A new study led by a University of Florida research team has identified a critical pathway used by an important sexually transmitted bacteria to acquire nutrients. The findings could contribute to developing new drug targets that starve the bacteria, if current antibiotic therapies fail.
The team is from the laboratory of senior author Tony Maurelli, a professor and associate chair of UF’s College of Public Health and Health Professions Department of Environmental and Global Health. Maurelli is also affiliated with UF’s Emerging Pathogens Institute, where his research agenda is deeply focused on the sexually transmitted disease commonly known as Chlamydia. Their study published in the July issue of Infection and Immunity.
Chlamydia is caused by a bacterium named Chlamydia trachomatis which causes sexually transmitted infections worldwide mostly among young, sexually active adults, although rates are also rising in people age 55 and older. Most Chlamydia infections are asymptomatic, but untreated infections can result in serious complications, such as pelvic inflammatory disease and even infertility. Maurelli’s team has studied how Chlamydia survives within an infected individual by stealthily evading detection by its host’s immune system.
Bacteria like Chlamydia typically acquire nutrients by using an oligopeptide permease transporter system, also known as an Opp transporter. Unlike most bacteria, C. trachomatis can’t make its own amino acids, so it must sneak them away from its host in the form of linked chains of amino acids known as peptides. The Opp transporter system is C. trachomatis’ secret weapon for acquiring these peptides. Linked chains of 20 or fewer amino acids are called oligopeptides, and Opp transporters specialize in moving oligopeptides ranging in size from two to 18 amino acids.
Raghuveer Singh, a postdoctoral fellow in Maurelli’s lab and the lead author of the new study, performed a bioinformatics analysis of the Chlamydia genome to identify the genes needed for an Opp transporter system. He discovered genes that resembled a transport system but, surprisingly, he also found multiple different genes for a protein called OppA that binds the peptides shuttled by the system.
Singh used a mutant of another bacteria, Escherichia coli, to demonstrate how the chlamydial transporter worked. Singh engineered the mutant bacteria to lack its native transporter system—named OppABCDF with each letter reflecting one of five components that comprise the system—and then swapped in the chlamydial OppABCDF. The swapped transporter was then used to transport a compound toxic to E. coli. If the chlamydial transporter system was not functional, the E. coli would survive; but if it was functional, it would essentially feed the toxin to the E. coli and kill it, which it did—proving that the chlamydial transporter did indeed function to acquire nutrients.
After verifying its function in the E. coli mutant, Singh worked with co-author Jessica Slade, also a member of Maurelli’s lab, and used a similar experimental strategy to show that the OppABCDF system functioned as an oligopeptide transporter in Chlamydia.
In the micrograph at right, C. trachomatis are stained green and the nuclei of the HeLa cells they have infected are stained blue. To show that the Opp transporter in C. trachomatis was functional, the researchers engineered it to ferry a toxic molecule. The bacteria took up the toxin which then reduced their growth, as evidenced by the smaller size of their inclusion bodies (the forms that look like green bubbles) in the second and third panels.
The team then turned their attention to one remaining puzzle. Why does Chlamydia have multiple genes for OppA, the protein that binds the oligopeptide? Evolution drives bacteria to only keep the genes that they need, and unused genes are typically eliminated by mutation. The fact that Chlamydia had several genes for one function raised a red flag.
Bacteria like Chlamydia that invade and grow inside cells of a mammalian host have developed wily strategies that hide them from their host’s immune system. One strategy used by Chlamydia involves peptidoglycan which is a substance made by the bacteria to build its cell wall, but to which its host’s immune system is highly sensitive.
As bacteria snip the peptidoglycan chain to expand their cell wall, they constantly churn out peptidoglycan fragments. Some bacteria use their Opp transporter to capture and recycle peptidoglycan fragments that are released during the normal process of cell wall growth. Recapturing these cell wall pieces saves the bacterium the energy of synthesizing new components from scratch, and it also helps the bacterium avoid triggering an immune response.
While it was reasonable to assume that Chlamydia also recycled its cell wall components, the fate of peptidoglycan fragments in C. trachomatis was unknown. Singh collaborated with coauthor George Liechti at the Uniformed Services University to solve the mystery. They showed that OppA3—one of the seemingly “extra” peptide-binding proteins of the chlamydial Opp transport system—also works with the transport system to bind and transport peptidoglycan pieces.
They used a combination of genetic, biochemical and immunological tools to show that the chlamydial OppABCDF system transports a tri-peptide fragment of peptidoglycan in both an E. coli mutant and in C. trachomatis. This means that Chlamydia evolved a remarkable dual-function system that both transports essential amino acid chains for growth and also recycles cell wall fragments to save energy and avoid stimulating the immune system.
“The Opp transporter in Chlamydia acts like a Swiss Army knife,” Singh says. “It not only provides nutrition but also protects the bacterium from host immune system attack.”
The team notes that the peptidoglycan fragment transport systems of many intracellular pathogens have yet to be identified. The experimental strategies they used to discover the dual function transporter of Chlamydia can be applied to other intracellular pathogens to identify similar subunits that play a role in peptidoglycan recycling.
This work was supported by a grant from the U.S. National Institute of Allergy and Infectious Diseases, of the National Institutes of Health to Dr. Maurelli.
Written by: DeLene Beeland