Mycobacterial Genetic Technologies for Probing the Host-Pathogen Microenvironment

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the oldest and most successful pathogens in the world. Diverse selective pressures encountered within host cells have directed the evolution of unique phenotypic traits, resulting in the remarkable evolutionary success of this largely obligate pathogen. Despite centuries of study, the genetic repertoire utilized by Mtb to drive virulence and host immune evasion remains to be fully understood. Various genetic approaches have been and continue to be developed to tackle the challenges of functional gene annotation and validation in an intractable organism such as Mtb. In vitro and ex vivo systems remain the primary approaches to generate and confirm hypotheses that drive a general understanding of mycobacteria biology. However, it remains of great importance to characterize genetic requirements for successful infection within a host system as in vitro and ex vivo studies fail to fully replicate the complex microenvironment experienced by Mtb. In this review, we evaluate the employment of the mycobacterial genetic toolkit to probe the host-pathogen interface by surveying the current state of mycobacterial genetic studies within host systems, with a major focus on the murine model. Specifically, we discuss the different ways that these tools have been utilized to examine various aspects of infection, including bacterial survival/virulence, bacterial evasion of host immunity, and development of novel antibacterial/vaccine strategies.

Keywords: host-pathogen interactions; infection microenvironment; mycobacterial genetics; tuberculosis.

FIG 1

Gene editing techniques currently utilized in the genetic manipulation of Mtb. Researchers use various techniques to assess the contribution of specific Mtb genes to virulence and survival. Highlighted here are the broad categories of technologies contained within the mycobacterial toolbox used to individually target single genes, simultaneously target multiple genes or collectively assess genetic mutant pools targeted on a genome-wide scale. (A) The majority of the technologies available for targeting Mtb genes enable the creation of single genetic mutants (gene knockout) or the modulation of single gene expression (gene knockdown) by both repression and regulated induction. Due to the intractable nature of Mtb, efforts continue in technology development within the listed categories. These include endeavors to broaden recombineering substrate leading to the development of tools such as oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting (ORBIT), as well as the assessment of the utility of various CRISPR-Cas systems for use in Mtb. (B) While many of the tools developed for single gene targeting can be harnessed to sequentially target multiple genes, these processes remain laborious and time-consuming. Fewer tools have been developed that can be used to simultaneously target multiple genes, and the available ones are based on various CRISPR-Cas systems for gene knockout or CRISPR interference for gene knockdown. (C) Unbiased genome-wide approaches that allow the analysis of many mutants within a single experiment are valuable but currently limited in developed technologies. Transposon mutagenesis has seen the most use, particularly within animal models, but CRISPR interference systems are being developed that have and will continue to further expand mycobacterial genetic analyses.