteria besides plague. The worm is an ideal model for genetic studies, he said, because it takes only three days to develop from an embryo to an adult capable of reproducing. Also, scientists can easily manipulate specific genes in the worm, and in contrast to other animal models, large quantities of the worms can be grown quickly and can even be frozen and used later.
"C elegans lives in the soil, so it continually comes into contact with bacteria and other microbes," Aballay said. "It has a highly developed system for not only recognizing bacteria, but also responding to them. The ability of its innate immune system to respond appropriately to specific bacteria is very similar to that of mammals."
Aballay tested Y. pestis in his model because another research team recently reported that the bacterium killed C. elegans by creating a "biofilm" over the worm's pharynx, causing it to die of starvation. Since mammals infected with Y. pestis do not die in this manner, Aballay believed that other virulence factors were involved in infecting the worm.
"We thought that a Y. pestis strain (known as KIM5) that lacks the genes (hmsHFRS operon) required for biofilm formation could still enter the worm's digestive system and eventually kill it by using a method different from food blockage," Aballay explained. "We did in fact show that as Y. pestis lacking the hmsHFRS operon accumulated in the intestine causing a persistent and lethal infection."
The researchers then screened a library of almost 1,000 Y. pestis mutants and found that six virulence factors are crucial for the bacterium to have full virulence. Of the six virulence factors, three are also required for infections in mammals. One of these factors is similar to an exported protein of Salmonella enterica. In a study published last year, Abalallay showed that Salmonella uses similar virulence factors to infect both mammals and C. elegans.
"The protein produced by this new Y. pestis virulence-related gene belongs to a family of uncharacterized proteins found exclusively in pathogenic enterobacteria," Aballay said. "This work links for the first time this particular family of bacterial proteins to virulence and we have done so by using a C. elegans pathogenesis system and a new mouse model of plague."
Aballay said that these types of conserved virulence factors may regulate innate immunity in a broad variety of hosts, including C. elegans, fleas and mammals.
"The importance of our work is that it will permit us to use a model whose genetics can be easily manipulated as a viable alternative not only for the identification of novel Y. pestis virulence factors but also to study conserved innate immune responses to the bacterium," Aballay said. "This may help us in developing strategies to protect humans from the plague."
Since the KIM5 strain of Y. pestis is well characterized and has a lower biosafety ranking than other infectious or toxic agents, Aballay believes that it will much easier for research laboratories to conduct research on virulence and innate immunity.
Other members of the research team were Duke's Katie Styer, Gregory Hopkins, and Richard Frothingham, as well as Sara Schessar Bartra and Gregory Plano, University of Miami School of Medicine.