Application of β-lactamase Reporter Fusions as an Indicator of Effector Protein Secretion During Infections with the Obligate Intracellular Pathogen \u3cem\u3e Chlamydia trachomatis \u3c/em\u3e

Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that C. trachomatis CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in Chlamydia

Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in \u3cem\u3eChlamydia trachomatis\u3c/em\u3e and Can Reverse Cassette-Induced Polar Effects

As obligate intracellular bacteria, Chlamydia spp. have evolved numerous, likely intricate, mechanisms to create and maintain a privileged intracellular niche. Recent progress in elucidating and characterizing these processes has been bolstered by the development of techniques enabling basic genetic tractability. Florescence-reported allelic exchange mutagenesis (FRAEM) couples chromosomal gene deletion with the insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Similar to other bacteria, many chlamydial genes exist within polycistronic operons, raising the possibility of polar effects mediated by insertion cassettes. Indeed, FRAEM-mediated deletion of Chlamydia trachomatis tmeA negatively impacts the expression of tmeB. We have adapted FRAEM technology by employing a gfp-bla cassette flanked by loxP sites. Conditional expression of Cre recombinase in Chlamydia tmeA containing a floxed cassette resulted in deletion of the marker and restoration of tmeB expression

Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in \u3cem\u3eChlamydia trachomatis\u3c/em\u3e

Although progress in Chlamydia genetics has been rapid, genomic modification has previously been limited to point mutations and group II intron insertions which truncate protein products. The bacterium has thus far been intractable to gene deletion or more-complex genomic integrations such as allelic exchange. Herein, we present a novel suicide vector dependent on inducible expression of a chlamydial gene that renders Chlamydia trachomatis fully genetically tractable and permits rapid reverse genetics by fluorescence-reported allelic exchange mutagenesis (FRAEM). We describe the first available system of targeting chlamydial genes for deletion or allelic exchange as well as curing plasmids from C. trachomatis serovar L2. Furthermore, this approach permits the monitoring of mutagenesis by fluorescence microscopy without disturbing bacterial growth, a significant asset when manipulating obligate intracellular organisms. As proof of principle, trpA was successfully deleted and replaced with a sequence encoding both green fluorescent protein (GFP) and β-lactamase. The trpA-deficient strain was unable to grow in indole-containing medium, and this phenotype was reversed by complementation with trpA expressed in trans. To assess reproducibility at alternate sites, FRAEM was repeated for genes encoding type III secretion effectors CTL0063, CTL0064, and CTL0065. In all four cases, stable mutants were recovered one passage after the observation of transformants, and allelic exchange was limited to the specific target gene, as confirmed by whole-genome sequencing. Deleted sequences were not detected by quantitative real-time PCR (qPCR) from isogenic mutant populations. We demonstrate that utilization of the chlamydial suicide vector with FRAEM renders C. trachomatis highly amenable to versatile and efficient genetic manipulation

Analysis of Farm Vehicle Crashes and Safety Belt Usage

The use of safety belts and child safety seats has been shown to be an effective means of reducing injuries to motor vehicle occupants involved in traffic crashes. There have been various methods used in efforts to increase safety belt and safety seat usage. Past efforts have included public information campaigns, local followed by statewide legislation, and enforcement of the legislation. The objectives of the study were to collect data and review related Kentucky data to determine the following information: usage rate specifically for occupants of farm vehicles, the number of registered farm vehicles and their percentage of all registered vehicles, the number of crashes in which a farm vehicle was involved and their percentage of all crashes, and the number of injuries and fatalities for occupants of farm vehicles and their percentage of all injuries and fatalities

2015 Safety Belt Usage Survey in Kentucky

The objective of the survey summarized in this report was to establish a statewide safety belt usage rate in Kentucky for 2015. This rate can be compared to those determined from previous surveys. The 2015 statewide survey documents the continued increase in usage associated with the change in the law to allow primary enforcement and related education and enforcement

2016 Safety Belt Usage Survey in Kentucky

The use of safety belts and child safety seats is a proven means of reducing injuries to motor vehicle occupants involved in traffic crashes. There have been various methods used in efforts to increase safety belt and safety seat usage. Past efforts have included public information campaigns, local and statewide legislation, and enforcement of the legislation. The objective of the survey summarized in this report was to establish a statewide safety belt usage rate in Kentucky for 2016. This rate can be compared to those determined from previous surveys. The 2016 statewide survey documents the continued increase in usage associated with the change in the law to allow primary enforcement and related education and enforcement

Analysis of Traffic Crash Data in Kentucky (2010-2014)

This report documents an analysis of traffic crash data in Kentucky for the years of 2010 through 2014. A primary objective of this study was to determine average crash statistics for Kentucky highways. Rates were calculated for various types of highways and for counties and cities. Difference criteria were used for exposure

Analysis of Traffic Crash Data in Kentucky (2010-2014)

This report documents an analysis of traffic crash data in Kentucky for the years of 2010 through 2014. A primary objective of this study was to determine average crash statistics for Kentucky highways. Rates were calculated for various types of highways and for counties and cities. Difference criteria were used for exposure

Fluorescence-Reported Allelic Exchange Mutagenesis Reveals a Role for \u3cem\u3eChlamydia trachomatis\u3c/em\u3e TmeA in Invasion That Is Independent of Host AHNAK

Development of approaches to genetically manipulate Chlamydia is fostering important advances in understanding pathogenesis. Fluorescence-reported allelic exchange mutagenesis (FRAEM) now enables the complete deletion of specific genes in C. trachomatis L2. We have leveraged this technology to delete the coding sequences for a known type III effector. The evidence provided here indicates that CT694/CTL0063 is a virulence protein involved in chlamydial invasion. Based on our findings, we designate the gene product corresponding to ct694-ctl0063 translocated membrane-associated effector A (TmeA). Deletion of tmeA did not impact development of intracellular chlamydiae. However, the absence of TmeA manifested as a decrease in infectivity in both tissue culture and murine infection models. The in vitro defect was reflected by impaired invasion of host cells. TmeA binds human AHNAK, and we demonstrate here that AHNAK is transiently recruited by invading chlamydiae. TmeA, however, is not required for endogenous AHNAK recruitment. TmeA also impairs AHNAK-dependent actin bundling activity. This TmeA-mediated effect likely does not explain impaired invasion displayed by the tmeA strain of Chlamydia, since AHNAK-deficient cells revealed no invasion phenotype. Overall, our data indicate the efficacy of FRAEM and reveal a role of TmeA during chlamydial invasion that manifests independently of effects on AHNAK

Analysis of Traffic Crash Data in Kentucky (2009-2013)

This report documents an analysis of traffic crash data in Kentucky for the years of 2009 through 2013. A primary objective of this study was to determine average crash statistics for Kentucky highways. Rates were calculated for various types of highways and for counties and cities. Difference criteria were used for exposure. Average and critical numbers and rates of crashes were calculated for various types of high ways in rural and urban areas. These rates used crashes identified on highways where traffic volumes were available. Improved methods of identifying crash locations have resulted in higher rates for the last couple of years. The crash rate data can be used in Kentucky’s procedure to identify locations that have abnormal rates or numbers of crashes. The other primary objective of this study was to provide data that can be used in the preparation of the problem identification portion of Kentucky’s Annual Highway Safety Plan. County and city crash statistics were analyzed. A summary of results and recommendations in several problem identification areas is presented. These general areas include; alcohol involvement, occupant protection, speed, teenage drivers, pedestrians, bicycles, motorcycles, trucks, and vehicle defects. Other areas included in the analysis for which specific recommendations were not made include, school bus crashes and train crashes. The crash data are contained in the Collision Report Analysis for Safer Highways (CRASH) data base. This data base is updated daily so the number of crashes in a given calendar year will continue to change for a substantial time after the end of that year