Host Cell Types Research Articles

504. Latent HHV7 Infection Attenuates Beneficial Host-Microbe Interactions in the Human Gut

Abstract Background Human Herpes Virus 7 (HHV7) infection results in a lifelong latent infection with over 95% of adults seropositive for HHV7. Lymphocytes are the best understood host cell type for latent HHV7, with the gastric mucosa another site of latent infection. Herpesviridae latency affects host processes like interferon signaling, antigen presentation, and proliferation—the same host pathways are implicated in the beneficial responses to commensal microbes. Methods Adult human colonic organoids with or without latent HHV7 infection were co-cultured with B. longum in asymmetric oxygen conditions that allow for live interaction (F1). The transcriptional state after 24h of interactions was determined with RNA-sequencing. The transcriptional state was compared between axenic (no microbes) or after co-culture and with or without latent HHV7. F1: Establishing oxygen gradients across epithelial monolayers (A) Schematic of oxygen gradient apparatus (B) Closeup view of individual well (C) Growth of the anaerobe B. longum was identical in the apparatus with hypoxic epithelial compared to full anaerobic growth (D) Butyrate administration protects the epithelium from inflammatory effects of deoxycholate. Results Screening human intestinal organoids (that lack lymphocytes and are strictly of the endodermal lineage) we have generated evidence that intestinal stem cells (ISC) and IEC can be a host cell for latent HHV7 infection in some but not all people (T1). RNA-sequencing reveals that this is true latency, with ISC and IEC containing HHV7 without transcripts from any of the protein-coding genes in the HHV7 genome. Further we have observed that latent HHV7 is mosaic in the gut, not affecting all of the ISC in an individual (T1). In organoid-derived human IEC we have noted co-culture with the commensal bacteria B. longum suppresses key executors of apoptosis and pyroptosis (F2). These same effects are no longer observed with the B. longum is interacting with IEC latently infected with HHV7. A similar pattern was observed with NFkB regulators (F3). T1: Latent HHV7 is found in some intestinal organoids derived from healthy adults From the University of Michigan Translational Tissue Modeling Laboratory (TTML) organoid registry. Each donor is assigned a unique numerical ID. Viral panel is from a commercial multiplex PCR screen. Notably, donor 87 has colonic organoids with and without latent HHV7. F2: Co-culture with the commensal microbe B. longum suppresses most apoptosis and pyroptosis executors only in an organoid without latent HHV7 infection. Fragments per kilobase per millions of reads (FPKM) from RNA-sequencing of fully-differentiated organoid-derived human intestinal epithelium after 24 hours of co-culture with B.longum (Orange) or remaining axenic (without microbes; Blue). Results from cell without (left) or with (right) latent HHV7. F3: Stimulation of the NFkB inhibitor NFKBIA by co-culture with the commensal microbe B. longum is absent in organoids with latent HHV7 infection. Fragments per kilobase per millions of reads (FPKM) from RNA-sequencing of fully-differentiated organoid-derived human intestinal epithelium after 24 hours of co-culture with B.longum (Orange) or remaining axenic (without microbes; Blue). Results from cell without (left) or with (right) latent HHV7. Conclusion Latent HHV7 in the human gut epithelium may attenuate the effect of beneficial microbes like B. longum (F4). The evidence for mosaicism of latent HHV7 in the human gut adds a spatial aspect to these attenuating effects that could be relevant for specific disease processes (e.g. skip lesions in Crohn’s disease). Confirmation of these findings, estimating the true prevalence of latent HHV7 in the gut, and identification of the HHV7 latency mechanism will be required to understand the full implications of these findings. F4: A Model of Latent HHV7 Infection Attenuating Responses to Commensal Microbes. Based on our findings, latent HHV7 infection in the intestinal epithelium may leave the gut mucosa at increased risk for injury by impairing the beneficial response to commensal microbes in a spatially complex manner due to the apparent mosaic nature of latent HHV7 infection in the gut. Disclosures Jonathan L. Golob, MD/PhD, Loxo Oncology: Employment of Spouse.

Microscopy-based phenotypic profiling of infection by Staphylococcus aureus clinical isolates reveals intracellular lifestyle as a prevalent feature

Staphylococcus aureus is increasingly recognized as a facultative intracellular pathogen, although the significance and pervasiveness of its intracellular lifestyle remain controversial. Here, we applied fluorescence microscopy-based infection assays and automated image analysis to profile the interaction of 191 S. aureus isolates from patients with bone/joint infections, bacteremia, and infective endocarditis, with four host cell types, at five times post-infection. This multiparametric analysis revealed that almost all isolates are internalized and that a large fraction replicate and persist within host cells, presenting distinct infection profiles in non-professional vs. professional phagocytes. Phenotypic clustering highlighted interesting sub-groups, including one comprising isolates exhibiting high intracellular replication and inducing delayed host death in vitro and in vivo. These isolates are deficient for the cysteine protease staphopain A. This study establishes S. aureus intracellular lifestyle as a prevalent feature of infection, with potential implications for the effective treatment of staphylococcal infections.

Quantitative single-cell analysis of Leishmania major amastigote differentiation demonstrates variably extended expression of the lipophosphoglycan (LPG) virulence factor in different host cell types.

Immediately following their deposition into the mammalian host by an infected sand fly vector, Leishmania parasites encounter and are engulfed by a variety of cell types. From there, parasites may transit to other cell types, primarily macrophages or dendritic cells, where they replicate and induce pathology. During this time, Leishmania cells undergo a dramatic transformation from the motile non-replicating metacyclic stage to the non-motile replicative amastigote stage, a differentiative process that can be termed amastigogenesis. To follow this at the single cell level, we identified a suite of experimental 'landmarks' delineating different stages of amastigogenesis qualitatively or quantitatively, including new uses of amastigote-specific markers that showed interesting cellular localizations at the anterior or posterior ends. We compared amastigogenesis in synchronous infections of peritoneal and bone-marrow derived macrophages (PEM, BMM) or dendritic cells (BMDC). Overall, the marker suite expression showed an orderly transition post-infection with similar kinetics between host cell types, with the emergence of several amastigote traits within 12 hours, followed by parasite replication after 24 hours, with parasites in BMM or BMDC initiating DNA replication more slowly. Lipophosphoglycan (LPG) is a Leishmania virulence factor that facilitates metacyclic establishment in host cells but declines in amastigotes. Whereas LPG expression was lost by parasites within PEM by 48 hours, >40% of the parasites infecting BMM or BMDC retained metacyclic-level LPG expression at 72 hr. Thus L. major may prolong LPG expression in different intracellular environments, thereby extending its efficacy in promoting infectivity in situ and during cell-to-cell transfer of parasites expressing this key virulence factor.

The Antiviral Effects of 2-Deoxy-D-glucose (2-DG), a Dual D-Glucose and D-Mannose Mimetic, against SARS-CoV-2 and Other Highly Pathogenic Viruses.

Viral infection almost invariably causes metabolic changes in the infected cell and several types of host cells that respond to the infection. Among metabolic changes, the most prominent is the upregulated glycolysis process as the main pathway of glucose utilization. Glycolysis activation is a common mechanism of cell adaptation to several viral infections, including noroviruses, rhinoviruses, influenza virus, Zika virus, cytomegalovirus, coronaviruses and others. Such metabolic changes provide potential targets for therapeutic approaches that could reduce the impact of infection. Glycolysis inhibitors, especially 2-deoxy-D-glucose (2-DG), have been intensively studied as antiviral agents. However, 2-DG’s poor pharmacokinetic properties limit its wide clinical application. Herein, we discuss the potential of 2-DG and its novel analogs as potent promising antiviral drugs with special emphasis on targeted intracellular processes.

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells.

Stress granule formation is induced by numerous environmental stressors, including sodium arsenite treatment and viral infection. Accordingly, stress granules can inhibit viral propagation and function as part of the antiviral host response to numerous viral infections. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antagonizes stress granule formation, in part, via interaction between SARS-CoV-2 nucleocapsid (N) protein and Ras-GTPase-activating SH3-domain-binding protein 1 (G3BP1). However, it is unclear whether there are differential effects in different cell types. In this study, we assessed interaction between the N protein of SARS-CoV-2 S clade and G3BP1/2 in Vero and Calu-3 cells and investigated the effect of various SARS-CoV-2 strains on sodium arsenite-induced stress granule formation. Our data show that SARS-CoV-2 S clade N protein interacts with both G3BP1 and G3BP2 more strongly in Calu-3 vs. Vero cells. Consistent with this observation, infection with SARS-CoV-2 S clade induces stress granule formation in Vero but not in Calu-3 cells. However, infection with SARS-CoV-2 S clade, as well as other SARS-CoV-2 variants, inhibits sodium arsenite-induced stress granule formation in both cell lines. Taken together, our results show differential effects of SARS-CoV-2 infection on stress granule formation that is dependent on host cell type, rather than virus strain type.

In vitro evaluation of Resveratrol as a potential pre-exposure prophylactic drug against Trypanosoma cruzi infection

Chagas' disease or American trypanosomiasis, caused by Trypanosoma cruzi infection, is an endemic disease in Latin America, which has spread worldwide in the past years. The drugs presently used for treatment have shown limited efficacy due to the appearance of resistant parasites and severe side effects. Some of the most recent studies on anti-parasitic drugs have been focused on protein acetylation, a reversible reaction modulated by Acetyl Transferases (KATs) and Deacetylases (KDACs). We have previously reported the anti-parasite activity of resveratrol (RSV), an activator of KDACs type III (or sirtuins), and showed that this drug can reduce the growth of T. cruzi epimastigotes and the infectivity of trypomastigotes. Since RSV is now widely used in humans due to its beneficial effects as an antioxidant, it has become an attractive candidate as a repurposing drug. In this context, the aim of the present study was to evaluate the ability of this drug to protect three different types of host cells from parasite infection. RSV treatment before parasite infection reduced the percentage of infected cells by 50–70% depending on the cell type. Although the mammalian cell lines tested showed different sensitivity to RSV, apoptosis was not significantly affected, showing that RSV was able to protect cells from infection without the activation of this process. Since autophagy has been described as a key process in parasite invasion, we also monitored this process on host cells pretreated with RSV. The results showed that, at the concentrations and incubation times tested, autophagy was not induced in any of the cell types evaluated. Our results show a partial protective effect of RSV in vitro, which justifies extending studies to an in vivo model to elucidate the mechanism by which this effect occurs.

Fit to dwell in many places - The growing diversity of intracellular Salmonella niches.

Salmonella enterica is capable of invading different host cell types including epithelial cells and M cells during local infection, and immune cells and fibroblasts during the subsequent systemic spread. The intracellular lifestyles of Salmonella inside different cell types are remarkable for their distinct residential niches, and their varying replication rates. To study this, researchers have employed different cell models, such as various epithelial cells, immune cells, and fibroblasts. In epithelial cells, S. Typhimurium dwells within modified endolysosomes or gains access to the host cytoplasm. In the cytoplasm, the pathogen is exposed to the host autophagy machinery or poised for rapid multiplication, whereas it grows at a slower rate or remains dormant within the endomembrane-bound compartments. The swift bimodal lifestyle is not observed in fibroblasts and immune cells, and it emerges that these cells handle intracellular S. Typhimurium through different clearance machineries. Moreover, in these cell types S. Typhimurium grows withing modified phagosomes of distinct functional composition by adopting targeted molecular countermeasures. The preference for one or the other intracellular niche and the diverse cell type-specific Salmonella lifestyles are determined by the complex interactions between a myriad of bacterial effectors and host factors. It is important to understand how this communication is differentially regulated dependent on the host cell type and on the distinct intracellular growth rate. To support the efforts in deciphering Salmonella invasion across the different infection models, we provide a systematic comparison of the findings yielded from cell culture models. We also outline the future directions towards a better understanding of these differential Salmonella intracellular lifestyles.

The In Vitro Replication, Spread, and Oncolytic Potential of Finnish Circulating Strains of Herpes Simplex Virus Type 1.

Herpes simplex virus type 1 (HSV-1) is the only FDA- and EMA- approved oncolytic virus, and accordingly, many potential oncolytic HSVs (oHSV) are in clinical development. The utilized oHSV parental strains are, however, mostly based on laboratory reference strains, which may possess a compromised cytolytic capacity in contrast to circulating strains of HSV-1. Here, we assess the phenotype of thirty-six circulating HSV-1 strains from Finland to uncover their potential as oHSV backbones. First, we determined their capacity for cell-to-cell versus extracellular spread, to find strains with replication profiles favorable for each application. Second, to unfold the differences, we studied the genetic diversity of two relevant viral glycoproteins (gB/UL27, gI/US7). Third, we examined the oncolytic potential of the strains in cells representing glioma, lymphoma, and colorectal adenocarcinoma. Our results suggest that the phenotype of a circulating isolate, including the oncolytic potential, is highly related to the host cell type. Nevertheless, we identified isolates with increased oncolytic potential in comparison with the reference viruses across many or all of the studied cancer cell types. Our research emphasizes the need for careful selection of the backbone virus in early vector design, and it highlights the potential of clinical isolates as backbones in oHSV development.

Disruption of Toxoplasma gondii-Induced Host Cell DNA Replication Is Dependent on Contact Inhibition and Host Cell Type.

ABSTRACTThe protozoan Toxoplasma gondii is a highly successful obligate intracellular parasite that, upon invasion of its host cell, releases an array of host-modulating protein effectors to counter host defenses and further its own replication and dissemination. Early studies investigating the impact of T. gondii infection on host cell function revealed that this parasite can force normally quiescent cells to activate their cell cycle program. Prior reports by two independent groups identified the dense granule protein effector HCE1/TEEGR as being solely responsible for driving host cell transcriptional changes through its direct interaction with the cyclin E regulatory complex DP1 and associated transcription factors. Our group independently identified HCE1/TEEGR through the presence of distinct repeated regions found in a number of host nuclear targeted parasite effectors and verified its central role in initiating host cell cycle changes. Additionally, we report here the time-resolved kinetics of host cell cycle transition in response to HCE1/TEEGR, using the fluorescence ubiquitination cell cycle indicator reporter line (FUCCI), and reveal the existence of a block in S-phase progression and host DNA synthesis in several cell lines commonly used in the study of T. gondii. Importantly, we have observed that this S-phase block is not due to additional dense granule effectors but rather is dependent on the host cell line background and contact inhibition status of the host monolayer in vitro. This work highlights intriguing differences in the host response to reprogramming by the parasite and raises interesting questions regarding how parasite effectors differentially manipulate the host cell depending on the in vitro or in vivo context.IMPORTANCE Toxoplasma gondii chronically infects approximately one-third of the global population and can produce severe pathology in immunologically immature or compromised individuals. During infection, this parasite releases numerous host-targeted effector proteins that can dramatically alter the expression of a variety of host genes. A better understanding of parasite effectors and their host targets has the potential to not only provide ways to control infection but also inform us about our own basic biology. One host pathway that has been known to be altered by T. gondii infection is the cell cycle, and prior reports have identified a parasite effector, known as HCE1/TEEGR, as being responsible. In this report, we further our understanding of the kinetics of cell cycle transition induced by this effector and show that the capacity of HCE1/TEEGR to induce host cell DNA synthesis is dependent on both the cell type and the status of contact inhibition.

Host cell targeting of novel antimycobacterial 4-aminosalicylic acid derivatives with tuftsin carrier peptides

Mycobacterium tuberculosis is an intracellular pathogen and the uptake of the antimycobacterial compounds by host cells is limited. Novel antimycobacterials effective against intracellular bacteria are needed. New N-substituted derivatives of 4-aminosalicylic acid have been designed and evaluated. To achieve intracellular efficacy and selectivity, these compounds were conjugated to tuftsin peptides via oxime or amide bonds. These delivery peptides can target tuftsin- and neuropilin receptor-bearing cells, such as macrophages and various other cells of lung origin. We have demonstrated that the in vitro antimycobacterial activity of the 4-aminosalicylic derivatives against M. tuberculosis H37Rv was preserved in the peptide conjugates. The free drugs were ineffective on infected cells, but the conjugates were active against the intracellular bacteria and have the selectivity on various types of host cells. The intracellular distribution of the carrier peptides was assessed, and the peptides internalize and display mainly in the cytosol in a concentration-dependent manner. The penetration ability of the most promising carrier peptide OT5 was evaluated using Transwell-inserts and spheroids. The pentapeptide exhibited time- and concentration-dependent penetration across the non-contact monolayers. Also, the pentapeptide has a fair penetration rate towards the center of spheroids formed of EBC-1 cells.

Targeting mucin hypersecretion in COVID-19 therapy.

Targeting mucin hypersecretion in COVID-19 therapy.

SARS-CoV-2: Receptor and Co-receptor Tropism Probability.

The recent pandemic which arose from China, is caused by a pathogenic virus named “severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2)”. Its rapid global expansion has inflicted an extreme public health concern. The attachment of receptor-binding domains (RBD) of the spike proteins (S) to the host cell’s membrane, with or without the help of other cellular components such as proteases and especially co-receptors, is required for the first stage of its pathogenesis. In addition to humans, angiotensin-converting enzyme 2 (ACE2) is found on a wide range of vertebrate host’s cellular surface. SARS-CoV-2 has a broad spectrum of tropism; thus, it can infect a vast range of tissues, organs, and hosts; even though the surface amino acids of the spike protein conflict in the receptor-binding region. Due to the heterogeneous ACE2 distribution and the presence of different domains on the SARS-CoV-2 spike protein for binding, the virus entry into diverse host cell types may depend on the host cells’ receptor presentation with or without co-receptors. This review investigates multiple current types of receptor and co-receptor tropisms, with other molecular factors alongside their respective mechanisms, which facilitate the binding and entry of SARS-CoV-2 into the cells, extending the severity of the coronavirus disease 2019 (COVID-19). Understanding the pathogenesis of COVID-19 from this perspective can effectively help prevent this disease and provide more potent treatment strategies, particularly in vulnerable people with various cellular-level susceptibilities.

Design, synthesis, and biological evaluation of novel 2′-methyl-2′-fluoro-6-methyl-7-alkynyl-7-deazapurine nucleoside analogs as anti-Zika virus agents

Zika virus (ZIKV) is a mosquito-borne flavivirus and outbreaks of ZIKV have been reported in Africa, Americas and other parts of the world lately. The ZIKV epidemic has received extensive attention due to its ability to cause serious medical consequences and complications such as microcephaly and Guillain-Barre syndrome in recent years. Up to now, there are no specific treatments or vaccines available for ZIKV infection, which highlights the urgent need for developing new therapies. In this work, we designed and synthesized a series of novel 6-methyl-7-acetylenenyl-7-deazapurine nucleoside analogs as potential inhibitors of ZIKV replication. The biological activities against ZIKV replication were evaluated and the structure-activity relationship (SAR) was also studied. Among the compounds evaluated, nucleoside analog 38 (EC50 = 2.8 ± 0.8 μM, EC90 = 6.8 ± 2.3 μM) showed the most potent anti-ZIKV activity with low cytotoxicity (CC50 = 54.1 ± 6.9 μM) in an A549 based cellular model. The inhibitory activity of 38 was about 5 times more potent than the positive control NITD008. Notably, 38 showed similar inhibition potency against different ZIKV strains (ZG-01 and MR766) in a variety of host cell types including SNB19, A549, Huh7, Vero. In addition, 38 (Kd = 1.87 μM) has a stronger affinity to ZIKV RNA-dependent RNA polymerase (RdRp) protein than NITD008 (Kd = 3.43 μM) in the non-phosphorylation assay. These results indicated that compound 38 may serve as a promising candidate in future anti-ZIKV drug discovery.

Type and abundance of sialic acid receptors on host cell membrane affect infectivity and viral titer of different strains of Newcastle disease virus

Cell-based vaccine manufacture is a very cost-effective approach for animal vaccine production. Newcastle disease virus (NDV) can infect a wide range of host cells, but the viral titers of cell culture are too low to meet the vaccine manufacture. In this study, we explored the selectivity of NDV vaccine strains LaSota and Mukteswar to type of sialic acid receptors and demonstrated the relationship between receptor expression levels on cell membrane and viral titers of cell culture. The results suggested that NDV strain LaSota preferentially binds to Neu5Ac-2-S-α-2,6 Gal10Me receptor (SAα2,6 Gal) and strain Mukteswar selectively binds to Neu5Ac-α-2,3 Gal-β-1,4Glc (SAα2,3 Gal) receptor. Subsequently, the expression levels of SAα2,3 Gal and SAα2,6 Gal receptors on BHK-21 cell membrane were adjusted by overexpression and RNAi assays. The results indicated that the viral titers of NDV strains LaSota and Mukteswar in cell culture were positively correlated with the expression levels of SAα2,6 Gal and SAα2,3 Gal receptors on host cell membrane respectively. In conclusion, our studies provide an understanding of the relationship between infectivity of NDV different strains and receptor types of host cell, and provide a method to increase viral titer of NDV for cell-based vaccine production.

Roles of Glycans and Non-glycans on the Epithelium and in the Immune System in H1-H18 Influenza A Virus Infections.

The large variation of influenza A viruses (IAVs) in various susceptible hosts and their rapid evolution, which allows host/tissue switching, host immune escape, vaccine escape, and drug resistance, are difficult challenges for influenza control in all countries worldwide. Access and binding of the IAV to actual receptors at endocytic sites is critical for the establishment of influenza infection. In this chapter, the progress in identification of and roles of glycans and non-glycans on the epithelium and in the immune system in H1-H18 IAV infections are reviewed. The first part of the review is on current knowledge of H1-H16 IAV receptors on the epithelium including sialyl glycans, other negatively charged glycans, and annexins. The second part of the review focuses on H1-H16 IAV receptors in the immune system including acidic surfactant phospholipids, Sia on surfactant proteins, the carbohydrate recognition domain (CRD) of surfactant proteins, Sia on mucins, Sia and C-type lectins on macrophages and dendritic cells, and Sia on NK cells. The third part of the review is about a possible H17-H18 IAV receptor. Binding of these receptors to IAVs may result in inhibition or enhancement of IAV infection depending on their location, host cell type, and IAV strain. Among these receptors, host sialyl glycans are key determinants of viral hemagglutinin (HA) lectins for H1-H16 infections. HA must acquire mutations to bind to sialyl glycans that are dominant on a new target tissue when switching to a new host for efficient transmission and to bind to long sialyl glycans found in the case of seasonal HAs with multiple glycosylation sites as a consequence of immune evasion. Although sialyl receptors/C-type lectins on immune cells are decoy receptors/pathogen recognition receptors for capturing viral HA lectin/glycans protecting HA antigenic sites, some IAV strains do not escape, such as by release with neuraminidase, but hijack these molecules to gain entry and replication in immune cells. An understanding of the virus-host battle tactics at the receptor level might lead to the establishment of novel strategies for effective control of influenza.

Photochemical Internalization as a New Strategy to Enhance Efficacy of Antimicrobial Agents Against Intracellular Infections.

Pathogens such as Staphylococcus aureus are able to survive in many types of host cells including phagocytes such as neutrophils and macrophages, thereby resulting in intracellular infections. Treatment of intracellular infections by conventional antimicrobials (e.g., antibiotics) is often ineffective due to low intracellular efficacy of the drugs. Thus, novel techniques which can enhance the activity of antimicrobials within cells are highly demanded. Our recent studies have shown that photochemical internalization (PCI) is a promising approach for improving the efficacy of antibiotics such as gentamicin against intracellular staphylococcal infection. In this chapter, we describe the protocols aiming to study the potential of PCI-antibiotic treatment for intracellular infections in vitro and in vivo using a RAW 264.7 cell infection model and a zebrafish embryo infection model. Proof of concept of this approach is demonstrated. The protocols are expected to prompt further development of PCI-antimicrobial based novel therapies for clinically challenging infectious diseases associated with intracellular survival of pathogens.

Toxoplasma gondii Induces Apoptosis via Endoplasmic Reticulum Stress-Derived Mitochondrial Pathway in Human Small Intestinal Epithelial Cell-Line.

Toxoplasma gondii, an intracellular protozoan parasite that infects one-third of the world’s population, has been reported to hijack host cell apoptotic machinery and promote either an anti- or proapoptotic program depending on the parasite virulence and load and the host cell type. However, little is known about the regulation of human FHs 74 small intestinal epithelial cell viability in response to T. gondii infection. Here we show that T. gondii RH strain tachyzoite infection or ESP treatment of FHs 74 Int cells induced apoptosis, mitochondrial dysfunction and ER stress in host cells. Pretreatment with 4-PBA inhibited the expression or activation of key molecules involved in ER stress. In addition, both T. gondii and ESP challenge-induced mitochondrial dysfunction and cell death were dramatically suppressed in 4-PBA pretreated cells. Our study indicates that T. gondii infection induced ER stress in FHs 74 Int cells, which induced mitochondrial dysfunction followed by apoptosis. This may constitute a potential molecular mechanism responsible for the foodborne parasitic disease caused by T. gondii.

The Redox Homeostasis of Skeletal Muscle Cells Regulates Stage Differentiation of Toxoplasma gondii.

Toxoplasma gondii is an obligatory intracellular parasite that causes persistent infections in birds and mammals including ~30% of the world’s human population. Differentiation from proliferative and metabolically active tachyzoites to largely dormant bradyzoites initiates the chronic phase of infection and occurs predominantly in brain and muscle tissues. Here we used murine skeletal muscle cells (SkMCs) to decipher host cellular factors that favor T. gondii bradyzoite formation in terminally differentiated and syncytial myotubes, but not in proliferating myoblast precursors. Genome-wide transcriptome analyses of T. gondii-infected SkMCs and non-infected controls identified ~6,500 genes which were differentially expressed (DEGs) in myotubes compared to myoblasts, largely irrespective of infection. On the other hand, genes related to central carbohydrate metabolism, to redox homeostasis, and to the Nrf2-dependent stress response pathway were enriched in both infected myoblast precursors and myotubes. Stable isotope-resolved metabolite profiling indicated increased fluxes into the oxidative branch of the pentose phosphate pathway (OxPPP) in infected myoblasts and into the TCA cycle in infected myotubes. High OxPPP activity in infected myoblasts was associated with increased NADPH/NADP+ ratio while myotubes exhibited higher ROS levels and lower expression of anti-oxidants and detoxification enzymes. Pharmacological reduction of ROS levels in SkMCs inhibited bradyzoite differentiation, while increased ROS induced bradyzoite formation. Thus, we identified a novel host cell-dependent mechanism that triggers stage conversion of T. gondii into persistent tissue cysts in its natural host cell type.

Uropathogenic Escherichia coli Shows Antibiotic Tolerance and Growth Heterogeneity in an In Vitro Model of Intracellular Infection.

Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can invade different types of host cells. To compare the pharmacodynamic properties of antibiotics against intra- and extracellular UPEC, an in vitro model of intracellular infection was established in J774 mouse macrophages infected by the UPEC strain CFT073. We tested antibiotics commonly prescribed against urinary tract infections (gentamicin, ampicillin, nitrofurantoin, trimethoprim, sulfamethoxazole, and ciprofloxacin) and the investigational fluoroquinolone finafloxacin. The metabolic activity of individual bacteria was assessed by expressing the fluorescent reporter protein TIMERbac within CFT073. Concentration-response experiments revealed that all tested antibiotics were much less effective against intracellular bacteria than extracellular ones. Most antibiotics, except fluoroquinolones, were unable to reach a bactericidal effect intracellularly at clinically achievable concentrations. Ciprofloxacin and finafloxacin killed 99.9% of extracellular bacteria at concentrations around the MIC, while for intracellular bacteria, concentrations more than 100× over the MIC were required to achieve a bactericidal effect. Time-kill curves showed that finafloxacin was more rapidly bactericidal in acidic medium than at neutral pH, while the reverse observation was made for ciprofloxacin. Intracellularly, kill curves showed biphasic kinetics for both fluoroquinolones, suggesting the presence of drug-tolerant subpopulations. Flow cytometry analysis of TIMERbac fluorescence revealed a marked heterogeneity in intracellular growth of individual bacteria, suggesting that the presence of subpopulations reaching a state of metabolic dormancy was the main reason for increased antibiotic tolerance of intracellular UPEC.

Versatile human in vitro triple coculture model coincubated with adhered gut microbes reproducibly mimics pro-inflammatory host-microbe interactions in the colon.

The colonic epithelial barrier is vital to preserve gut and host health by maintaining the immune homeostasis between host and microbes. The mechanisms underlying beneficial or harmful host-microbe interactions are poorly understood and impossible to study in vivo given the limited accessibility and ethical constraints. Moreover, existing in vitro models lack the required cellular complexity for the routine, yet profound, analysis of the intricate interplay between different types of host and microbial cells. We developed and characterized a broadly applicable, easy-to-handle in vitro triple coculture model that combines chemically-induced macrophage-like, goblet and epithelial cells covered by a mucus layer, which can be coincubated with complex human-derived gut microbiota samples for 16h. Comparison with a standard epithelial monolayer model revealed that triple cocultures produce thicker mucus layers, morphologically organize in a network and upon exposure to human-derived gut microbiota samples, respond via pro-inflammatory cytokine production. Both model systems, however, were not suffering from cytotoxic stress or different microbial loads, indicating that the obtained endpoints were caused by the imposed conditions. Addition of the probiotic Lactobacillus rhamnosus GG to assess its immunomodulating capacity in the triple coculture slightly suppressed pro-inflammatory cytokine responses, based on transcriptomic microarray analyses. TNF conditioning of the models prior to microbial exposure did not cause shifts in cytokines, suggesting a strong epithelial barrier in which TNF did not reach the basolateral side. To conclude, the triple coculture model is tolerable towards manipulations and allows to address mechanistic host-microbe research questions in a stable in vitro environment.