High Throughput, em tempo real, Teste Dual-leitura dos intracelular Actividade Antimicrobiana e citotoxicidade celular eucariótica

Pathogens that grow or reside temporarily in intracellular compartments are difficult to therapeutically eradicate. Obligate or relatively obligate intracellular pathogens such as Legionella pneumophila, Coxiella burnetii, Brucella spp., Francisella tularensis, and Mycobacterium spp. often require prolonged courses of antimicrobial therapy for cure that may range from months to even years. Furthermore, extracellular pathogens may transiently occupy intracellular niches and in this way escape clearance by normal courses of antimicrobial therapy and later emerge to start new rounds of virulent infection. Staphylococcus aureus¹ and uropathogenic Enterobacteriaceae^2,3 infections are two increasingly recognized examples. Therefore, a fundamental drug discovery goal is to identify novel antimicrobials that penetrate into intracellular compartments. Optimal therapy to quickly eradicate intracellular organisms and prevent development of resistance through sub-inhibitory antimicrobial exposure is especially desirable.

To this end, we developed a high-throughput screening technology to identify intracellular-penetrant antimicrobials targeting the intracellular growth of the model pathogen, Legionella pneumophila.⁴ Previous clinical observations indicate that standard antimicrobial susceptibility testing did not accurately predict in vivo therapeutic efficacy against this organism.⁵ Specifically, this was because major classes of antimicrobials such as β-lactams and aminoglycosides, although highly effective against axenically grown Legionella, do not sufficiently penetrate into the intracellular compartments where Legionella resides.^5,6 Later evidence suggested that technically more complex intracellular growth assays effectively predicted clinical efficacy.⁷ Unfortunately, these assays were extremely laborious endpoints assays, requiring infected macrophages, treated with antimicrobials, to be lysed at different times points for colony forming unit enumeration. Such assays are impractical to do on a large scale and are unsuitable for high-throughput drug discovery.

Therefore, we developed technology for real-time determination of intracellular bacterial growth.⁶ This was accomplished through use of a bacterial strain modified through integration of either a bacterial luciferase operon⁸ (first generation assay, described previously)⁴ or fluorescent protein⁹ reporters (second generation, orthogonal assay, described here) into the bacterial chromosome. In this way, luminescent or fluorescent signal provides a surrogate, real-time readout of bacterial number.

However, these attributes do not address a major confounder in intracellular infection assays, off-target effects on host cells. In particular, the death of the host cell inherently limits intracellular growth and leads to false positive identification of antimicrobial effect. As many compounds in screening libraries are eukaryotic cell toxic, such false positives would overwhelm true antimicrobials, necessitating a large number of follow-up, endpoint cytotoxicity assays for resolution.

Thus, it was of great interest to be able to assess eukaryotic cell viability and intracellular growth simultaneously. Notably, a characteristic of non-viable eukaryotic cells is loss of cell membrane integrity. Probes that test the permeability of the cell membrane may therefore be used to assess cell viability. We previously characterized the ability of a series of putatively cell membrane-impermeant, fluorescent, DNA-binding dyes to access and stain nuclear DNA of dead cells.⁴ On binding nuclear DNA, these dyes display a large increase in quantum fluorescent yield resulting in increased signal over background solution fluorescence. As such, these dyes provided a quantitative readout of eukaryotic cell death.⁴ Notably, we found that several were non-toxic themselves during prolonged co-incubation with J774 macrophages. When added during initial infection, they provided a real-time, fluorescent readout of eukaryotic cell death that can be measured by a microplate fluorimeter or observed microscopically.

Therefore, by combining use of a bacterial reporter and non-toxic, membrane-impermeant, DNA-binding dyes, we were able to develop a simple, non-destructive, real-time assay to measure both bacterial load and eukaryotic cell cytotoxicity simultaneously. This assay has allowed us to screen in 384-well plate format ~10,000 known bioactives including ~250 antimicrobials and >240,000 small molecules with functionally uncharacterized activity for the ability to inhibit intracellular growth of Legionella pneumophila, while at the same time generating eukaryotic cell cytotoxicity data for each compound.⁶ Our analysis of known antimicrobials against intracellular growth of Legionella was the most comprehensive exploration of this type to date.⁶

Based on the efficiency of our assay format, we also subsequently explored the potentially synergistic effects of known antimicrobials when used in combination. One of the most common synergy tests, the so-called checkerboard assay, is standardly performed by assessing combinatorial effects of two-fold serial dilutions of two or more antimicrobials.¹⁰ In these assays, synergy is defined by the observation of greater effect when two or more antimicrobials are applied together than the sum of the effects of each applied separately. Of note, heretofore, only focused and selective synergy testing was performed against intracellular Legionella pneumophila because of the great effort involved in traditional endpoint assays multiplied by the combinatorial permutations required.

To facilitate synergy testing, we made use of our real-time intracellular growth/eukaryotic cytotoxicity assay in combination with automated digital dispensing technology⁶. This automation permitted us to dispense serial dilutions of compounds dissolved in DMSO or aqueous solution alone or in combination in 384-well format.¹¹ Furthermore, such robust liquid handling technology permitted us to easily perform higher resolution, square-root-of-two (rather than the standard, lower resolution, doubling) dilution combinations to achieve higher levels of specificity in our two-dimensional, checkerboard synergy analysis. This resolution was especially valuable in addressing concerns in the synergy field about reproducibility when using two-fold dilution series¹². Lastly, our assay was quantitative and also therefore measured gradations of inhibition. As a result, the assay captured the entirety of inhibitory information, expressible in isocontour isobolograms in which isocontours connect combinatorial concentrations with similar levels of growth inhibition.⁶ This plotting strategy allowed visualization of combinatorial dose-response curves. To illustrate our methodology, we describe our protocol for performing these assays and show representative results.