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MICROBIOLOGY Cloak Partly Lifted on Tiny ChlamydiaScience Moves Closer to Effective Vaccine In this era of sex education and ample health information, teenagers in the U.S. probably grow up more mindful of the dangers of sexually transmitted diseases (STDs) than any generation before them. After all, HIV is looming large as they come of age. In spite of all that, they remain woefully unaware of less deadly threats. Consider Chlamydia trachomatis, the most common bacterial STD in the developed world. Since infection with Chlamydia is often silent, it goes mostly undetected even though it is the leading cause of preventable infertility in this country. In the Jan. 30 Proceedings of the National Academy of Sciences, Michael Starnbach, HMS assistant professor of microbiology and molecular genetics, and colleagues here and at the Seattle biotech company Corixa Corporation, present data that advance researchers' understanding of this mysterious pathogen and, at the same time, move them closer to developing a vaccine. The study also presents a new method for identifying bacterial T cell antigens.
Chlamydia, which can cause infertility when left untreated, has evolved a stealth operation to grow while evading the host's immune system. Its infectious form enters the host cell sheathed in a protective layer of host cell membrane. It then changes into a replicative form and multiplies. In the process, it somehow imports all necessary amino acids, energy, and other supplies from the host cell's cytoplasm, possibly with the help of the newly discovered protein Cap1 (black dots). Chlamydia reverts to its infectious form when the cell can no longer tolerate the growing vacuole (see light micrograph on right). The cell finally bursts, releasing infectious particles. Micrograph by Zarine Balsara The need is pressing. The Boston Public Health Commission last year released 1999 statistics showing 2 percent of the city's 15- to 19-year-olds have chlamydia. Boston's minority girls were reported to have infection rates of almost 6 percent, and a 1998 Journal of the American Medical Association study of sexually active teenage girls in Baltimore found infection rates approaching 30 percent. In the short term, this problem could be controlled by annually screening sexually active girls; once detected, chlamydia responds well to antibiotic treatment. Yet a better solution would be crushing chlamydia's prevalence in the population with a vaccine, said Starnbach. Chlamydia can infect people many times, so those at high risk would have to be repeatedly screened and treated. Moreover, in the developing world, chlamydia causes trachoma, the leading cause of preventable blindness in the world. There, too, a vaccine might provide effective protection. Attack from WithinBefore that can happen, researchers need to learn much more about Chlamydia's unusual biology. This is what drew Starnbach, a microbial immunologist interested in CD8+—or cytotoxic—T cell responses, to studying this pathogen. Once Chlamydia has infected a host cell, it hides inside a membranous bubble called the vacuole (see image, p. 6). There it multiplies up to 1,000-fold until the cell can no longer tolerate the expanding bubble and bursts, spewing infectious particles.This reproductive cycle poses a riddle because Chlamydia—almost a viruslike particle with no more than 900 genes—is a complete scavenger. It takes from the host cell all that it needs for its replication, including all amino acids and even the basic energy unit ATP. How can it purloin all these things while being walled off from the cytoplasm where the supplies are? "This has got to be an enormous transport challenge," said Starnbach, "and virtually nothing is known about it." The logistics of Chlamydia's stealth operation may have remained invisible to researchers, but the immune system has taken notice. Seven years ago, Starnbach first discovered that cytotoxic T cells arise during a Chlamydia infection in mice. That was intriguing because this arm of the immune system responds to peptides that come from a host cell's cytosol, where Chlamydia is not present. If cytotoxic T cells "see" Chlamydia, at least one of its proteins must have access to the cytoplasm. Starnbach realized that with these CD8+ T cells he might be able to kill two birds with one stone. Used as probes, they could reveal Chlamydia proteins that are key to improving basic knowledge of the bug's pathogenicity. At the same time, the T cells could help identify Chlamydia antigens capable of triggering a cytotoxic immune response, one possible component of a future vaccine. Yet identifying these proteins proved technically difficult. Methods for manipulation of Chlamydia's genome are only just emerging. Scientists for decades have been able to grow bacteria like E. coli in broth culture by the billions, allowing them to perfect methods for the cloning of its DNA. By comparison, Chlamydia grows only inside eukaryotic cells that scientists must culture for them. Shrouded inside the vacuole, its DNA is hard to reach for any vector DNA investigators may wish to introduce into the organism.
Postdoc Sarah D'Orazio, graduate student Lisa Steele, Michael Starnbach (l to r), and others discovered the first cytotoxic T cell antigen in Chlamydia trachomatis, cause of the most common sexually transmitted disease in the developed world. Photo by Pam Murray The break came when Starnbach teamed up with scientists from Corixa, who had developed a retroviral expression cloning system. It allowed them to transfer snippets of Chlamydia DNA into a retroviral vector, creating a library that could be used to infect cultured cells. The cells containing the library were then probed with Starnbach's Chlamydia-specific T cells. They were able to isolate DNA from these cells encoding the protein fragment responsible for activating the T cells. Once they had a string of about 60 amino acids, a search of the Chlamydia genome—sequenced by others in 1998—identified the elusive protein as a novel one that has no homology with any other known protein outside of the Chlamydia genus. Researchers in Starnbach's lab are currently trying to find out what this protein, dubbed Cap1, might be doing. Does it perhaps divert nutrients from the cell's metabolism toward the vacuole or corrupt the cell in some other way? Many new proteins have common motifs in their sequence that hint at a certain function, such as secretion or proteolysis. Cap1's sequence, by contrast, gives away nothing, leaving much to be discovered about the molecular war-game interplay between Chlamydia and its host, says Starnbach. Cap1 shows promise as an antigen candidate for a future vaccine. An experimental Cap1 vaccine reduced the number of Chlamydia in the spleens of mice about threefold, the study shows. Wish ListTo develop a human vaccine, re-searchers need to understand the relative contributions each arm of the immune system can make to an effective immune response. Research into cytotoxic T cells has lagged behind work into stimulating antibodies or T helper cells, in part because no effective CD8+ T cell antigens were known and in part because cytotoxic T cells were thought never to see antigen from a bacterial pathogen ensconced in a vacuole.Ideally, said Starnbach, a human vaccine should contain antigens that stimulate an antibody and T helper cell response to eliminate Chlamydia before it has entered host cells, as well as antigens like Cap1 that stimulate a cytotoxic T cell response to destroy those that do manage to infect cells. Experimental vaccines based on antibodies and CD4+ T cells exist at the preclinical stage. In addition, vaccine developers always try to tailor their vaccine away from causing an inflammatory reaction. This is relevant to chlamydia, too, because of the pelvic inflammatory disease followed by scarring and eventual occlusion of the fallopian tubes that occurs in many women with persistent infection. Yet so far, researchers know little about which aspects of the body's immune response are necessary to fight chlamydia and where it begins to tip over into self-destruction. These kinds of reaction are difficult to stop once set in motion, and they are a major reason experimental vaccines often fail. "To me, the immediate benefit is to better understand these bugs," said Starnbach. —Gabrielle Strobel |
