Tuberculosis, the disease caused by Mycobacterium tuberculosis (Mtb), is the number one cause of death by an infectious agent. Mtb, an obligate pathogen, must compete with the host for nutrients, including for the essential metal, iron. The inability to acquire iron leads to a loss of virulence for Mtb. On the other hand, acquiring excess iron leads to oxidative stress that can damage the bacterium. Therefore, the pathways governing iron homeostasis must be precisely balanced for optimal fitness. Though we know many of the proteins participating in this pathway, we still do not understand several aspects, such as how iron transits the cell envelope or how other stress response pathways affect iron homeostasis. The numerous uncharacterized genes in Mtb, including those required for infection, represent potential participants in this pathway and potential drug targets. In this thesis, we describe two novel players required for iron homeostasis in Mtb. We describe a new role for intramembrane proteolytic signaling and sigma factor regulation in iron homeostasis through the finding that Rip1 and SigL are required for growth in low iron. In addition, we describe a role for the previously uncharacterized protein, Rv3193c, in iron homeostasis. We demonstrate that it is required for infection in mice and a conserved residue of the protein contributes to its function. By characterizing essential pathways such as iron homeostasis, we can better understand the requirements for Mtb growth in the host and develop new treatments to address the global burden of tuberculosis.

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes.

In 2017 over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB) occurred, emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for drug-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, an inflammatory pathway important for early immunity during M. tuberculosis infection. However, IL-1 can contribute to pathology and disease severity late in TB progression. Since IL-1 may contribute to LZD toxicity and does influence TB pathology, we targeted this pathway with a potential host-directed therapy (HDT). We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce bone marrow toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, a TB-susceptible mouse model and clinically relevant cynomolgus macaques. Antagonizing IL-1 in mice with established infection reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of bone marrow suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 4 weeks (the FDA approved regimen at the time of study), we observed sterilization of the majority of granulomas regardless of co-administration of the FDA-approved IL-1 receptor antagonist (IL-1Rn), also known as Anakinra. However, total lung inflammation was significantly reduced in macaques treated with IL-1Rn and LZD compared to LZD alone. Importantly, IL-1Rn administration did not impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB and the need for further research in this area.

In 2017, there were over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB), emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for antibiotic-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, a pathway important for early immunity during M. tuberculosis infection that later contributes to pathology. We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce BM toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, mice and cynomolgus macaques. Antagonizing IL-1 in chronically-infected mice reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of BM suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 1 month, the majority of granulomas were sterilized with reduced inflammation (assessed by PET/CT) in animals treated with both LZD and IL-1 receptor antagonist (IL-1Rn). However, overall lung inflammation was significantly reduced in macaques treated with both IL-1Rn and LZD, compared to LZD alone. Importantly, IL-1Rn administration did not noticeably impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB.

Two efficient recombination systems were combined to produce a versatile method for chromosomal engineering that obviates the need to prepare double-stranded DNA (dsDNA) recombination substrates. A synthetic "targeting oligonucleotide" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annealase. This oligonucleotide contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a "payload plasmid" that contains a cognate recombination site and a selectable marker. The targeting oligonucleotide and payload plasmid are cotransformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate gene knockouts, promoter replacements, or C-terminal tags. This new system is called ORBIT (for "oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting") and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions, or fusions in a bacterial chromosome. We demonstrate the utility of this "drag and drop" strategy by the construction of insertions or deletions in over 100 genes in Mycobacterium tuberculosis and M. smegmatis IMPORTANCE We sought to develop a system that could increase the usefulness of oligonucleotide-mediated recombineering of bacterial chromosomes by expanding the types of modifications generated by an oligonucleotide (i.e., insertions and deletions) and by making recombinant formation a selectable event. This paper describes such a system for use in M. smegmatis and M. tuberculosis By incorporating a single-stranded DNA (ssDNA) version of the phage Bxb1 attP site into the oligonucleotide and coelectroporating it with a nonreplicative plasmid that carries an attB site and a drug selection marker, we show both formation of a chromosomal attP site and integration of the plasmid in a single transformation. No target-specific dsDNA substrates are required. This system will allow investigators studying mycobacterial diseases, including tuberculosis, to easily generate multiple mutants for analysis of virulence factors, identification of new drug targets, and development of new vaccines.

Current methods for genome engineering in mycobacteria rely on relatively inefficient recombination systems that require the laborious construction of a long double-stranded DNA substrate for each desired modification. We combined two efficient recombination systems to produce a versatile method for high-throughput chromosomal engineering that obviates the need for the preparation of double-stranded DNA recombination substrates. A synthetic targeting oligonucleotide is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annelase. This oligo contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a payload plasmid that contains a cognate recombination site and selectable marker. The targeting oligo and payload plasmid are co-transformed into a RecT- and Int- expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate knockouts and promoter replacements, or to fuse the C-terminal-encoding regions of target genes with tags of various functionalities. This new system is called ORBIT (Oligo-mediated Recombineering followed by Bxb1 Integrase Targeting) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions or fusions in a target bacterium. We demonstrate the utility of ORBIT by the construction of insertions or deletions in over 100 genes in M. tuberculosis and M. smegmatis. The report describes the first genetic engineering technique for making selectable chromosomal fusions and deletions that does not require the construction of target- or modification-specific double-stranded DNA recombination substrates.