UC San Diego

We are in a constant battle against pathogens. Millions of individuals die every year due to inadequate diagnosis and emerging resistance to current therapeutics. Improvements in diagnosis and treatment strategies are needed, but these can be hindered by a lack of knowledge regarding a particular pathogen’s molecular and resistance mechanisms. Plasmodium and Leptospira are two such examples of widespread pathogenic organisms whose genomes are still poorly understood and additional knowledge is desperately needed to improve therapeutic strategies. This dissertation presents two evolution-based strategies that when coupled with whole genome sequencing can be used to identify virulence and resistance associated genes. These include a “forward” approach, which studies the development of resistance, and a “reverse” approach, which examines the loss of virulence.

In Chapter 2, the forward approach is used to examine novel targetable pathways in Plasmodium falciparum. Selectively evolving resistance to 50 novel antimalarial compounds, we successfully identify potential targets to 21 compounds and eight novel gene targets. Additionally, this chapter examines resistance development patterns against the compound set, and identifies fast-killing compounds may result in a slower onset of clinical resistance.

Chapter 3 focuses on PfCARL, one potential target identified in Chapter 2, which has been previously described as the target for KAF156, a drug currently in clinical trials. Our data demonstrate that pfcarl mutations confer resistance to two distinct compound classes – benzimidazolyl piperidines and imidazolopiperazines. However, these two classes appear to have different timing of action in the asexual blood stage and different potencies against the liver and sexual blood stages, suggesting pfcarl is a multidrug resistance gene rather than a common target.

Finally, using the reverse approach, Chapter 4 identifies virulence-related genes in Leptospira by observing cumulative genomic changes occurring after serial in vitro passaging of a highly virulent Leptospira interrogans strain into a nearly avirulent isogenic derivative. Comparison between these two polyclonal strains identifies 15 non-synonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased. These frequency changes likely contribute to the loss of virulence, and suggest new virulence-associated genes whose role in Leptospira pathogenesis should be further studied.