key: cord-0817951-llfqgf8j authors: Ramírez-Flores, Carlos J.; Knoll, Laura J. title: Breakthroughs in microbiology made possible with organoids date: 2021-11-24 journal: PLoS Pathog DOI: 10.1371/journal.ppat.1010080 sha: 63892d69be8ac293a1fae575e557cec24c992314 doc_id: 817951 cord_uid: llfqgf8j nan Zika virus (ZAU : PleasenotethattheabbreviationZIKVhasbeenintroducedforZikavirusinthesentenceZika IKV) spreads by the bite of an infected Aedes spp. mosquito causing mild flu symptoms in adults and congenital Zika syndrome in newborns. Congenital Zika syndrome includes several birth defects but is most known for small infant head size called microcephaly. Zika infection of HBO allowed the mechanism by which ZIKV affects the neurons to be determined [1, 2] as well as the evaluation of potential treatments [3] . ZIKV-infected HBOs have shown that replication of the virus killed neural precursors that led to microcephaly [1, 2] . The microcephaly model of ZIKV in HBO further showed thinning of the cortices and impaired cortical expansion, which also lead to HBO size reduction [4] . Neurosphere and HBO infection with Brazilian and African ZIKV strains showed larger reductions in the proliferative zones, greater disruption of the cortical layers, and an increased number of apoptotic cells with the Brazilian compared to the African ZIKV strain [5] . Other advantages and limitations of the HBO to study ZIKV over other in vitro models as well as advances to understand the affections in HBO that resemble microcephaly were recently summarized [6] . The study of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) marked a breakthrough in the viral pathogenesis field with the rapid modeling of the infection using organoids. Kidney, small intestine, lung, and cerebral organoids have helped to studyAU : Pleasenotetha the multi-organ infection/damage observed by SARS-CoV-2 [7] [8] [9] [10] . SARS-CoV-2 causes Coronavirus Disease 2019 (COVID-19). COVID-19 shows influenza-like symptoms and injury in the airways, gastrointestinal tract, and the central nervous system (CAU : PleasenotethatCNShasbeendefine NS), causing meningitis/ encephalitis. Modeling of these tissues in organoids allowed researchers to determine the differential expression of cytokines and chemokines in colonic organoids, neuronal death, hypermetabolic state and hypoxia in HBO, and chemokine production and responsiveness to drug treatment in lung organoids [9, 10] . SARS-CoV-2 infection of human kidney organoids and HBO confirmed viral binding to the angiotensin converting enzyme 2 receptor in 3D culture [7, 10] . These examples show how organoid technology allowed researchers to efficiently recapitulate host-pathogen interactions to study the physiopathology of viruses and how we can cope with future pandemics. The study of human enteric viruses, such as rotavirus, norovirus, adenovirus, and astrovirus, have all been advanced using intestinal organoid technology [11] . Rotavirus and norovirus are responsible for food and waterborne diarrhea, causing together >1 billion cases and >500,000 deaths per year in children <6 years old (World Human Organization). A breakthrough for gastrointestinal viruses was obtained by the reproducible cultivation of stool-isolated rotavirus in human intestinal organoids (HIOs) [12] . This study showed rotavirus replication in mesenchymal as well as the epithelial cells. In human biliary liver organoids, robust rotavirus replication was blocked by antiviral drugs and neutralizing antibodies [13] . For previously uncultivable norovirus, HIO supported replication and modeling multiple human variants [14] . During norovirus infection of HIO, researchers discovered essential cofactors (e.g., bile acids, histo-blood group antigens, and divalent cations), overexpression of genes, and response to type I and III interferons [14] [15] [16] . Enteric bacterial infections remain a health challenge. The study of pathology caused by bacteria in immortalized 2D cultures, ex vivo tissues, and animal models has contributed to the knowledge in this field. However, 2D cell lines lack the structural complexity of tissues, ex vivo tissues have a limited life span, and animal models have many pathophysiological and immune response differences from humans. Because of these limitations, HIOs that resemble the intestinal environment are emerging as models for bacterial infections. Multiple enteric bacteria, such as Escherichia coli, Salmonella, and Listeria, have been extensively studied using organoids to provide valuable insights and expand our knowledge of bacteria-host interaction [17, 18] . Foundational experiments creating an anaerobic environment for examining interactions of anaerobic bacteria with host epithelium have been performed in HIOs. Clostridium difficile, the agent responsible for 25% of the nosocomial diarrhea cases, is an obligate anaerobe and notoriously difficult to study. Microinjection of C. difficile into the lumen of the HIO resulted in infection, toxin production, and consequent paracellular barrier disfunction and altered mucus oligosaccharide composition [19, 20] . As another example, Helicobacter pylori colonize the gastric mucosa and is the main cause of peptic ulcers, chronic gastritis, and gastric cancer. Microinjection of H. pylori into gastric organoids can emulate changes and pathological events during its infection, such as inflammation and regulation of tight junctions [21, 22] . Cryptosporidium is an intracellular parasite that causes diarrhea and gastroenteritis in vertebrates including humans. In the past, short-term (up to 5 days) infection and incomplete propagation was supported in a 2D culture of human intestinal epithelial cells [23] . Recently, 3D systems have been used to successfully sustain the infection from oocysts and generate damage in colon explants, providing evidence that the parasite could induce cancer [24] . Cryptosporidium parvum can infect human small intestinal and lung organoids where it can successfully develop for up to 28 days [25] . Addition of an air-liquid interface to the culture system allowed for life cycle completion, including the production of oocysts that were infectious to mice [26] . Some parasites have strict species specificity to their life cycles, which has hindered their study in vitro. Organoids have opened an avenue for those parasites with host-specific requirements. Toxoplasma gondii can infect any warm-blooded vertebrate, but its sexual cycle is restricted to the feline intestine. The specificity of T. gondii sexual development in cat intestinal organoid-derived monolayers was determined. Cat intestinal cells supplemented with linoleic acid supported early sexual development of T. gondii in vitro [27] . To model T. gondii human brain infection, HBOs were infected with the rapidly replicating form called a tachyzoite, which then spontaneously transformed into the chronic cyst form called a bradyzoite [28] . Changes in transcriptomics related to parasite invasion and replication were also detected in these HBOs. Recently, a multispecies organoid platform was successfully modeled to coinfect T. gondii with Giardia duodenalis by using organoids or organoid-derived monolayers of various host species [29] . The study of fungi in traditional 2D cultures can miss key signaling events that occur during host-fungus interactions. To capture the complexity of the lung, researchers used human lung organoids to develop a model of the bronchiole including an airway, vascular, and extracellular matrix components [30] . This model contained a clickable extension to facilitate volatile compound communication between the microbes and host. Immune cell recruitment and leukocyte extravasation were examined in this model after coinfection with the fungal pathogen Aspergillus fumigatus and the bacteria Pseudomonas aeruginosa. Greater inflammatory responses were seen in these bronchioles when they were in contact with volatiles from both pathogens, compared to either monoculture, showing volatile communication between the kingdoms [30] . Organoid cultures of Plasmodium spp., which are well known as the causative agents of malaria, are examples of some progress but continued limitations that must be addressed to model the infection successfully. These limitations include long-term culture, poor infectability, size of the organoids for consistent infection, cell function, presence of immune cells, difficulty to test drugs, and parasites being trapped within the matrix [31] . To date, the liver stage has been studied using rodent-infecting parasite Plasmodium berghei in liver spheroids, resulting in infective liver stages called merozoites [32] . Liver spheroids derived from simian and human hepatocytes supported the complete liver stage of Plasmodium cynomalgi and Plasmodium vivax, starting with the sporozoite stage and finishing with the release of merozoites capable of invading erythrocytes in vitro [33] . Those results have shown that the use of organoids in the malaria field is promising for the study of Plasmodium species that infect humans. Organoids are still in their infancy and under constant development. Improvements to the system are required to keep revolutionizing the field. Integration of immune cells, low oxygen conditions, and microbiota to emulate the host microenvironment are progressing as well as adding variability of the structure and size. Organoid-pathogen interaction of other microbes should be investigated further to fill the gaps from 2D systems. Modeling infection will be useful to know mechanisms and factors of host/pathogen specificity during the life cycle of microbes. Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure Genetic Ablation of AXL Does Not Protect Human Neural Progenitor Cells and Cerebral Organoids from Zika Virus Infection Self-Organized Cerebral Organoids with Human-Specific Features Predict Effective Drugs to Combat Zika Virus Infection Organoid modeling of Zika and herpes simplex virus 1 infections reveals virus-specific responses leading to microcephaly The Brazilian Zika virus strain causes birth defects in experimental models Using brain organoids to understand Zika virusinduced microcephaly Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2 SARS-CoV-2 productively infects human gut enterocytes Neuroinvasion of SARS-CoV-2 in human and mouse brain Identification of SARS-CoV-2 inhibitors using lung and colonic organoids Gastrointestinal organoid technology advances studies of enteric virus biology Stem cell-derived human intestinal organoids as an infection model for rotaviruses Rotavirus Infection and Cytopathogenesis in Human Biliary Organoids Potentially Recapitulate Biliary Atresia Development Replication of human noroviruses in stem cell-derived human enteroids Human intestinal organoids express histo-blood group antigens, bind norovirus VLPs, and support limited norovirus replication Norovirus Replication in Human Intestinal Epithelial Cells Is Restricted by the Interferon-Induced JAK/STAT Signaling Pathway and RNA Polymerase II-Mediated Transcriptional Responses. mBio Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions Human Clostridium difficile infection: Altered mucus production and composition Clostridioides difficile infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection Helicobacter pylori targets cancer-associated apical-junctional constituents in gastroids and gastric epithelial cells Human primary intestinal epithelial cells as an improved in vitro model for cryptosporidium parvum infection Three-dimensional (3D) culture of adult murine colon as an in vitro model of cryptosporidiosis: Proof of concept Modelling Cryptosporidium infection in human small intestinal and lung organoids A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability Intestinal delta-6-desaturase activity determines host range for Toxoplasma sexual reproduction Modelling Toxoplasma gondii infection in human cerebral organoids Harmonization of Protocols for Multi-Species Organoid Platforms to Study the Intestinal Biology of Toxoplasma gondii and Other Protozoan Infections Microbial volatile communication in human organotypic lung models Next-generation human liver models for antimalarial drug assays Flexible 3d cell-based platforms for the discovery and profiling of novel drugs targeting plasmodium hepatic infection Hepatic spheroids used as an in vitro model to study malaria relapse The authors like to thank Gina M. Gallego-López and Andrés M. Tibabuzo-Perdomo (University of Wisconsin-Madison) for their helpful comments.