Dielectrophoresis-based purification of antibiotic-treated bacterial subpopulations

Persistence of bacteria during antibiotic therapy is a widespread phenomenon, of particular importance in refractory mycobacterial infections such as leprosy and tuberculosis. Persistence is characterized by the phenotypic tolerance of a subpopulation of bacterial cells to antibiotics. Characterization of these "persister" cells is often difficult due to the transient, non-heritable nature of the phenotype and due to the presence of contaminating material from non-persisting cells, which usually comprise the larger fraction. In this study, we use 3D carbon-electrode arrays for dielectrophoresis-based separation of intact cells from damaged cells, revealed by differential staining with propidium iodide, and we use this procedure to purify intact cells from cultures of Mycobacterium smegmatis treated with isoniazid, a frontline anti-tuberculosis drug. The method presented in this study could be used for rapid label-free enrichment of intact persister cells from antibiotic-treated cultures while preserving the metastable persister phenotype. This approach would facilitate the downstream analysis of low-frequency subpopulations of cells using conventional omics techniques, such as transcriptomic and proteomic analysis.

Single-Cell Analysis of Mycobacterial Persistence Using Microfabricated Tools

Meltem Elitas

Bacterial cells behave as individuals despite being genetically identical and subject to the same environment. Although the underlying mechanisms of cellular individuality are not well understood, temporal variation of gene expression and protein levels in individual cells could act as sources for phenotypic heterogeneity of populations. Bacterial persistence in fluctuating environments relies on such diversity, allowing some individuals, by chance, to survive a challenge that would kill an unadapted and phenotypically homogeneous population. Bacterial persistence is a non-genetic, metastable phenomenon that is readily lost upon subculture of surviving cells in the absence of the environmental stress. A medically relevant example of persistence is the fractional killing of bacteria by antibiotics, whereby a subpopulation of cells survives indefinitely despite prolonged exposure to a bactericidal drug. It is hypothesized in the case of tuberculosis (TB) that drug therapy may require administration of multiple drugs for 6-9 months in order to eliminate a subpopulation of "persister" cells. The focus of this thesis is to understand how persisters tolerate antibiotics that kill their genetically identical siblings. To address this question, automated time-lapse fluorescence microcopy was applied in conjunction with microfluidic and microelectromechanical systems (MEMS) to study bacterial responses to antibiotics in real time at the single-cell level. A genetic approach was employed to identify mutants of Mycobacterium smegmatis with altered rates of persistence against the first-line anti-TB drug isoniazid (INH). A microfluidic platform was designed for high-throughput screening of transposon (Tn) mutant libraries. A pilot screen of 1,100 Tn mutants revealed 11 mutants that showed increased (persister-up, PU) or decreased (persister-down, PD) survival when exposed to isoniazid. These mutants were further characterized in comparison to wild-type cells using the single-cell microfluidics platform. This single-cell analysis revealed distinct modes of persistence within each category, which were otherwise masked at the population level. The results indicate that persistence behavior was the cumulative result of the dynamics of cell division and cell lysis. Mutants with decreased persistence exhibited behavior at single-cell level that challenges the accepted mechanism of isoniazid-mediated killing. Therefore, these results yield new insights into antibiotic persistence and isoniazid's mode of action. Persisters cannot be easily studied in non-fractionated populations of cells where they represent a small percentage of the total. As a complementary approach, a method was developed based on dielectrophoretic (DEP) purification of naturally occurred low frequency persister cells from an antibiotic-treated bacterial culture. Purification of persister cells would allow for downstream analysis using conventional bulk/population level transcriptomic and proteomic approaches. The crossover frequencies for live and dead cells were determined to enable selective trapping or release from the device. This approach enabled a proof-of-concept enrichment of live bacteria in the mixture of live and dead cells after INH treatment, based on their dielectric properties. Quantitative confirmation of the enriched/isolated cell populations was attempted using flow cytometry. The high-throughput microfluidic screening and single-cell approaches developed in this thesis could, in principal, be applied to the genetic analysis of a wide variety of dynamic cellular processes that can be imaged by time- lapse fluorescence microscopy, such as gene expression, cell division, chromosome replication and segregation, and protein localization. The biochemical analysis of live cells isolated by dielectrophoresis-based separation would provide useful information about the underlying persistence mechanism. Dielectrophoresis could also be a valuable analytical tool for mutant characterization based on their intrinsic dielectric properties, either at single-cell or population levels. In conclusion, this thesis presents several new microfabricated tools to identify and understand bacterial persistence. The thesis illustrates how integration of microfluidic tools with automated time-lapse microscopy allows hidden characteristics that are present but invisible in the macro- world of batch cultures to be examined in a quantitative and high-throughput manner in the micro-world of single-cell analysis.

EPFL2012

Separation of antibiotic-treated mycobacterial subpopulations using multiple-frequency dielectrophoresis

Thomas Michael Braschler, John McKinney, Philippe Renaud, Ana Valero

Exposure of a bacterial culture to an antibiotic or other lethal stress typically results in the generation of sub-populations of live cells ("persisters") and dead cells, and, in some cases, viable but non-culturable cells. Studies aimed at understanding the mechanisms of antibiotic killing and bacterial persistence will require the purification and analysis of each of these sub-populations in isolation. This paper reports a method based on continuous-flow dielectrophoresis to separate live and dead cells from antibiotic-treated bacterial cultures. Our experiments were carried out on Mycobacterium smegmatis, a model organism that is closely related to the etiological agent of human tuberculosis.

2009

Single-cell studies of Mycobacterium smegmatis cell cycle using time-lapse fluorescence microscopy

Joëlle Xiao Yuan Ven

Cell division is one of the most primordial cellular processes and is essential for life propagation. It is composed of crucial events, namely genome duplication, chromosomes segregation and membrane separation. Failure of these processes will have dramatic impacts on the development of any organisms, including cell death, polyploidy, or cell cycle arrest. In the case of bacterial pathogens, blocking cell division efficiently using targeted drugs is of interest to cure bacterial infections. However, despite its ancestral origin, the mechanisms of cell division have diverged amongst organisms. Given its crucial role, as well as the potential therapeutic applications, cell division is an intensive field of research and many aspects are yet to be fully understood. Amongst bacteria, the phyla Proteobacteria and Firmicutes have been intensively studied using their respective model organisms Escherichia coli and Bacillus subtilis. However, there is a paucity of research on cell division in Actinobacteria, the phylum that contains the worldâs leading pathogen, Mycobacterium tuberculosis. In particular, the molecular players involved in the process of chromosome segregation and the timing of cell division have not been fully investigated. In the well-studied bacteria E. coli and B. subtilis the Min system determines the division site localization, while the nucleoid occlusion system (Noc or SlmA, respectively) determines the timing of division. In mycobacteria, however, no homologs for the Min or Noc systems have been identified thus far. However, the partitioning (par) genes are currently thought to be responsible for symmetric chromosome segregation. In addition, FtsK has been identified as a homolog of the B. subtilis protein SpoIIIE, which is responsible for rescuing DNA in the forming septum and pulling it in the forespore during starvation-induced sporulation. Overall, the role and dynamics of DNA, where FtsK might play a role, during mycobacterial cell division, still remain to be uncovered. Current methods to study DNA localization using snapshot microscopy typically utilise DNA dyes, such as DAPI, Hoechst, or SYTO. However, these compounds impair cell growth, preventing the dynamic study of mycobacterial division. To circumvent this shortcoming, I constructed a Mycobacterium smegmatis reporter strain expressing a Dendra2 fusion protein in-frame to the histone-like protein Hlp, and compared it to conventional DNA dyes. Additionally, I characterized FtsK and, for the first time, demonstrated its essentiality in M. smegmatis. As FtsK is essential I adapted a dual repression system to knock down FtsK, which proved to be a synthetic rescue with the par genes deletion. Using a custom-made microfluidic device and long-term time-lapse microscopy, the distribution and dynamics of both Hlp and FtsK fluorescent reporter proteins were followed at the single-cell level. This approach allowed the spatial and temporal characterization of these proteins with respect to other septal genes in wild-type cells as well as in Îpar mutants. Finally, I propose a new model where the par genes are important for chromosome orientation and FtsK is important for chromosome segregation. Altogether, this thesis represents a step forward towards a better understanding of the mechanisms underlying cell division in mycobacteria and in the long-term, a potential source of novel treatments to control the division of M. tuberculosis, for instance by targeting FtsK.