generation-mouse-prostate-organoid-based-model-for-studying-host

Generation of a Mouse Prostate Organoid-Based Model for Studying Host-Pathogen Interactions

Aging men are highly affected by prostate diseases, such as benign prostatic hyperplasia, prostatitis, and prostate cancer. Together, these conditions ...

University of Würzburg

MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.

Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia.

Salmonellosis is a gastrointestinal disease caused by non-typhoidal Salmonella serovars such as Salmonella Typhimurium. This pathology is a zoonosis, and food animals with subclinical infection constitute a vast reservoir for disease. After intestinal colonization, Salmonella Typhimurium reaches mesenteric lymph nodes (MLN), where infection is controlled avoiding systemic spread. Although the molecular basis of this infection has been extensively studied, little is known about how microRNA (miRNA) regulate the expression of proteins involved in the Salmonella-host interaction. Using small RNA-seq, we examined expression profiles of MLN 2 days after infection with Salmonella Typhimurium, and we found 110 dysregulated miRNA. Among them, we found upregulated miR-21, miR-155, miR-150, and miR-221, as well as downregulated miR-143 and miR-125, all of them previously linked to other bacterial infections. Integration with proteomic data revealed 30 miRNA potentially regulating the expression of 15 proteins involved in biological functions such as cell death and survival, inflammatory response and antigenic presentation. The inflammatory response was found increased via upregulation of miRNA such as miR-21 and miR-155. Downregulation of miR-125a/b, miR-148 and miR-1 were identified as potential regulators of MHC-class I components PSMB8, HSP90B1 and PDIA3, respectively. Furthermore, we confirmed that miR-125a is a direct target of immunoproteasome component PSMB8. Since we also found miR-130 downregulation, which is associated with upregulation of HSPA8, we suggest induction of both MHC-I and MHC-II antigen presentation pathways. In conclusion, our study identifies miRNA that could regulate critical networks for antigenic presentation, inflammatory response and cytoskeletal rearrangements.

Regulatory role of microRNA in mesenteric lymph nodes after Salmonella Typhimurium infection.

While mucosal inflammation is a major source of stress during enteropathogen infection, it remains to be fully elucidated how the host benefits from this environment to clear the pathogen. Here, we show that host stress induced by different stimuli mimicking inflammatory conditions strongly reduces the binding of  to epithelial cells. Mechanistically, stress activates acid sphingomyelinase leading to host membrane remodeling. Consequently, knockdown or pharmacological inhibition of the acid sphingomyelinase blunts the stress-dependent inhibition of  binding to host cells. Interestingly, stress caused by intracellular  replication also results in remodeling of the host cell membrane,  and , which precludes re-infection by this and other non-motile pathogens. In contrast,  Typhimurium overcomes the shortage of permissive entry sites by gathering effectively at the remaining platforms through its flagellar motility. Overall, our findings reveal host membrane remodeling as a novel stress-responsive cell-autonomous defense mechanism that protects epithelial cells from infection by non-motile bacterial pathogens.

Stress-induced host membrane remodeling protects from infection by non-motile bacterial pathogens.

MicroRNAs are a class of small noncoding RNAs that act as major post-transcriptional regulators of gene expression. They are currently recognized for their important role in the intricate interaction between host and bacterial pathogens, either as part of the host immune response to neutralize infection, or as a molecular strategy employed by bacteria to hijack host pathways for their own benefit. Here, we summarize recent advances on the function of miRNAs during infection of mammalian hosts by bacterial pathogens, highlighting key cellular pathways. In addition, we discuss emerging themes in this field, including the participation of miRNAs in host-microbiota crosstalk and cell-to-cell communication.

MicroRNAs at the Host-Bacteria Interface: Host Defense or Bacterial Offense.

MicroRNAs (miRNAs) are a well-characterized class of small noncoding RNAs that act as major posttranscriptional regulators of gene expression. Accordingly, miRNAs have been associated with a wide range of fundamental biological processes and implicated in human diseases. During the past decade, miRNAs have also been recognized for their role in the complex interplay between the host and bacterial pathogens, either as part of the host response to counteract infection or as a molecular strategy employed by bacteria to subvert host pathways for their own benefit. Importantly, the characterization of downstream miRNA targets and their underlying mechanisms of action has uncovered novel molecular factors and pathways relevant to infection. In this article, we review the current knowledge of the miRNA response to bacterial infection, focusing on different bacterial pathogens, including , ,  spp., and , among others.

Multifaceted Roles of microRNAs in Host-Bacterial Pathogen Interaction.

MicroRNAs (miRNAs) are increasingly recognized for their role in infection by bacterial pathogens, although the effect of each individual miRNA remains largely unknown. Here, we used a comparative genome-wide microscopy-based functional screening approach to identify miRNAs controlling infection by two bacterial pathogens-Salmonella enterica serovar Typhimurium and Shigella flexneri. Despite the similarities between these pathogens, we found infections to be controlled by largely non-overlapping subsets of miRNAs, seemingly reflecting different requirements prompted by their distinct intracellular lifestyles. By characterizing a small subset of miRNAs chosen among the strongest inhibitors of Shigella infection, we discovered that miR-3668, miR-4732-5p and miR-6073 exert a selective effect on Shigella infection by impairing bacterial actin-based motility by downregulating N-WASP. Additionally, by identifying let-7i-3p miRNA as a strong inhibitor of Salmonella replication and performing in-depth analysis of its mechanisms of action, we showed that this miRNA specifically inhibits Salmonella infection via modulation of endolysosomal trafficking and the vacuolar environment by targeting the host RGS2 protein. These findings illustrate two paradigms underlying miRNA-mediated regulation of bacterial infection, acting as part of the host response to infection, or as part of bacterial strategies to modulate the host environment and favour pathogenesis.

Functional screenings reveal different requirements for host microRNAs in Salmonella and Shigella infection.

Cells infected with pathogens can contribute to clearing infections by releasing signals that instruct neighbouring cells to mount a pro-inflammatory cytokine response, or by other mechanisms that reduce bystander cells' susceptibility to infection. Here, we show the opposite effect: epithelial cells infected with Salmonella Typhimurium secrete host factors that facilitate the infection of bystander cells. We find that the endoplasmic reticulum stress response is activated in both infected and bystander cells, and this leads to activation of JNK pathway, downregulation of transcription factor E2F1, and consequent reprogramming of microRNA expression in a time-dependent manner. These changes are not elicited by infection with other bacterial pathogens, such as Shigella flexneri or Listeria monocytogenes. Remarkably, the protein HMGB1 present in the secretome of Salmonella-infected cells is responsible for the activation of the IRE1 branch of the endoplasmic reticulum stress response in non-infected, neighbouring cells. Furthermore, E2F1 downregulation and the associated microRNA alterations promote Salmonella replication within infected cells and prime bystander cells for more efficient infection.

Reprogramming of microRNA expression via E2F1 downregulation promotes Salmonella infection both in infected and bystander cells.

Infection with Salmonella Typhimurium (S. Typhimurium) is a common cause of food-borne zoonosis leading to acute gastroenteritis in humans and pigs, causing economic losses to producers and farmers, and generating a food security risk. In a previous study, we demonstrated that S. Typhimurium infection produces a severe transcriptional activation of inflammatory processes in ileum. However, little is known regarding how microRNAs regulate this response during infection. Here, small RNA sequencing was used to identify 28 miRNAs differentially expressed (DE) in ileum of S. Typhimurium-infected pigs, which potentially regulate 14 target genes involved in immune system processes such as regulation of cytokine production, monocyte chemotaxis, or cellular response to interferon gamma. Using in vitro functional and gain/loss of function (mimics/CRISPR-Cas system) approaches, we show that porcine miR-194a-5p (homologous to human miR-194-5p) regulates TLR4 gene expression, an important molecule involved in pathogen virulence, recognition and activation of innate immunity in Salmonella infection.

Study of microRNA expression in Salmonella Typhimurium-infected porcine ileum reveals miR-194a-5p as an important regulator of the TLR4-mediated inflammatory response.

The human gastric epithelium forms highly organized gland structures with different subtypes of cells. The carcinogenic bacterium Helicobacter pylori can attach to gastric cells and subsequently translocate its virulence factor CagA, but the possible host cell tropism of H. pylori is currently unknown. Here, we report that H. pylori preferentially attaches to differentiated cells in the pit region of gastric units. Single-cell RNA-seq shows that organoid-derived monolayers recapitulate the pit region, while organoids capture the gland region of the gastric units. Using these models, we show that H. pylori preferentially attaches to highly differentiated pit cells, marked by high levels of GKN1, GKN2 and PSCA. Directed differentiation of host cells enable enrichment of the target cell population and confirm H. pylori preferential attachment and CagA translocation into these cells. Attachment is independent of MUC5AC or PSCA expression, and instead relies on bacterial TlpB-dependent chemotaxis towards host cell-released urea, which scales with host cell size.

Helicobacter pylori shows tropism to gastric differentiated pit cells dependent on urea chemotaxis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection primarily affects the lung but can also cause gastrointestinal (GI) symptoms. In vitro experiments confirmed that SARS-CoV-2 robustly infects intestinal epithelium. However, data on infection of adult gastric epithelium are sparse and a side-by-side comparison of the infection in the major segments of the GI tract is lacking. We provide this direct comparison in organoid-derived monolayers and demonstrate that SARS-CoV-2 robustly infects intestinal epithelium, while gastric epithelium is resistant to infection. RNA sequencing and proteome analysis pointed to angiotensin-converting enzyme 2 (ACE2) as a critical factor, and, indeed, ectopic expression of ACE2 increased susceptibility of gastric organoid-derived monolayers to SARS-CoV-2. ACE2 expression pattern in GI biopsies of patients mirrors SARS-CoV-2 infection levels in monolayers. Thus, local ACE2 expression limits SARS-CoV-2 expression in the GI tract to the intestine, suggesting that the intestine, but not the stomach, is likely to be important in viral replication and possibly transmission.

SARS-CoV-2 tropism to intestinal but not gastric epithelial cells is defined by limited ACE2 expression.

The innate immune response of an infected host is an essential defense mechanism to fight influenza virus infections in the respiratory tract. This response is essential to limit virus replication and spread. However, an exacerbated response may cause severe immune-pathologies. Therefore, it is very important to better understand innate immune responses at the level of its molecular networks in the context of viral infections.

The host response to influenza infections in human lung and macrophages cell lines.

Bacterial prostatitis caused by uropathogenic Escherichia coli (UPEC) strains is a highly prevalent and recurrent infection responsible for significant morbidity in men. The molecular pathogenesis of prostatitis remains poorly understood, partly due to a lack of suitable in vitro models. Here we developed a 2D mouse stem cell-derived prostate epithelial organoid model. In the organoid model, 5α-dihydrotestosterone promoted differentiation of basal into luminal cells, while transcriptomic analyses validated the model in comparison to 3D models and mouse prostate tissue. Infection analyses revealed that UPEC preferentially attached to, invaded and replicated within luminal prostate cells. Experiments with a UPEC mutant strain lacking the bacterial adhesin, FimH, alongside immunoprecipitation, mass spectrometry, biochemistry and infection experiments with host gene knockouts revealed that FimH-prostatic acid phosphatase (PPAP) binding interactions promote UPEC invasion of luminal prostate cells. ᴅ-Mannose competitively inhibited FimH-PPAP interactions. Findings were validated using ex vivo human prostate tissue. These data highlight the adaptability of FimH in engaging host receptors and the potential for FimH-targeting strategies to reduce bacterial prostatitis.

Article	Year
Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia. PLoS pathogens\| PubMed ID: 28394930	2017
Regulatory role of microRNA in mesenteric lymph nodes after Salmonella Typhimurium infection. Veterinary research\| PubMed ID: 29391047	2018
Stress-induced host membrane remodeling protects from infection by non-motile bacterial pathogens. The EMBO journal\| PubMed ID: 30389666	2018
MicroRNAs at the Host-Bacteria Interface: Host Defense or Bacterial Offense. Trends in microbiology\| PubMed ID: 30477908	2019
Multifaceted Roles of microRNAs in Host-Bacterial Pathogen Interaction. Microbiology spectrum\| PubMed ID: 31152522	2019
Functional screenings reveal different requirements for host microRNAs in Salmonella and Shigella infection. Nature microbiology\| PubMed ID: 31792428	2020
Reprogramming of microRNA expression via E2F1 downregulation promotes Salmonella infection both in infected and bystander cells. Nature communications\| PubMed ID: 34099666	2021
Study of microRNA expression in Salmonella Typhimurium-infected porcine ileum reveals miR-194a-5p as an important regulator of the TLR4-mediated inflammatory response. Veterinary research\| PubMed ID: 35598011	2022
Helicobacter pylori shows tropism to gastric differentiated pit cells dependent on urea chemotaxis. Nature communications\| PubMed ID: 36198679	2022
SARS-CoV-2 tropism to intestinal but not gastric epithelial cells is defined by limited ACE2 expression. Stem cell reports\| PubMed ID: 38670110	2024
The host response to influenza infections in human lung and macrophages cell lines. BMC genomic data\| PubMed ID: 40629279	2025
Uropathogenic Escherichia coli invade luminal prostate cells via FimH-PPAP receptor binding. Nature microbiology\| PubMed ID: 41507584	2026

Carmen Aguilar

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