Microbiology

In the expanding field of intestinal organoid research, various protocols for three- and two-dimensional organoid-derived cell cultures exist. Two-dimensional organoid-derived monolayers are used to overcome some limitations of three-dimensional organoid cultures. They are increasingly used also in infection research, to study physiological processes and tissue barrier functions, where easy experimental access of pathogens to the luminal and/or basolateral cell surface is required. This has resulted in an increasing number of publications reporting different protocols and media compositions for organoid manipulation, precluding direct comparisons of research outcomes in some cases. With this in mind, here we describe a protocol aimed at the harmonization of seeding conditions for three-dimensional intestinal organoids of four commonly used research species onto cell culture inserts, to create organoid-derived monolayers that form electrophysiologically tight epithelial barriers. We give an in-depth description of media compositions and culture conditions for creating these monolayers, enabling also the less experienced researchers to obtain reproducible results within a short period of time, and which should simplify the comparison of future studies between labs, but also encourage others to consider these systems as alternative cell culture models in their research.

Graphic abstract:

Schematic workflow of organoid-derived monolayer generation from intestinal spheroid cultures. ECM, extracellular matrix; ODM, organoid-derived monolayer.

Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma’s environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the pathogen. In addition to the incorporation of a physiological extracellular matrix, this model has the singular advantage of recapitulating dormant-like Mtb features, as well as reproducing Mtb resuscitation observed under immunomodulatory treatments, which have not been reported in other published protocols to generate in vitro granulomas.

The ability of the human fungal pathogen Candida albicans to disseminate into tissues is promoted by a switch from budding to invasive hyphal growth. This morphological transition is stimulated by multiple environmental factors that can vary at different sites of infection. To identify genes that promote invasive growth, C. albicans mutants can be screened for defects in growing invasively into solid agar medium as a substitute for studying tissue invasion. This in vitro approach has advantages in that it permits the media conditions to be varied to mimic different host environments. In addition, the concentration of agar can be varied to determine the effects of altering the rigidity of the matrix into which the cells invade, as this provides a better indicator of invasive growth than the ability to form hyphae in a liquid culture. Testing under multiple conditions can be used to identify mutant cells with the strongest defects. Therefore, protocols and media for analyzing invasive growth of C. albicans under different conditions will be described that are appropriate for testing a single strain or high-throughput analysis of a collection of mutant C. albicans strains.

The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected erythrocyte. The task remains delicate despite the use of enzymes or detergents theoretically capable of degrading specific membranes inside the infected cell.

The remit of this protocol is to propose a method to isolate the erythrocyte cytosol and ghosts from the other compartments of the infected cell via a percoll gradient. Also, the lysis of the erythrocyte membrane is done using equinatoxin II, which has proven to be more effective at erythrocyte lysis regardless of the cell infection status, compared to the commonly used streptolysin. The parasitophorous vacuole (PV) content is collected after saponin lysis, before recovering membrane and parasite cytosol proteins by Triton X-100 lysis. The lysates thus obtained are analyzed by Western blot to assess the accuracy of the various extraction steps. This protocol allows the separation of the host compartment from the parasite compartments (PV and parasite), leading to potential studies of host proteins as well as parasite proteins exported to the host cell.

Oral mucositis is a common complication of cancer chemotherapy treatment. Because of the lack of relevant oral mucositis experimental models, it is not clear how chemotherapeutic agents injure the oral mucosa and if commensal microorganisms accelerate tissue damage. We developed an organotypic oral mucosa model that mimics cellular responses commonly associated with cytotoxic chemotherapy. The organotypic model consists of multilayer oral epithelial cells growing over a collagen type I matrix, with embedded fibroblasts. Treatment of organotypic constructs with the chemotherapeutic agent, 5-fluorouracil (5-FU), leads to major histopathologic changes resembling mucositis, such as DNA synthesis inhibition, increased apoptosis and cytoplasmic vacuolation. Candida albicans formed mucosal biofilms on these tissues and augmented the inflammatory responses to 5-FU. This model can be used in further mechanistic studies of oral mucositis and associated opportunistic infections.

Alcohol consumption has diverse and well-documented effects on the human immune system and its ability to defend against infective agents. While pulmonary related infections can occur in healthy humans, binge alcohol use is recognized as a major health risk factor (Nelson et al., 1991). Although binge alcohol consumption has been considered as a risk factor for the development of pulmonary infections, no experimental studies have investigated the outcomes of a single binge alcohol exposure during infection. A key assay to assess the effects of a single binge alcohol exposure on the interactions between bacteria and alveolar macrophage is a binge alcohol intracellular invasion and killing assay. MH-S alveolar macrophages (AMs) are exposed to a single binge alcohol dose prior to infection for 3 h. The macrophage monolayer is then infected to allow for engulfment, followed by removal of extracellular bacteria to assess the intracellular killing capacity of infected macrophages over time. We have utilized this assay to demonstrate that low alcohol exposure significantly suppressed the uptake and killing of less virulent Burkholderia thailandensis (B. thailandensis) by AMs. More recently we found that activated AMs with interferon (IFN)-γ incubated in alcohol (0.08%) for 3 h prior to infection showed significantly lower bacterial uptake at 2 and 8 h post infection, which lead to B. thailandensis survival and a ~2.5-fold replication increase compared to controls (Jimenez et al., 2017). These results provide insights into binge alcohol consumption, a culturally prevalent risk factor, as a predisposing factor for pulmonary bacterial infections. This assay can be adapted to other bacterial species and host cell types to assess tissue specific effects of alcohol during infection.

Salmonella is a Gram-negative bacterium causing a gastro-enteric disease called salmonellosis. During the first phase of infection, Salmonella uses its flagella to swim near the surface of the epithelial cells and to target specific site of infection. In order to study the selection criteria that determine which host cells are targeted by the pathogen, and to analyze the relation between infecting Salmonella (i.e., cooperation or competition), we have established a high-throughput microscopic assay of HeLa cells sequentially infected with fluorescent bacteria. Using an automated pipeline of image analysis, we quantitatively characterized a multitude of parameters of infected and non-infected cells. Based on this, we established a predictive model that allowed us to identify those parameters involved in host cell vulnerability towards infection. We revealed that host cell vulnerability has two origins: a pathogen-induced cellular vulnerability emerging from Salmonella uptake and persisting at later stages of the infection process; and a host cell-inherent vulnerability linked with cell inherent attributes, such as local cell crowding, and cholesterol content. Our method forecasts the probability of Salmonella infection within monolayers of epithelial cells based on morphological or molecular host cell parameters. Here, we provide a detailed description of the workflow including the computer-based analysis pipeline. Our method has the potential to be applied to study other combinations of host-pathogen interactions.

Rubella virus (RuV) is an enveloped, positive-sense single-stranded RNA virus that is pathogenic to humans. RuV binds to the target cell via the viral envelope protein E1, but the specific receptor molecules on the target cell are yet to be fully elucidated. Here, we describe a protocol for liposome flotation assay to study direct interactions between RuV particles and lipid membranes in a qualitative manner. Interactions are examined by a Nycodenz density gradient fractionation using UV-inactivated RuV particles and fluorescent-labeled liposomes consisting of pure lipids. Fractionated RuV particles are detected using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis for viral proteins. On the Nycodenz gradient, RuV particles bound to liposomes shift to lower density fractions than unbound RuV particles. Using this protocol, we provide compelling evidence that, at neutral pH in a calcium-dependent manner, RuV particles bind to lipid membranes containing both sphingomyelin (SM) and cholesterol in certain cell types.

The stable HBV-transfected cell lines, which based on stable integration of replication-competent HBV genome into hepatic cells, are widely used in basic research and antiviral drug evaluation against HBV. However, previous reported strategies to generate HBV-replicating cell lines, which primarily rely on random integration of exogenous DNA by plasmid transfection, are inefficient and time-consuming. We newly developed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable HBV-replicating cell lines of different genotype. The pTSMP-HBV vector contains HBV 1.3-copy genome and dual selection markers (mCherry and puromycin resistance gene), allowing rapid enrichment of stably-transfected cells via red fluorescence-activated cell sorting and puromycin antibiotic selection. In this protocol, we described the detailed procedure for constructing the HBV-replicating stable cells and systematically evaluating HBV replication and viral protein expression profiles of these cells.

Botrytis cinerea (B. cinerea) attacks many crops of economic importance, represents one of the most extensively studied necrotrophic pathogens. Inoculation of B. cinerea and phenotypic analysis of plant resistance are key procedures to investigate the mechanism of plant immunity. Here we describe a protocol for B. cinerea inoculation on medium and planta based on our study using the tomato-B. cinerea system.