Lottchristensen1414

With increasing prevalence of antimicrobial resistance, a fundamental goal of antibiotic discovery is to uncover new small molecules that prevent growth of pathogenic bacteria through diverse mechanisms of action. This goal is particularly pertinent for tuberculosis, caused by Mycobacterium tuberculosis. In this chapter, we describe the application of a chemical-genetic method, PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), for sensitively detecting small molecule bioactivity using a pooled panel of hypomorphs (strains depleted in a particular essential gene) of M. tuberculosis. We describe statistical and heuristic approaches to assign small molecule mechanism of action from the resulting chemical-genetic interaction profiles.Phage recombination systems have been instrumental in the development of gene modification technologies for bacterial pathogens. In particular, the Che9 phage RecET system has been used successfully for over 10 years for making gene knockouts and fusions in Mycobacterium tuberculosis. This "recombineering" technology typically uses linear dsDNA substrates that contain a drug-resistance marker flanked by (up to) 500 base pairs of DNA homologous to the target site. Less often employed in mycobacterial recombineering is the use of oligonucleotides, which require only the action of the RecT annealase to align oligos to ssDNA regions of the replication fork, for subsequent incorporation into the chromosome. Despite the higher frequency of such events relative to dsDNA-promoted recombineering, oligo-mediated changes generally suffer from the disadvantage of not being selectable, thus making them harder to isolate. This chapter discusses steps and methodologies that increase the frequencies of finding oligo-mediated events, including the transfer of single nucleotide polymorphisms (SNPs) to mycobacterial chromosomes, and the use of oligos in conjunction with the mycobacterial phage Bxb1 site-specific recombination system for the easy generation of knockouts, insertion, and fusions, in a protocol known as ORBIT.The identification of essential genes is of major importance to mycobacterial research, and a number of essential genes have been identified in mycobacteria, however confirming essentiality is not straightforward, as deletion of essential genes results in a lethal phenotype. In this chapter, protocols are described which can be used to confirm gene essentiality using gene switching, following the construction of a strain carrying its only functional copy on an integrated plasmid (Δ'int). Since deletion mutants cannot be created for essential genes, a second gene copy is introduced via an integrating vector, which allows the chromosomal gene copy to be deleted. The integrated vector can then be replaced using the gene switching method, where no transformants are obtained, essentiality is confirmed. This technique can also be used to confirm functionality of gene homologs and to easily identify essential operon members.The introduction of DNA into bacterial cells is one of the foundational methods of bacterial genetics. Transformation of mycobacterial species is complicated due to the structure of the cell wall, which has a complex outer layer with low permeability. Electroporation has become a routine procedure in genetic studies. ALK assay In this process, cells are subjected to a brief high-voltage electrical impulse which allows the entry of DNA. It can be used to introduce plasmid DNA, phage DNA, or oligonucleotides. This chapter presents methods for introducing DNA into a representative slow-growing species, M. tuberculosis, and a representative fast-growing species, M. smegmatis. Other mycobacteria can be transformed using variations of these methods, although the efficiency of transformation will vary.Flow cytometry enables the measurement of tens of features on individual cells from complex mixtures. Flow cytometry enables high-throughput quantification of cell size, gene and protein expression. In the case of studies of host-pathogen interactions, this tool provides a facile way of identifying cells that have been successfully infected by a pathogen. Several recent technological advances have greatly improved throughput and the number of features that can be simultaneously monitored by this technique. Here, we describe common workflows to study Mycobacterium tuberculosis heterogeneity and host-M. tuberculosis interactions using flow cytometry and related technologies.Non-replicating persistence (NRP) is a functional adaptation that mycobacteria undergo in response to the stresses of the granuloma, facilitating antibiotic tolerance and long-term infection. These stresses, or NRP-inducing factors, include hypoxia, nutrient deprivation, and nitric oxide assault, which mycobacteria are well evolved to tolerate through a series of metabolic and physiological adaptations producing the NRP state. Most attempts to replicate these conditions in vitro have focused on only one of these factors at a time for ease and simplicity, but as a result, do not necessarily produce physiologically relevant phenotypes. Here, we provide the methods for two different in vitro NRP strategies that are useful for drug susceptibility testing and high-throughput screening.Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux.Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe a method that allows (1) the measuring of permeability and the quantification of general efflux activity of mycobacteria, by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (2) the screening of compounds in search of agents that increase the permeability of the cell wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.Studies on cell-to-cell phenotypic variation in microbial populations, with individuals sharing the same genetic background, provide insights not only on bacterial behavior but also on the adaptive spectrum of the population. Phenotypic variation is an innate property of microbial populations, and this can be further amplified under stressful conditions, providing a fitness advantage. Furthermore, phenotypic variation may also precede a latter step of genetic-based diversification, resulting in the transmission of the most beneficial phenotype to the progeny. While population-wide studies provide a measure of the collective average behavior, single-cell studies, which have expanded over the last decade, delve into the behavior of smaller subpopulations that would otherwise remain concealed. In this chapter, we describe approaches to carry out spatiotemporal analysis of individual mycobacterial cells using time-lapse microscopy. Our method encompasses the fabrication of a microfluidic device; the assembly of a microfluidic system suitable for long-term imaging of mycobacteria; and the quantitative analysis of single-cell behavior under varying growth conditions. Phenotypic variation is conceivably associated to the resilience and endurance of mycobacterial cells. Therefore, shedding light on the dynamics of this phenomenon, on the transience or stability of the given phenotype, on its molecular bases and its functional consequences, offers new scope for intervention.The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three microscopy methods that allow the precise identification and quantification of very diverse phenotypes arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was established to study the infection dynamics with high-throughput imaging of a large number of cells at the different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning disc confocal microscope.Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages, HoxB8 conditionally immortalized myeloid cells, Max Planck Institute alveolar macrophage-like cells, and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging.Zinc starvation in Mycobacterium smegmatis and Mycobacterium tuberculosis induces ribosome remodeling and hibernation. Remodeling involves replacement of C+ ribosomal (r-) proteins containing the zinc-binding CXXC motif with their C- paralogues without the motif. Hibernation is characterized by binding of mycobacterial-specific protein Y (Mpy) to 70S C- ribosomes, stabilizing the ribosome in an inactive state that is also resistant to kanamycin and streptomycin. We observed that ribosome remodeling and hibernation occur at two different concentrations of cellular zinc. Here, we describe the methods to purify hibernating and active forms of C- ribosomes from zinc-starved mycobacteria, along with purification of C+ ribosomes from zinc-rich mycobacterial cells. In vitro analysis of these distinct types of ribosomes will facilitate screening of small molecule inhibitors of ribosome hibernation for improved therapeutics against mycobacterial infections.