In vitro and ex vivo modeling of enteric bacterial infections

Abstract

Enteric bacterial infections contribute substantially to global disease burden and mortality, particularly in the developing world. In vitro 2D monolayer cultures have provided critical insights into the fundamental virulence mechanisms of a multitude of pathogens, including Salmonella enterica serovars Typhimurium and Typhi, Vibrio cholerae, Shigella spp., Escherichia coli and Campylobacter jejuni, which have led to the identification of novel targets for antimicrobial therapy and vaccines. In recent years, the arsenal of experimental systems to study intestinal infections has been expanded by a multitude of more complex models, which have allowed to evaluate the effects of additional physiological and biological parameters on infectivity. Organoids recapitulate the cellular complexity of the human intestinal epithelium while 3D bioengineered scaffolds and microphysiological devices allow to emulate oxygen gradients, flow and peristalsis, as well as the formation and maintenance of stable and physiologically relevant microbial diversity. Additionally, advancements in ex vivo cultures and intravital imaging have opened new possibilities to study the effects of enteric pathogens on fluid secretion, barrier integrity and immune cell surveillance in the intact intestine. This review aims to present a balanced and updated overview of current intestinal in vitro and ex vivo methods for modeling of enteric bacterial infections. We conclude that the different paradigms are complements rather than replacements and their combined use promises to further our understanding of host-microbe interactions and their impacts on intestinal health.

Keywords: Organotypic culture; infection models; intestine; microphysiological systems; monolayer cell culture.

Figure 1.

Overview of key infectious mechanisms of enteric bacteria revealed using 2D monolayer cultures. Illustration showing the mechanism of invasion and bacteria-host interactions for Vibrio cholerae, Campylobacter jejuni, Shigella flexneri and Salmonella Typhimurium. AC = adenylate cyclase; CdtB = virulence factor with DNase I activity; CjeCas9 = virulence factor causing unspecific DNA damage; CtxA = cholera toxin A; GPCR = G-protein coupled receptor; Mφ = macrophage; PMN = polymorphonuclear leukocyte; T3SS = type III secretion system.

Figure 2.

Schematic overview of typhoid toxin secretion and export, and intracellular trafficking of the typhoid toxin. A) Typhoid toxin expression commences upon invasion of S. Typhi into host cells. Specifically, typhoid toxin consists of two enzymatic subunits, PltA and CdtB, which bind to pentamers of PltB or PltC. Upon assembly, the toxin subunits are secreted into the bacterial periplasm (inset). The bacterial transpeptidase YcbB and muramidase TtsA are required for typhoid toxin secretion into the lumen of the Salmonella containing vacuole. Subsequently, typhoid toxin is packaged into vesicle carrier intermediates, which transport the toxin to the plasma membrane where it gets released into the extracellular space. B) The fully assembled PltB toxin binds to the N-acetylneuraminic acid cell surface receptor (Neu5Ac) which results in the endocytosis and retrograde trafficking to the Golgi complex and endoplasmic reticulum (ER) where the CdtB component is released from its pentameric structure and proceeds into the nucleus to induce DNA damage via its DNase I activity. T3SS = type III secretion system.

Figure 3.

Examples of organotypic and microphysiological culture methods to mimic enteric infections. A) Schematic of a fluidic device based on dissociated intestinal organoids. Inset shows the hydrogel-based microchannel. Immunofluorescence of C. parvum undergoing its major epicellular stages in the mini-guts (right-top) and scanning electron microscopy image of distinct stages of the C. parvum life cycle at 72 h post infection (right-bottom). Figure obtained with permission from. B) Illustration showing a 3D villi model infected with S. Typhimurium. Scanning electron microscopy image of the fabricated villi (top right), and fluorescence micrographs showing S. Typhimurium in 2D (bottom left), the crypt section of 3D villi (bottom-middle) and the villus tips of 3D villi (bottom right) 20 days after infection. Image obtained with permission from. C) Schematic of a cross-section of an intestinal microphysiological system (left top). Micrographs showing cross-sections of uninfected (control) and chips infected with GFP expressing Shigella (bottom left). Phase contrast images, and fluorescence confocal micrographs (vertical cross-sectional views) of villi showing intestinal villus damage upon infection with enteropathogenic E. coli (right). Images in this panel were obtained with permission from.^, D) Examples of intravital microscopy (IVM). Top: IVM analyses of TCRγδ^GFP reporter mice after infection with S. Typhimurium. Arrows show tracked flossing movements on one villus (left) and 4D tracking of TCRγδ^GFP cells (right). The intraepithelial compartment is outlined. Reproduced from with permission. Bottom: IVM analyses of Salmonella uptake by CD103⁺ dendritic cells showing that the immune cells extend dendrites through the epithelium while crawling above the basement membrane (arrow on left); the dendrites engulf Salmonella (circled on right), and retract them toward the cell’s soma. Images adopted with permission from.

Figure 4.

Representative schematic of biological models applicable to study enteric bacterial infections and host-pathogen interactions in the intestine. The specific features, as well as advantages and limitations of intestinal model systems are highlighted and compared with respect to complexity, physiological relevance and scalability. Note that transwells, engineered scaffolds and microphysiological systems are compatible with the culture of cell lines, primary intestinal cells as well as dissociated organoids.