The obligate intracellular bacteria chlamydiae cause serious diseases in both humans and animals. Human infections by Chlamydia trachomatis number in the millions per year and lead to serious pathologies in the eye and genital tract. Several unique characteristics define the genus Chlamydia, one of which is the formation of large, polyploid, nonculturable persistent cell forms- termed aberrant forms- that occur when the growing bacteria are exposed to stress. The biological reasons that Chlamydia spp. have evolved this unusual growth form are completely unknown. Genomic and laboratory-based studies demonstrate that clinical C. trachomatis undergoes a high level of lateral gene transfer (LGT) within the species, but essentially no genetic exchange with organisms outside the species. Any mechanism associated with chlamydial LGT remains undiscovered, as does the biological reason for this process. This proposal will explore possible interactions between the formation of aberrant forms and the process of lateral gene transfer/recombination in the chlamydial system. Our overarching hypothesis is that that the polyploid aberrant form facilitates intraspecies LGT and recombination, leading to recovery from stress-induced mutagenesis during persistence inside cells. Intraspecies LGT can be modeled very effectively in the laboratory using differently antibiotic resistant parents with modest levels of genetic diversity. This proposal expands on that model, using in vitro conditions to mimic stresses and selective forces that might be encountered in vivo.
In Aim 1 we will use culture and genome sequencing to explore the breadth and depth of LGT in cocultures of different C. trachomatis, and evaluate qualitatively and quantitatively the effects of different stressors on the process. The conditions to be tested include tryptophan starvation, beta-lactam exposure, and culture in the presence of neutralizing antibodies.
In Aim 2, we will use plasmid constructs to explore recombination within individual C. trachomatis developmental forms, with constructs that carry untranslatable resistance markers transformed into antibiotic-sensitive strains. We will examine the phenotypic shift to antibiotic resistance within these strains, in the presence and absence of stressors.
This aim will test the hypothesis that recombination within individual developmental forms is affected by the polyploidy introduced by a stress-induced transformation to aberrancy. Collectively, these experiments will explore different aspects of how stress and aberrancy affect the rate of lateral gene transfer and recombination in C. trachomatis. We will use these data to examine the possible roles that chlamydial LGT and recombination serve in stress survival during persistent infections of patients.
The chlamydiae are obligate intracellular bacteria that cause serious disease in humans and many animal species. Chlamydia trachomatis infections in humans affect millions of people worldwide, affecting the reproductive health of women and causing the disease trachoma. There are many interesting and unique aspects of chlamydial biology, one of which is lateral gene transfer within species. Genomics-based and laboratory analysis have identified an apparent paradox: genetic exchange is very common between strains of C. trachomatis, although the bacterium never acquires DNA from other species. Experiments described in this proposal will explore the how and the why of chlamydial lateral gene transfer, with a long-term goal of identifying currently unknown opportunities for therapeutic intervention. The studies will also uncover novel aspects of chlamydial basic biology which may help us understand how other important bacteria exchange DNA.
