Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), is transmitted through aerosols and successfully replicates in the human lung. Before the SARS-CoV-2 pandemic, Mtb was the leading cause of death worldwide from a single infectious agent. In 2021, 11 million people were infected with Mtb resulting in 1.3 million deaths. The WHO estimates that globally 3.3% of new cases and 18% of previously treated cases were multidrug resistant TB in 2021. A general goal in our lab is to understand essential biological systems of Mtb so that we can develop new ways to block these essential systems and treat Mtb infections.

Iron is an essential nutrient for all living organisms because iron is used for DNA replication, energy production and numerous other essential biological processes. Therefore, to successfully colonize the human host and cause disease Mtb is completely dependent on acquiring iron from within the human host. In the human body, >80% of the iron is stored in a molecule called heme and heme is then stored in the form of hemoglobin, which is a major component in our blood. This type of iron complexing is purposely done by the human immune system to prevent pathogens from acquiring the essential iron nutrient. Not surprisingly, most bacterial pathogens have developed elegant ways to acquire iron from heme, which is a rich source of iron. A specific goal in the Mitra Lab is to identify Mtb proteins that are used to capture host heme and to understand how these Mtb heme capturing proteins function.

The Mitra Lab has identified several potential candidate genes that could be required for Mtb heme acquisition. An example of such a gene is eccC4. A previous experiment showed the growth of the parent strain and the eccC4 mutant strain. The two strains are grown in the presence of two different iron sources, ferric citrate (FeCi) and heme (Hm). FeCi is a non-heme iron source and serves as a control condition. When eccC4 is grown in heme, the growth is reduced compared to the parent strain grown in heme. The growth experiment showed that deleting the gene eccC4 reduces growth of Mtb only when heme is the iron source.

My goal of the project was to assess a heme biosensor function and construct gene deletions. To construct gene deletions, we employed molecular biology techniques such as PCR, cloning, ligation, transformation, and homologous recombination. To assess effect of gene deletions, I performed several growth experiments. I measured both the optical density of the bacteria and the fluorescence. For example, based on previous observations we can reason that the slow growth of the eccC4 mutant in heme iron could be for two reasons: cells are not taking up enough heme into the cell and the cells are dying from a lack of iron nutrient or heme is accumulating within cells because not enough heme is being expelled out of the cell and they are dying from heme overload toxicity (because excess heme is poisonous to all organisms). To test these hypotheses, we used a molecular heme sensor which produces green fluorescing light (GFL) in response to heme. The level of GFL produced by the sensor is inversely proportional to the levels of heme. Thus, the level of GFL will be low in the presence of heme and high in the absence of heme. First, we produced this biosensor protein in both parent and mutant cells. Then, we grew the parent and mutant strains in medium with heme, measure cell density and GFL level in the strains. Based on our hypotheses, if the eccC4 mutant cells have lower levels of heme than the parent cells, then the GFL level will be higher in the eccC4 mutant compared to the parent strain and vice versa.

Once we had performed the experiments, we analyzed the results by graphing the fluorescence levels of the ppe64 mutant and eccC4 mutant against our wild type in both ferric citrate and heme. Both mutant cells had more fluorescence within the heme, suggesting that heme levels were lower than in the mutant. From this, we can assume that the deletion of eccC4 and ppe64 reduces the heme uptake in Mtb. The conclusions of my research are that an intracellular heme biosensor can be used in Mtb to compare the cytoplasmic heme levels and that the PPE64 and ESX-4 secretion system are involved in the uptake of heme in Mtb. Future experiments should repeat the same tests to check for validity with more replicates and more timepoints in the growth curves and analyze other Mtb mutants.