Microbiology

The mitochondrial electron transport chain (ETC) is a multi-component pathway that mediates the transfer of electrons from metabolic reactions that occur in the mitochondrion to molecular oxygen (O₂). The ETC contributes to numerous cellular processes, including the generation of cellular ATP through oxidative phosphorylation, serving as an electron sink for metabolic pathways such as de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential. Proper functioning of the mitochondrial ETC is necessary for the growth and survival of apicomplexan parasites including Plasmodium falciparum, a causative agent of malaria. The mitochondrial ETC of P. falciparum is an attractive target for antimalarial drugs, due to its essentiality and its differences from the mammalian ETC. To identify novel P. falciparum ETC inhibitors, we have established a real-time assay to assess ETC function, which we describe here. This approach measures the O₂ consumption rate (OCR) of permeabilized P. falciparum parasites using a Seahorse XFe96 flux analyzer and can be used to screen compound libraries for the identification of ETC inhibitors and, in part, to determine the targets of those inhibitors.

Key features

• With this protocol, the effects of candidate inhibitors on mitochondrial O₂ consumption in permeabilized asexual P. falciparum parasites can be tested in real time.

• Through the sequential injection of inhibitors and substrates into the assay, the molecular targets of candidate inhibitors in the ETC can, in part, be determined.

• The assay is applicable for both drug discovery approaches and enquiries into a fundamental aspect of parasite mitochondrial biology.

Graphical overview

Seahorse assay experimental workflow. Prior to the assay, coat the cell culture microplate with Cell-Tak to help adhere the parasites to the wells; hydrate the cartridge wells to ensure proper sensor functionality and design the assay template using the Agilent Seahorse Wave Desktop software (Analyze Seahorse data files, Seahorse Wave desktop software|Agilent). On the day of the assay, prepare the inhibitors/substrates that are to be injected into the ports. Then, separate 3 × 10⁸ trophozoite-stage parasites from the uninfected red blood cells (RBCs) and ring-stage parasites using a MACS^® magnetic column. Check the purity of the parasites with Giemsa-stained smears. Determine the concentration of infected RBCs in the sample using a hemocytometer and dilute to approximately 5 × 10⁷ parasites per milliliter. Treat infected RBCs with saponin to permeabilize the host cell membrane and seed approximately 5 × 10⁶ parasites (100 μL) per well in mitochondria assay solution (MAS) buffer. Supplement MAS buffer with digitonin to permeabilize the parasite plasma membrane. Load the ports with the prepared inhibitors/substrates and run the assay using a Seahorse XFe96 analyzer. Once the assay is completed, analyze the data using the Wave desktop software. Further data processing can be done using statistical analysis software.

The mitochondrial electron transport chain (ETC) performs several critical biological functions, including maintaining mitochondrial membrane potential, serving as an electron sink for important metabolic pathways, and contributing to the generation of ATP via oxidative phosphorylation. The ETC is important for the survival of many eukaryotic organisms, including intracellular parasites such as the apicomplexan Toxoplasma gondii. The ETC of T. gondii and related parasites differs in several ways from the ETC of the mammalian host cells they infect, and can be targeted by anti-parasitic drugs, including the clinically used compound atovaquone. To characterize the function of novel ETC proteins found in the parasite and to identify new ETC inhibitors, a scalable assay that assesses both ETC function and non-mitochondrial parasite metabolism (e.g., glycolysis) is desirable. Here, we describe methods to measure the oxygen consumption rate (OCR) of intact T. gondii parasites and thereby assess ETC function, while simultaneously measuring the extracellular acidification rate (ECAR) as a measure of general parasite metabolism, using a Seahorse XFe96 extracellular flux analyzer. We also describe a method to pinpoint the location of ETC defects and/or the targets of inhibitors, using permeabilized T. gondii parasites. We have successfully used these methods to investigate the function of T. gondii proteins, including the apicomplexan parasite-specific protein subunit TgQCR11 of the coenzyme Q:cytochrome c oxidoreductase complex (ETC Complex III). We note that these methods are also amenable to screening compound libraries to identify candidate ETC inhibitors.

The genus Flavivirus within the family Flaviviridae includes many viral species of medical importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cell-based molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirus-activatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG). When the viral protease cleaves the linker, the quenching peptide is removed, and the fluorescent protein adopts a conformation promoting fluorescence. Here we provide a detailed protocol for the generation, selection and implementation of stable BHK-21 cells expressing our flavivirus genetically-encoded molecular reporters, suitable to monitor the viral infection by live-cell imaging. We also describe the image analysis procedures and provide the required software pipelines. Our reporter cells allow the implementation of single-cell infection kinetics as well as plaque assays for both reference and native strains of flaviviruses by live-cell imaging.

Graphic abstract:

Workflow for the generation and implementation of reporter BHK-21 cells for live imaging of flavivirus infection.

The latent HIV-1 viral reservoir in resting CD4⁺ (rCD4⁺) T cells represents a major barrier to an HIV-1 cure. There is an ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this reservoir. However, clinical investigators lack an assay to determine whether or not a decrease in the latent reservoir has been achieved. Therefore, it is critical to develop assays that can reproducibly quantify the reservoir size and changes therein, in participant’s blood during a therapeutic trial. Quantification of the latent HIV viral reservoir requires a highly sensitive, cost-effective assay capable of measuring the low frequency of rCD4⁺ T cells carrying functional provirus. Preferably, such an assay should be such that it can be adopted for high throughput and could be adopted under conditions for use in large-scale clinical trials. While PCR-based assays are commonly used to quantify pro-viral DNA or intracellular RNA transcript, they cannot distinguish between replication-competent and defective proviruses. We have recently published a study where a reporter cell-based assay (termed TZA or TZM-bl based quantitative assay) was used to quantify inducible replication-competent latent HIV-1 in blood. This assay is more sensitive, cost-efficient, and faster than available technology, including the quantitative viral outgrowth assay or the Q-VOA. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in virally suppressed participants on ART is approximately 70-fold larger than previous estimates. We describe here in detail an optimized method to quantitate latently infected cells using the TZA.

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that regulates distinct aspects of bacterial physiology. It is synthesized by diguanylate cyclases (DGCs) and hydrolyzed by phosphodiesterases (PDEs). To date, the activities of DGC and PDE are commonly assessed by phenotypic assays, mass spectrometry analysis of intracellular c-di-GMP concentration, or riboswitch-based fluorescent biosensors. However, some of these methods require cutting-edge equipment, which might not be available in every laboratory. Here, we report a new simple, convenient and cost-effective system to assess the function of DGCs and PDEs in E. coli. This system utilizes the high specificity of a riboswitch to c-di-GMP and its ability to regulate the expression of a downstream β-galactosidase reporter gene in response to c-di-GMP concentrations. In this protocol, we delineate the construction of this system and its use to assess the activity of DGC and PDE enzymes.

Exosomes have emerged as an important mediator of intercellular communication. They are present in extracellular milieu and therefore, easily accessible by neighboring or distant cells. They carry mRNA, microRNAs and proteins within their vesicles and once internalized by recipient cells; they can modulate multiple signaling pathways with pleiotropic effects from inducing antiviral state to disease progression. We have previously shown that hepatitis C virus (HCV) infected hepatocytes or hepatoma cells harboring genome-length replicon secrete exosomes in culture supernatants. These exosomes are taken up by hepatic stellate cells (HSC) and activate them to induce fibrosis during HCV infection. Here, we describe detailed protocols for exosomes isolation and uptake of BODIPY labeled exosomes by hepatic stellate cells.

Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. Here, we provide transwell permeability assay and electrical impedance sensing assay for studying endothelial permeability in vitro. With these methods, the effect of a molecule on endothelial permeability could be defined.

Semen contains amyloid fibrils that enhance HIV-1 infection (Münch et al., 2007; Kim et al., 2010; Roan et al., 2011; Arnold et al., 2012; Usmani et al., 2014; Roan et al., 2014). Positively charged semen amyloids capture negatively charged viral particles and increase their attachment rates to the cell surface resulting in enhanced fusion and infection (Roan et al., 2009). Since semen is highly cytotoxic, we developed an assay that allows quantification of the infection enhancing activity of semen while minimizing its cell damaging activity. Here, we describe two protocols that allow the quantification of the infectivity enhancing activity of semen using a reporter cell line (TZM-bl cells) or peripheral blood mononuclear cells (PBMCs).

β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (Fiksdal et al., 1994) and 96-well plate format. The fluorogenic substrates 4-Methylumbelliferyl β-D-galactoside (for β-galactosidase assays) (Ramsay et al., 2013) or 4-Methylumbelliferyl β-D-glucuronide (for β-glucuronidase assays) (Ramsay et al., 2011) are cleaved to produce the fluorescent product 4-methylumbelliferone. Cells are permeabilized by freeze-thawing and lysozyme, and the production of 4-methylumbelliferone is monitored continuously by a fluorescence microplate reader as a kinetic assay. The rate of increase in fluorescence is then calculated, from which relative gene-expression levels are extrapolated. Due to the high sensitivity fluorescence-based detection of 4-methylumbelliferone and the high density of time points collected, this assay may offer increased accuracy in the quantification of low-level gene expression. The assay requires small sample volumes and minimal preparation time. The permeabilisation conditions outlined in this protocol have been optimised for Gram-negative bacteria (specifically Escherichia coli and Serratia), but is likely suitable for other organisms with minimal optimisation.