The research, published in the September 26 issue of the journal Science, provides the first detailed insight into how bacteria physically manipulate host cells at the onset of an attack. The findings may help scientists develop a more general understanding of similar strategies used by a diverse array of deadly bacteria and lead to improved drugs for fighting them.
Collaborating with researchers at the University of Virginia Health Sciences Center, the Rockefeller team found that Salmonella injects proteins into host cells that staple together molecules of actin, the scaffolding protein that provides structure to a cell. Like welding girders to each other to construct a skyscraper, Salmonella proteins tack actin into long filaments that expand the size and change the contour of a cell so it can fold itself around the bacterium.
The protein that staples actin together - the Salmonella invasion protein A (SipA) - is unlike any molecule found in the human body, the researchers say.
"No protein in our cells is quite as potent, or elegant, as SipA," says the lead author, Assistant Professor C. Erec Stebbins, Ph.D., head of the Laboratory of Structural Microbiology at Rockefeller University. "One small protein is able to go into a host cell, hijack its biochemistry and rearrange its structure. That's a powerful example of host-pathogen co-evolution."
"Erec Stebbins and his colleagues have made a crucial discovery that may lead to the development of better drugs to treat this major threat to human health and well-being," says Rockefeller University's new president, Paul Nurse, Ph.D.
The research helps complete the picture of what happens when Salmonella infects the human intestinal tract - which it does frequently. Worldwide, Salmonella bacteria cause more than one billion new human infections each year, resulting in more than three million deaths. Most infections are due to errant consumption of food contaminated with animal feces, but because Salmonella has also been used as a biological weapon, the pathogen is classified as a food and water safety threat in biodefense by the National Institutes of Health.
Stebbins and his colleagues are among the first researchers to study the intricate interactions that occur between infectious microbes and host cells at the molecular level. Using techniques adapted from biochemistry, microbial cell biology and structural biology, Stebbins is developing a library that will detail the molecular composition of proteins, known as "virulence factors," that bacteria employ to infect host cells and use its biochemical machinery to replicate.
"Bacteria ranging from plant pathogens to the plague share a virulence system that is quite similar to the one Salmonella uses to gain entry into cells," he says. These bacteria use a protein "secretion system" called "type III" or "contact dependent" to inject virulence factors directly into host cells. "Bacteria have a whole armament of very sophisticated virulence proteins, and each pathogen can use these proteins in different combinations to manipulate a host cell in unique ways," says Stebbins.
The system used by Salmonella, which has been described as a "molecular syringe" with a needle attached, is one of the most complex protein secretion systems discovered, Stebbins says. Once Salmonella attaches to a cell in the gut it wants to invade, it uses the syringe and needle system to inject virulence factors into the host cell.
Researchers knew that SipA, one of the proteins injected into the host cell, could force actin proteins to join together into a strong filament that rearranged the cell's structure, but the way in which it did that was not known.
To find the answer, the researchers used biophysical imaging techniques that produce molecular pictures of the virulence molecules: the Rockefeller scientists used X-ray crystallography to get an atomic resolution structure of SipA, and the Virginia researchers developed an electron microscopy image of SipA-actin filaments - an arrangement too large and diverse to crystallize. The two images were then combined in such a way that the scientists could superimpose the SipA structure upon the larger and less detailed electron microscopy representation, and through extensive image processing, tease apart the role that SipA plays in forming filaments.
They found that SipA is shaped like a molecular staple, with a rounded middle and two extending arms that project from either side of the molecule. The middle core of the SipA molecule binds to actin and positions the long arms so that they reach about and tether together more distant actin molecules, holding the filament together. Removing the arms produced SipA proteins that lost this ability.
"Actin is dynamic, and can be floating as free molecules in the cell or in small chains. SipA drives all the actin to link together and form a lot of scaffolding underneath the attached bacterium," Stebbins says.
By building the actin girders, SipA forces the host cell to expand into a shape that resembles curtain ruffles, which then surrounds the bacterium. Through a process called macropinocytosis, Salmonella is taken inside the host cell and replicates itself to spread the infection.
"Salmonella is very well adapted, and quite sophisticated," Stebbins says. "It induces its own uptake into a host cell, but once inside, it sends out proteins that turn off the effects induced by SipA and other virulence factors."
Findings from the study will allow researchers to probe how other bacteria use virulence factors similar to SipA to change the shape of cells they infect.
"While SipA is specific to Salmonella, many bacterial pathogens use their own virulence proteins to manipulate actin in the cytoplasm," Stebbins says. "What we have learned about Salmonella is a first step in a coming molecular understanding of similar processes in many bacteria. This field of study is really coming of age."
Defining how pathogens invade human cells may ultimately aid in the design of novel antibiotics, which is increasingly important given the rise of antibiotic resistance, Stebbins says.
The study was funded by grants from the National Institutes of Health and a Burroughs-Welcome Investigators in Pathogenesis of Infectious Disease award to Stebbins. The first author is Mirjana Lilic, Ph.D., of Rockefeller University, and co-authors at the Department of Biochemistry and Molecular Genetics at the University of Virginia Health Sciences Center include: Vitold E. Galkin, Ph.D., Albina Orlova, Margaret S. VanLoock, and Edward H. Egelman, Ph.D.