Browsing by Author "Smith, Clare M"

Tuberculosis (TB) is considered the most lethal infectious disease throughout human history. Mycobacterium tuberculosis (Mtb), the causal agent of tuberculosis, has consistently infected human hosts for millennia, and today, approximately 10 million TB cases and over 1 million deaths are caused by Mtb annually. Infection with Mtb, most often in the lungs via airborne transmission, can produce a spectrum of disease outcomes. An unknown proportion of resistant hosts efficiently contain and sterilize Mtb infection. If a host cannot clear the bacterium, Mtb can lie dormant over the course of decades. From this latent state, there is a 5-15% risk that the case will progress to active disease, characterized by coughing, fever, and cachexic wasting, which can be fatal if left untreated. Although TB is treatable with courses of antibiotics that can range from 3 to 9 months in duration, the emergence of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains increases the urgency of understanding the genetic factors within the host and in the pathogen that give rise to these variable disease outcomes.

To explore the contributions of host and bacterial genetic variation to clinically divergent TB outcomes, we combined mammalian models of natural genetic diversity with next-generation models of engineered mycobacterial diversity. Together, these systems allowed quantitative trait locus (QTL) mapping of the host genome for phenotype-to-genotype associations underlying bacterial fitness and host disease phenotypes following Mtb infection. For over a century, mice have served as tractable mammalian models for the interrogation of Mtb pathogenesis. The studies presented in this dissertation utilize modern genetic reference populations to maximize host genetic diversity for identification of TB QTL and classical mouse breeding schemata to isolate these regions of interest for functional interrogation.

In Chapter 1, we review the history of QTL mapping in Mtb-infected mice. We discuss murine models of natural genetic diversity, QTL mapping as a statistical approach, and vignettes of QTL mapping in Mtb-infected mice. The chapter concludes by contextualizing the work presented in this dissertation as it relates to previous in vivo mapping studies.

In Chapter 2, we report an infection screen of recombinant inbred BXD strains alongside the BXD panel parental strains, Mtb-resistant C57BL/6J (B6) and Mtb-susceptible DBA/2J (D2) with a comprehensive Mtb mutant library (TnSeq). We identified 140 transposon mutant fitness QTL across the host genome as well as a cluster of 4 highly significant QTL on mouse chromosome 6. The work reported in this chapter reveals early bacterial predictors of host divergence in TB susceptibility.

In Chapter 3, we report the investigation of TB susceptibility QTL on chromosomes 7, 15, and 16 (named Tip1-Tip4), which were identified between CC001 and CC042 of the octoparental Collaborative Cross (CC) recombinant inbred panel. Tip2 was caused by a private deletion in CC042, but the genetic causes of Tip1, Tip3, and Tip4 remain unknown. We report bioinformatic and classical congenic approaches that serve to narrow these QTL causal intervals and identify putative candidate genes underlying these TB susceptibility loci.

In Chapter 4, we screened multiple CC strains that exhibited similar lung Mtb burden but divergent TB outcomes to identify markers of disease resilience, independent of Mtb burden. Despite our observation that CC030 mice are highly Mtb-susceptible with high lung burden and inflammation, the CC030 haplotype within a QTL on chromosome 7 (Tip8) is associated with low lung burden and CXCL1. We generated congenic mice with CC030 Tip8 on an isogenic background to identify biomarkers of TB disease resilience in a burden-controlled context, revealing restriction of lung IL-1β and CCL3 a pathway to TB resilience.

In Chapter 5, we explore a region on mouse chromosome 15 in which genomic inheritance from wild-derived CAST/EiJ mice was associated with TB resistance QTL (Tip3, Tip7, Tip10) in several independent infection screens. We report a ~0.4Mb region within the chromosome 15 resistance locus exhibiting non-synteny between all eight of the CC founders, suggesting evolutionary selection at this resistance locus. We further leverage novel RNA-Seq-based CC founder genome annotations to identify novel strain-specific genes within this locus that were previously obscured in reference genome alignment approaches, highlighting mouse apolipoprotein L (Apol) genes as putative promoters of host TB resistance.

In Chapter 6, we explore a potent TB susceptibility locus observed on chromosome 2 (Tip5) in our previous infection screen of 52 CC lines and the eight founder lines. Within this locus, cathepsin Z (Ctsz; also, Ctsx), which has been associated with TB susceptibility in multiple independent TB patient cohorts, was identified as the lead candidate that may underlie Tip5. We report in vivo and ex vivo infection studies that highlight a protective role for Ctsz during Mtb infection for the first time, offering mechanistic insights that may assist in the development of therapeutics for TB patients harboring deleterious SNPs in CTSZ.

In Chapter 7, we summarize the work reported in this dissertation and discuss remaining questions and future directions for each chapter. We conclude the chapter with perspectives on the advances in high-throughput phenotyping, pangenomics, mouse reference populations, and bioinformatic tools and analyses that could support future work in defining the host-pathogen interactions during Mtb infection.

Collectively, the body of work presented in this dissertation represents a multipronged approach to uncovering the host and microbial genetic factors that promote unique TB disease outcomes. To date, the spread of TB remains a public health crisis, profoundly impacting communities across the globe with reduced healthcare accessibility. This work will assist in the development of therapeutics for the eradication of a pathogen that has impacted an estimated one-fourth of the global population.