Legionella pneumophila is the causative agent of Legionnaires' Disease. L. pneumophila is ubiquitous in freshwater environments as well as in man-made water systems such as air conditioners and cooling towers. Legionella biofilms in these systems have been identified as the source of a number of outbreaks. Gene expression in planktonic phase L. pneumophila has been well characterized but little analysis has been conducted within biofilms. We hypothesize that gene expression in Legionella biofilms will exhibit unique expression patterns as compared to planktonic cells. To test this hypothesis Legionella were transformed with reporter gene vectors and biofilms grown on glass slides and imaged using confocal microscopy. Characterization of biofilm stages was conducted from attachment through dispersal. Gene expression of the global regulatory protein, CsrA, and the flagellar gene, FlaA, was quantified over 120hr of biofilm growth. Biofilms were imaged at five key time points in the biofilm development: 12 hr (initial attachment), 24hr (irreversible attachment), 48hr (early maturation), 72hr (late maturation), 96hr (mature biofilm) and 120hr (mature biofilm with dispersal). Whole biofilm fluorescence was measured with syto59 staining and compared to the percentage of cells that demonstrated GFP fluorescence from the reporter gene. DIC images clearly demonstrate that Legionella biofilms follow the typical biofilm developmental stages. Analysis of the CsrA expression showed upregulation in early biofilms but little to no CsrA expression in mature biofilms. FlaA was expressed in early biofilms and during late biofilms where dispersal was occurring. Planktonic cultures are often used to characterize cycles of gene expression which are often not identical to the patterns seen in biofilms. Legionella biofilms are not well characterized molecularly and here we present the first evidence showing gene expression patterns of essential genes over time within biofilms. Use of confocal microscopy for such assays provides a high resolution, specific image that allows for quantification and detailed analysis of gene expression. This research begins the opportunity to better understand biofilm gene expression that can lead to improved prevention and control of infectious biofilms.
Limbaugh, D. and McNealy, T., "A study of gene expression in Legionella pneumophila biofilms through the use of confocal microscopy" (2014). Focus on Creative Inquiry. 13.