Scientific Monogamy: Thirty Years Dancing with the Same Bug

I WOULD LIKE TO BEGIN by expressing my thanks to the ASTDA and the Thomas Parran Selection Committee for this wonderful honor. None of us walks alone during our careers, and we depend upon mentors, colleagues, students, fellows, and research scientists who work on our projects with us. We also depend upon our families who give us perspective and balance. I have been blessed by wonderful support from all of these, and I am very grateful. The Parran Lecture frequently provides a retrospective view of a career or a field, and I would like to take you on a chronological journey over the past 30-plus years of syphilis research, describing our efforts to unravel the very complex relationship between Treponema pallidum and the host. I was introduced to syphilis in 1973 and was immediately captivated by the fascinating bacterium that causes this infection. However, my research career began before that time, when I was an undergraduate at University of California, San Diego. My first research mentor was Willie C. Brown (Fig. 1), a Professor of Biology. Willie introduced me to microbiology and agreed to let me conduct an independent study project in his laboratory. I proposed to make L-forms of Bacillus subtilis, the organism under study in his laboratory. L-forms are bacteria that lack cell walls and they are very difficult to maintain in culture because of their extreme fragility (perhaps presaging my future career focus!). Under Willie’s guidance, I learned to design logical experiments, to critically evaluate my results, and to regroup when things did not work out as planned. Most importantly, I learned that I loved the challenge and the freedom of developing my own research plan. As I prepared to leave his laboratory to begin graduate school in microbiology at UCLA, Willie gave me some advice: “Work on a bug that can be grown in liter quantities overnight” and “Be sure you can put it in the freezer when you go on vacation.” Although I learned much from Willie, I am afraid that I totally ignored this advice, as I have spent the rest of my career working on an organism (T. pallidum) that does not even grow in culture!Fig. 1: Mentors Extraordinaire. Willie C. Brown, PhD, Professor of Biology, University of California, San Diego; James N. Miller, PhD, Professor of Microbiology Stewart Sell, MD, Professor of Pathology, University of California, San Diego; and King K. Holmes, MD PhD, Professor of Medicine, University of Washington. (Titles were current during the author’s training).T. pallidum must be propagated by passage in rabbits and therefore cannot be manipulated genetically for examination of putative virulence factors. Equally problematic is the unusual ultrastructure of T. pallidum in which the cell wall layer is more closely associated with the cytoplasmic membrane than with the outer membrane, resulting in a very fragile, easily damaged surface structure. Because of these impediments, syphilis research moves slowly compared with other fields. When I was a young investigator, a well-respected senior scientist (unnamed here) spoke words of dubious encouragement to me: “Watching the syphilis field is like watching a glacier move!” Nonetheless, spectacular progress has been made in the past 30 years, due to the passion and commitment of the small number of investigators who work on this organism. Critical to that progress is the support of the STD Branch of the National Institute of Allergy through the circulation the bacteria disseminate, potentially reaching every part of the body including the central nervous system. The skin lesions persist for weeks or sometimes months and then heal spontaneously without medical intervention. The patients then enter the latent stage, in which infection persists but there are no clinical manifestations; this stage can last for many decades or the remainder of the person’s life. In the preantibiotic era, approximately one-third of infected persons developed late, often serious, tertiary clinical manifestations involving skin, bone, the aorta, the brain, and spinal cord. How does this fragile bacterium cause the chronic and complex disease that has been termed the “great imitator”? My journey in helping to unravel this mystery began in 1973 when I went to the University of California, Los Angeles, to pursue a graduate degree. New graduate students rotated through different laboratories during their first year to identify a mentor and laboratory for their dissertation research; most students performed three laboratory rotations before making a decision. I knew, within a few weeks of beginning my first rotation with Dr. James Miller (Fig. 1), that I had found my scientific home. Dr. Miller is a distinguished investigator who, since training with Dr. Ruth Boak, had been working on syphilis for a number of years and had already made some seminal discoveries, the most important of which was his classic demonstration of the first and only completely protective vaccine for syphilis.2 This immunization protocol, though onerous and not translatable to humans, provided the proof of concept that all subsequent investigators have used as their gold standard. For this achievement and others, Dr. Miller would be selected to receive the Parran Award in 1985. He was, and still is, a wonderful mentor to me. In addition to the dynamic laboratory atmosphere that Dr. Miller provided, with a number of other graduate students, postdocs, and technologists, he taught me the incredible value of knowing the old literature. “Know what came before you.” I remember many times that we would be talking about a particular topic and he would reach up to his shelf and pull down (from his well-organized files) an old article that had important insights into the topic at hand. With his encouragement, I spent hours in the bowels of the UCLA library searching the old publications on syphilis. The early investigators were not constrained by editors who want to minimize figures, tables, and page length; these early and very lengthy publications were true scholarly works in which exquisite detail was provided. It is often only through the examination of the fine detail of the results that important insights can be gleaned. The discussions in these articles were full of speculation and argument, providing a clear window into the scientific thinking of the author. Through careful reading of these classic papers, we can still today find valuable information that, when put in the context of our modern understanding, can direct our current efforts. When Justin Radolf and I were working on our recent book, Pathogenic Treponema: Molecular and Cellular Biology,3 we spent many hours reading and rereading many of the old classic papers on syphilis, and we found numerous instances in which early scientists speculated on concepts that we considered to be modern. What did we believe about syphilis immunology in 1973? We believed that antibodies were the key element in the immune response. There was an active body of literature, based upon the study of peripheral blood lymphocytes, purporting that cellular immune function is generally suppressed during early syphilis. T. pallidum was believed to be resistant to phagocytosis by macrophages and neutrophils. It was also believed, based upon the vaccine studies of Miller, that protective immunity can be induced only by a labile and scarce surface antigen. There were, however, contradictions between some of these assertions and what we knew about the disease process. For example, antibody titers are very high during the secondary stage—a time at which millions of T. pallidum are found throughout the body of the infected person, seemingly oblivious to the antibodies swirling around them. The histopathology of primary and secondary skin lesions was already well recognized as a robust lymphocytic infiltration, which is not consistent with an hypothesis of generalized cellular immunity during early syphilis. So, I set out to dissect the immune mechanisms in early syphilis, with the goal of figuring out how T. pallidum are cleared from the chancre and rash before spontaneous resolution of the lesions. Cellular immunity was coming into its own at this time and I was in a department with a heavy focus on tumor immunology. Consequently, I spent years trying to show that lymphocytes from infected rabbits were directly cytotoxic to T. pallidum, just as lymphocytes could be cytotoxic to tumor cells. Many experiments later and being no closer to finishing my PhD degree, I decided to try a new approach. This was a period of very active work, by George Mackaness and others, on the role of macrophages in chronic infections, and I decided to see whether rabbit macrophages could ingest T. pallidum in a culture system. My very first experiment was a success and showed evidence of phagocytosis of the bacteria by macrophages—quite a thrill after years of disappointment! Dr. Miller, however, maintained an appropriate level of skepticism, and challenged me to convince him that my conclusions were correct. Eventually, he was persuaded. These cell-focused studies came full circle, back to the role of antibody in immunity, in that specific antibodies were opsonic for T. pallidum, significantly enhancing the phagocytosis of the organism by macrophages.4 Around this time, Dr. Stewart Sell (Fig. 1), a well-respected rabbit immunologist, became interested in working on syphilis using the rabbit model. He called the Miller laboratory looking for someone who was experienced with syphilis, so I moved down the road to UC San Diego to set up a syphilis research program as a postdoctoral fellow in his laboratory. Stew is a pathologist and the most important thing that he taught me was to “Look at the disease that you are studying.” Stew and I spent hours looking through a microscope observing that, during early infection, T. pallidum increased in numbers locally in concert with increasing numbers of infiltrating lymphocytes. As treponemes reached peak numbers, macrophages began to infiltrate the lesions and then the bacteria essentially disappeared—from billions to rarely seen within days.5 At this point, the only material that stained with anti-T. pallidum antibodies was apparently digested bacteria within the infiltrating macrophages, providing in vivo confirmation of the in vitro phagocytosis that we described earlier. After the bacteria were cleared by the immune response, the lesions resolved, just as in human syphilis. These results were certainly not consistent with a prevailing theory of syphilitic immunosuppression. Just before my arrival in the laboratory, Stew and his group had developed specific antisera to differentiate rabbit B and T lymphocytes, so we applied these reagents to the study of syphilis. Working with Sharon Baker-Zander, who became a friend and long-time colleague, we showed that splenic and regional lymph node T lymphocytes become sensitized to T. pallidum antigens very early during syphilis infection,6 again finding absolutely no evidence for either specific or generalized immunosuppression. In contrast, the cellular immune response is consistently robust and long lasting. In 1979, I traded sun and sand for the drizzly Pacific Northwest when I moved to Seattle to continue my postdoctoral training with Dr. King Holmes. Even at that time, King was recognized as a leader in the growing field of sexually transmitted diseases, and he received the Thomas Parran Award in 1983. King has truly been a great mentor to me. He has pushed me when I needed it, and has, from the beginning, fostered my my first in he me that I needed to a for a young to an independent King has also taught me to the laboratory and to study human syphilis to identify important research the years, have been of particular including the of the central nervous by T. pallidum and its there treatment with After work in my laboratory, Dr. has continued on developing for examination in syphilis and the of HIV infection on and response to In the laboratory, our group Sharon Baker-Zander, and continued our on the mechanisms of immune in early syphilis, with a particular focus on opsonic antibody and antibodies were to be antigens that are to the and not antigens with We also that sensitized T lymphocytes to be that the of macrophages to ingest and T. Although in vitro studies are very and it is to that in vitro are to the disease We began a with at University of and James at University of to the of the lymphocytic and the in early syphilis lesions in studies were in the rabbit with and This work that, in and with early syphilis, the infiltrating and T and that the is with as the These studies to a new of immune in early syphilis (Fig. in which T lymphocytes are sensitized to treponemal antigens and infiltrate the T. pallidum are The T that and as for B lymphocytes to specific anti-T. pallidum including with opsonic of the treponemes as the bacteria are by then and by the cellular and mechanisms of to be the in primary and secondary and the immune response is There is no evidence for or a from the primary to the secondary stage, as had been in the mechanisms in early syphilis. T lymphocytes become sensitized to T. pallidum antigens and infiltrate the T which and T also for of B to antibodies to T. pallidum These antibodies T. pallidum, the phagocytosis and of the treponemes by macrophages, resulting in resolution of primary and secondary you at the beginning that, after the early lesions the infected the latent stage in which treponemes persist clinical manifestations are the that I just made about a robust and immune response in early syphilis, how some treponemes to cause The is still under but we have made some and You remember that early is by macrophages and that only treponemes are there different about the treponemes that The is When we treponemes from rabbits that had already and these to our phagocytosis we found that these treponemes were resistant to phagocytosis even in the of immune These macrophages were to ingest so must have in the treponemes We speculated that the surface of the must have been so that opsonic antibody to The surface of T. pallidum to be a so the efforts of investigators to In addition to the fragility of the outer membrane, by that the outer membrane is of outer membrane approximately of the of as a This lack of antigens the that antibodies times to to T. pallidum in and and it also the of of T. the of surface we were of their and research set out to identify them. With and we took of our that opsonic antibodies are specific to T. used to identify that are to T. pallidum compared with a closely and used two antisera for of a T. pallidum library by infection of a compared with T. of these for that had some with the major of Treponema a that is associated with disease. As had been to be on T. to have and to and the with our was When the T. pallidum was we found that these to a called T. pallidum or that of the be in the outer We then began a of the using the efforts of the University of syphilis laboratories by and and found that some of T. and that rabbits infected with T. pallidum T and that most of the with the and most robust immune response. these studies that different of the are by different of T. pallidum, later by using In an of experiments, and also showed that the of a of the of I and the level of of This is hypothesized to be a of for a of the and to how T. pallidum its surface antigens to cause infection. Because of the high level of of and the early robust immune response induced by that we much of our early on It is to be on the outer membrane, and we that antibodies a were opsonic for T. of rabbits with significantly the of challenge with infectious T. Although we are still very by these results, used a different approach to immunization and to our the field is not on the surface of New however, our hypothesis of of and began to the in a number of T. pallidum and that the is not only but also within Even more the is to in the reading called This immediately made us about in which even could the by antibodies a different We proposed to three during What are the of How is these we developed a of T. pallidum that had a We this times in rabbits the before antibodies are then it times at which time the immune response and the of the treponemes from the At each of these we the in the treponemes and found that is during infection; importantly, the of during in which the developing immune response treponemes expressing the the hypothesis of immune is to be the immune must the a graduate the B and T cell in using and that, T in the of antibodies consistent with the that antibodies (perhaps through bacteria expressing the recognized New on the other would have a graduate the exquisite of that very small in can antibody that the were by or than so he for the of the new In searching the T. pallidum he the that over to of the that we had in the in our These to the new that are into the of the in the and it is to identify the specific of the new that during our Because the not even when the does the is to be than is that antibodies the of the and that these antibodies T. pallidum for phagocytosis by macrophages, resulting in of the of treponemes from the early few that have new are not recognized by the antibody and are to the immune This is however, only the of are on the surface of T. What we about the of of the of that it is a with significant this that many of the are that would be on the surface of the bacterium and in contact with this journey full circle, how does it with what we of the natural history of The treponemes that the of the lesions of primary and secondary syphilis are cleared by the host’s immune response, phagocytosis and of the stage for resolution of the early lesions. During the of that immune response, a small of treponemes its surface antigens so that it is no recognized by of some antigens and the antibody of the of The of is well over for many years of immune The bacteria are selected for by the immune and are to persist in the for the infection of the latent and tertiary T. pallidum is a for and it does not give up its Nonetheless, a of investigators to study this fascinating bacterium and, using new and will continue to make progress in understanding the relationship of syphilis. I the ASTDA again for this wonderful and I also the many with I have had the to work throughout the years. This award to the