A study of gene expression in Legionella pneumophila biofilms through the use of confocal microscopy

Legionella pneumophila is the causative agent of Legionnaires\u27 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

CU and the CDC

Legionella is a gram-negative genus of bacteria that is the cause of Legionnaires disease. Currently, 50 species and 70 serogroups of Legionella have been identified. The Center for Disease Control (CDC) in Atlanta maintains a bank of identified and unidentified Legionella samples. The availability of sequencing technologies has increased since many samples were collected allowing for identification of many previously unidentifiable isolates. We received 68 unidentified samples from the CDC. A sequence based typing scheme was used for characterization. Genomic DNA was extracted from samples and polymerase chain reactionwas performed on the 16S and mip genes. These samples were then sequenced at Clemson University Genomics Institute. Currently, we have identified several samples which were previously undescribed. Once a sample is identified as novel, characterization through sequencing other genes along with morphological and biochemical assays will be conducted. As a collaborative project, regular meetings occur with scientists from the CDC. Characterization of novel strains expands this lab\u27s ability to conduct outbreak analysis and risk assessment along with expanding our knowledge of the pathogen

Assessment of Virally Vectored Autoimmunity as a Biocontrol Strategy for Cane Toads

BACKGROUND: The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. METHODOLOGY/PRINCIPAL FINDINGS: The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. CONCLUSIONS/SIGNIFICANCE: While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach

A study of gene expression in Legionella pneumophila biofilms through the use of confocal microscopy

Legionella pneumophila is the causative agent of Legionnaires\u27 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