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Genetic Analysis of Susceptibility to Infectious Disease
Summary: William Dietrich is interested in identifying genes important for determining susceptibility and/or resistance to infectious disease.
Genetics is a powerful means to determine the molecular basis of biological function. In the absence of any specific a priori hypotheses about the molecular mechanisms of a heritable trait, we can define and isolate genes important for virtually any biological process. Accordingly, we are using the recently developed and expanding arsenal of tools now available to the mouse geneticist to study the genetics of variation in disease susceptibility.
Our current interest is studying the elaborate defenses against infection that are collectively called the immune system. The immune system consists of many different interrelated parts, and there are heritable, individual differences in the functioning of the different parts of the immune system that account for variations in disease susceptibility. We are working on three models of infectious disease for which there are heritable susceptibility differences among common laboratory mouse strains.
Chlamydial Infections
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes a variety of human pathologies, including trachoma, blindness, pneumonia, a host of urogenital syndromes (including infertility), and invasive lymphadenopathic disease. It is a major cause of blindness in endemic regions and may be the most common source of bacterial sexually transmitted disease in the world. Although effective treatment is available, controlling the incidence of infection remains a major public health effort.
Interestingly, different inbred strains of mice exhibit distinct courses of chlamydial infection. For example, C3H/HeJ (C3H) mice maintain a semichronic state of infection in which viable Chlamydia can be recovered from the spleen for at least several weeks. In contrast, C57BL/6J (B6) mice are able to clear the infection by day 12 postinfection.
Our greatest interest was in assessing the earliest stages of this bacterial infection to determine if the differences in resistance between C3H and B6 mice could be accounted for by early events after inoculation. Therefore, we used a quantitative PCR (polymerase chain reaction) assay to test for early differences in bacterial load between B6 and C3H mice. Throughout the earliest stages of the infection, all the mice have mostly equivalent numbers of Chlamydia. However, by 16 hours postinfection (hpi), C3H mice exhibit a marked increase in the amount of Chlamydia in the spleen relative to B6 mice. This trend becomes exaggerated by 29 hpi, when there is a 3- to 6-fold difference between B6 and C3H mice. Based on these data, it seems likely that the difference in Chlamydia resistance between B6 and C3H mice is not attributable to differences in initial colonization but rather to bacterial replication and/or survival.
We are interested in using genetics to identify the gene(s) responsible for this trait difference (a process called positional cloning). We have analyzed the infection resistance of cross progeny of the C3H and B6 strains. These data suggest that the inheritance of Chlamydia resistance is affected by multiple genes. Using this cross, we have mapped three quantitative trait loci (QTL) on chromosomes 2, 3, and 11 that affect this trait. In the future, we hope to understand the molecular nature of the gene differences that are responsible for these QTL effects and to study genital tract infection models to compare and contrast the resistance genes for each type of infection.
Legionnaire's Disease
Legionnaire's disease (LD) is a surprisingly common form of pneumonia, accounting for ~5–10 percent of community-acquired pneumonia cases. Unfortunately, even though effective treatments for LD exist, the overall mortality rate approaches 15 percent. LD is caused by infection with a gram-negative facultative intracellular bacterium called
Legionella pneumophila. An important aspect of the pathogenesis of L. pneumophila infection involves its ability to grow inside the macrophages of the lung. Macrophages are cells that typically phagocytose and destroy invading bacterial and fungal pathogens as part of their role in immunity. Paradoxically, L. pneumophila avoids destruction by the macrophage and utilizes the macrophage as a convenient host for growth.
Purified populations of macrophages from different inbred mouse strains exhibit differences in susceptibility to the intracellular replication of L. pneumophila. These differences can be dramatic: macrophages from the inbred mouse strain A/J allow virtually unchecked replication of the parasite, whereas inbred mouse strain C57BL/6J macrophages allow virtually none. The progeny of crosses between these two strains exhibit a susceptible or resistant phenotype, depending on the parental origin of a single gene on mouse chromosome 13 that we call Lgn1.
We have used positional cloning to identify the gene difference responsible for the Lgn1 effect. We have found two closely related candidate genes in the region, Naip2(Birc1b) and Naip5 (Birc1e), that exhibit many sequence differences between resistant and susceptible mouse strains. Furthermore, we and others have found that a transgene expressing Naip5 is able to make macrophages more resistant to the intracellular growth of Legionella. While these data support the idea that mutations in Naip5 underlie the Lgn1 phenotype, we are continuing our experiments to rule out the possibility that the closely related Naip2 gene may also play a role in resistance to Legionella.
The exact functions of Naip2 and Naip5 are unknown, although relatives of these genes have been implicated in fundamental cellular processes such as regulation of cell death, segregation of chromosomes during cell division, passing signals between cells of the immune system, and detecting the presence of infecting pathogens. In collaboration with Ralph Isberg (HHMI, Tufts University School of Medicine), we have shown that the mouse Lgn1 locus influences very early intracellular trafficking decisions made upon entry of Legionella into host macrophages. In light of these data, we consider it possible that Naip5 somehow exerts effects on the endocytic physiology of the cell, perhaps in response to attempts by Legionella to subvert that physiology to its own purposes. (Grants from the Muscular Dystrophy Association and the National Institutes of Health provided partial support for this project.)
Anthrax
The deadly disease anthrax is caused by infection with the gram-positive bacterium Bacillus anthracis. Although human anthrax is relatively infrequent, anthrax is an important disease of livestock in developing countries. In addition, B. anthracis has properties that make it well suited for development as a weapon of terrorism and war. One aspect of anthrax pathogenesis involves the production of toxins by B. anthracis that (as one of their many activities) act to kill macrophages. One of these toxins, lethal toxin (LeTx), is an extremely potent macrophage poison, but the mechanism whereby it kills these immune cells is unknown.
Purified populations of macrophages from different inbred mouse strains exhibit differences in susceptibility to the cytotoxic effects of LeTx. For example, C3H/HeJ macrophages are completely killed by minute amounts of LeTx, whereas C57BL/6J macrophages are impervious to even very large amounts of LeTx. We have recently discovered that the progeny of crosses between these two strains exhibit a susceptible or resistant phenotype, depending on the parental origin of a single gene on chromosome 11 called Kif1C.
Kif1C encodes a kinesin motor protein whose role in the cell likely involves the transport of vesicular or organellar "cargo." While the cargo of Kif1C is unknown, our data suggest that efficient levels of Kif1C function are required for macrophages to survive LeTx poisoning. Elucidation of the cargo and regulation of Kif1C function will provide information about the molecular events of this aspect of anthrax pathogenesis. (A grant from the National Institutes of Health provided support for this project.)
Last updated January 26, 2004
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