This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goals of our research is to determine if (a) fungal infections may have detrimental effects on CF patients and (b) if our in vitro model systems are predictive of in vivo microbial characteristics. The research proposed here will be performed in conjuction with studies currently supported as a Cystic Fibrosis Foundation pilot project focused on determining if P. aeruginosa produces higher levels of virulence determinants in the presence of fungi. These studies will be performed in collaboration with George O Toole who is examining antibiotic resistance and biofilm formation in CF sputum. In the research proposed here, we will expand our current studies to include additional analyses and a larger number of sputum samples.
Specific Aim I. It is known that both C. albicans and Aspergillus fumigatus are detected in between 10-20% of sputum samples, but enumeration by sensitive and quantitative molecular methods has not been reported in the literature. Using PCR primers designed to amplify the 18S region from all fungi, we will assess total fungal load in 40 sputum samples from different patients. Published primers that distinguish between Candida spp., Aspergillus spp., will be used to determine the abundance of these and other fungi in sputum samples. The levels of fungi will be correlated with the culture data, patient status, the presence of other microbes, and history of antibiotic therapy. This work will be published as preliminary studies designed to assess the impact of fungal colonization on lung function and to analyze the factors that may predispose to fungal colonization. The total sputum RNA isolated in Aim I will be saved for subsequent studies (described below) and for use by other CF researchers at Dartmouth.
Specific Aim II. In addition to isolating total RNA from the CF patients in Aim I, the individual bacterial strains will be purified and frozen. It is frequently observed that P. aeruginosa strains undergo changes over the course of a chronic infection. Furthermore, there is significant strain to strain variation among P. aeruginosa CF isolates. It is not yet known if this variation, which is usually assessed using in vitro phenotypes such as colony morphology or nucleic acid profiling techniques, is relevant to P. aeruginosa behavior in vivo. In this aim, we will examine the variability of P. aeruginosa CF isolates in terms of their transcript levels of five different secreted signaling molecules and virulence factors in both single species cultures and in co-culture with fungi. Transcript levels will be normalized to the amount of bacterial DNA present. We will determine if the production of signaling molecules in single species cultures correlates with the profiles observed upon co-culture with fungi to determine if the presence of fungi elicits a common response in different P. aeruginosa isolates.
Specific Aim III. Using the total RNA isolated from the sputum samples (Aim I) from which the CF P. aeruginosa strains were origincally isolated (strains used in Aim II), transcript levels for the five target genes will be measured and normalized to total bacterial DNA measured by PCR. We will compare the in vitro variability between strains to the variability of these transcripts observed in sputum. These studies will assess the value of P. aeruginosa liquid cultures and P. aeruginosa-C. albicans co-cultures as models for the study of P. aeruginosa in the CF lung. These data will also be incorporated in to the sputum analyses of biofilm-related and antibiotic resistance genes by George O Toole to evaluate the accuracy of our laboratory models. The study of chronic bacterial infections requires both the development of adequate model systems and the assessment of how well these model systems replicate the important environmental factors that impact microbial function in vivo. Our parallel in vitro and in vivo studies will contribute to the assessment and improvement of our laboratory model systems designed to enable us to predict microbial behavior in vivo. Ultimately, this knowledge may help researchers develop powerful screens for the identification of therapeutic agents that are potent against all P. aeruginosa strains in the complex environment found within the CF lung environment. Using the same set of samples, we will determine the levels of fungi in CF sputum samples with the goal of accumulating publishable preliminary data that will be necessary for determining whether the presence of fungi is detrimental to the health of individuals with CF
Showing the most recent 10 out of 133 publications
