Hemerythrin-like protein Rv2633c is a small 18 kDa protein that is expressed in Mycobacterium tuberculosis (Mtb). Sequence analysis of Rv2633c predicts the presence of a hemerythrin-like domain, which binds dioxygen using a µ-oxo-bridge (Fe-O-Fe), rather than a heme group. Though it is noticeably upregulated during macrophage infection and during in vitro acidification, the role of Rv2633c in Mtb survival has yet to be elucidated. This project aims to characterize the function of Rv2633c by studying the in vitro response of the recombinant protein to conditions present in the macrophage lysosome, such as reduced oxygen levels or the presence of reactive oxygen species. UV-visible spectroscopy is used to observe these changes, as the spectrum shows a characteristic peak at 330 nm that likely corresponds to the diiron cofactor in its native state. Our results show this spectrum shifts in response to hydrogen peroxide addition, showing the proposed environmental conditions can affect the active site. Bioinformatics techniques, such as the 3D modeling program SWISS-MODEL, have been used to hypothesize possible structure and function. Determining the function of Rv2633c may help explain how Mtb so readily evades the human immune system to reside in the macrophage.

According to the Centers for Disease Control and Prevention (CDC), 8 million people each year are infected with Mycobacterium tuberculosis (Mtb) leading to 1.5 million deaths annually. This staggering number calls for advancements in understanding this bacterium so progress can be made in treating and preventing the disease. It is particularly important to understand mechanisms by which TB survives inside hostile host immune cells known as macrophages and within hypoxic granuloma lesions of the lung. Preliminary microarray data has shown that a TB gene known as Rv2633c is induced upon macrophage invasion. Bioinformatic analysis of Rv2633c coding sequence shows the product of Rv2633c has homology with hemerythrin-like proteins. Hemerythrins are a class of proteins commonly used to bind oxygen and sense nitric oxide and iron, leading us to hypothesize a role for Rv2633c in surviving hypoxic or nitrosative stress encountered within macrophages and granulomas. My first aim will be to generate a reporter strain of Mycobacterium smegmatis (Msm) expressing the mCherry fluorescent protein driven by the Rv2633c promoter. This tool will allow us to determine the stress conditions (i.e. hypoxia, nitric oxide treatment, acid pH) that activate expression of this gene by measuring the change in fluorescence. Linking the regulation of Rv2633c to specific environmental cues relevant to infections in vivo will provide insight into the role of this unique protein. Secondly, a knockout mutant of Rv2633c in the attenuated M. bovis BCG will be constructed and characterized to determine the importance and function of this protein during TB infections.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a debilitating disease that affects the health of millions annually. Understanding its ability to persist within host and resist eradication by antibiotics is of utmost importance in the effort to develop new interventions. This study will focus on the transcriptional activator WhiB7 and its regulation of the multidrug Tap efflux pump encoded by Rv1258c. WhiB7 is thought to respond to redox stress induced by antibiotics and a variety of in vivo stresses by activating multiple genes including Rv1258c. Much remains to be determined regarding the role of WhiB7 and downstream genes in Mtb virulence and drug resistance. We will create a tool for studying WhiB7-mediated gene regulation by engineering a strain of the nonpathogenic bacterium Msm expressing the mCherry fluorescent protein controlled by the Rv1258c promoter. Knocking out the native WhiB7 gene in Msm via homologous recombination will allow clear introduction of wild type and mutant versions of Mtb WhiB7. Changes in the fluorescent activity of Rv1258c promoter fusion to mCherry will indicate the effects of WhiB7 mutagenesis. Secondly, we can also use this system to confirm additional genes identified by microarray analysis that are potentially regulated by WhiB7. This will be done by cloning other promoters in front of mCherry in the Msm strain containing wild-type Mtb WhiB7. Understanding WhiB7’s role in Mycobacterium tuberculosis macrophage survival and antibiotic resistance may provide new strategies for developing drugs that can lead to a cure.

The genus Mycobacterium contains many pathogenic bacteria that are known to cause serious diseases in humans. One of the most well-known of these bacteria is Mycobacterium tuberculosis, or Mtb, which is the causative agent of tuberculosis. It infects nearly one-third of the world’s population and kills 1.4 million people annually. Another important mycobacterial pathogen is Mycobacterium abscessus, or Mabs, which causes respiratory infections in cystic fibrosis patients. One of the biggest difficulties in combating these pathogens is the lack of effective diagnostics, as current strategies hold many pitfalls and can be unreliable. One common method used is sputum smear microscopy which involves acid fast staining of the bacteria present in a patient’s sputum. This method of detection fails to detect more than 50% of infections and is unable to differentiate between species of mycobacterium. This project introduces a novel method of mycobacterial diagnostics using binary deoxyribozymes (DNAzymes). Binary DNAzymes recognize bacteria-specific nucleic acid sequences and bind to them, forming a catalytic core which cleaves a substrate molecule. This cleavage separates a quencher molecule from a fluorophore, which results in a fluorescent output. This flexible assay platform has great potential for the detection of Mtb or Mabs. Our data shows the specificity of the DNAzymes allowing for a differential diagnosis of various species of Mycobacteria. It also shows the limit of detection of this technology and its additional utility in molecular typing of Mtb clinical isolates as well as drug resistance characterization. This multipurpose tool can contribute to disease management in multiple ways.

One common limitation of enzymatic reactions is the diffusion of a substrate to the enzyme active site and/or the release of the reaction products. These reactions are known as diffusion –controlled. Overcoming this limitation may enable faster catalytic rates, which in the case of catalytic biosensors can potentially lower limits of detection of specific analyte. Here we created an artificial system to enable deoxyribozyme (Dz) 10-23 based biosensor to overcome its diffusion limit. The sensor consists of the two probe strands, which bind to the analyzed nucleic acid by Watson-Crick base pairs and, upon binding re-form the catalytic core of Dz 10-23. The activated Dz 10-23 cleaves the fluorophore and quencher-labeled DNA-RNA substrate which separates the fluorophore from the quencher thus producing high fluorescent signal. This system uses a Dz 10-23 biosensor strand associated to a DNA antenna tile, which captures the fluorogenic substrate and channels it to the reaction center where the Dz 10-23 cleaves the substrate. DNA antenna tile captures fluorogenic substrate and delivers it to the activated Dz 10-23 core. This allows for lower levels of analyte to be detected without compromising the specificity of the biosensor. The results of this experiment demonstrated that using DNA antenna, we can create a synthetic environment around the Dz 10-23 biosensor to increase its efficiency and allow for lower levels of analyte to be detected without using amplification techniques like PCR.

No description available.

No description available.

No description available.

No description available.

No description available.