Biology subjects

O'Riordan, M.

Publications and source records attributed to O'Riordan, M..

7 recordsLinked to original sources

bioRxiv2020

Macrophage aggresome-like induced structures are flexible organizing platforms for immune signaling

Macrophages adopt a pro-inflammatory phenotype in response to environmental challenges in a process that often coincides with the formation of transient cytosolic p62/SQSTM1 inclusions containing ubiquitinated proteins in structures known as aggresome-like induced structures (ALIS). Although described as stress-induced inclusions that accumulate aggregated proteins, little direct evidence supports their hypothesized structural role in the context of immune stimulation. Here, we showed that these structures in primary macrophages are induced by multiple microbialbased ligands, including exposure to cytosolic double-stranded DNA. Rather than accumulating aggregated proteins, we observed that ubiquitinated proteins form a ring-shaped structure around the perimeter of these circular foci. We identified that different microbial stimuli induced the formation of ubiquitin-positive foci with distinct characteristics and we observed selective recruitment of context-dependent immune regulators. Our findings are consistent with a model where these ubiquitin-containing structures act as adaptable organizing centers for innate immune signaling. SUMMARYCharbonneau et al. demonstrate that ubiquitin- and p62-containing cytosolic ring-shaped structures induced by bacterial infections, microbial ligands and cytosolic double-stranded DNA contain context-dependent immune regulators, revealing an important insight on the cellular architecture required to coordinate signal transduction in macrophage.

immunology↗

bioRxiv2020

Comparative transcriptional profiling of the early host response to infection by typhoidal and non-typhoidal Salmonella serovars in human intestinal organoids

Salmonella enterica represents over 2500 serovars associated with a wide-ranging spectrum of disease; from self-limiting gastroenteritis to invasive infection caused by non-typhoidal serovars (NTS) and typhoidal serovars, respectively. Host factors strongly influence infection outcome as malnourished or immunocompromised individuals can develop invasive infections from NTS, however, comparative host responses to individual serovars have been difficult to perform due to reliance on poorly representative model systems. Here we used human intestinal organoids (HIOs), a three-dimensional "gut-like" in vitro system derived from human embryonic stem cells, to elucidate similarities and differences in host responses to NTS and typhoidal serovars. HIOs discriminated between the two most prevalent NTS, Salmonella enterica serovar Typhimurium (STM) and Salmonella enterica serovar Enteritidis (SE), and typhoidal serovar Salmonella enterica serovar Typhi (ST) in epithelial cell invasion, replication and transcriptional responses. Pro-inflammatory signaling and cytokine output was reduced in ST-infected HIOs compared to NTS infections, reflecting early stages of NTS and typhoidal diseases. While we predicted that ST would induce a distinct transcriptional profile from the NTS strains, more nuanced expression profiles emerged. Notably, pathways involved in cell cycle, metabolism and mitochondrial functions were downregulated in STM-infected HIOs and upregulated in SE-infected HIOs. These results correlated with elevated levels of reactive oxygen species production in SE-infected HIOs compared to mock-infected HIOs. Collectively, these results suggest that the HIO model is well suited to reveal host transcriptional programming specific to individual Salmonella serovars, and that individual NTS may provoke unique host epithelial responses during intestinal stages of infection. Author SummarySalmonella enterica is the major causative agent of bacterial infections associated with contaminated food and water. Salmonella enterica consists of over 2500 serovars of which Typhimurium (STM), Enteritidis (SE) and Typhi (ST) are the three major serovars with medical relevance to humans. These serovars elicit distinctive immune responses and cause different diseases in humans, including self-limiting diarrhea, gastroenteritis and typhoid fever. Differences in the human host response to these serovars are likely to be a major contributing factor to distinct disease outcomes but are not well characterized, possibly due to the limitations of human-derived physiological infection models. Unlike immortalized epithelial cell culture models, human intestinal organoids (HIOs) are three-dimensional structures derived from embryonic stem cells that differentiate into intestinal mesenchymal and epithelial cells, mirroring key organizational aspects of the intestine. In this study, we used HIOs to monitor transcriptional changes during early stages of STM, SE and ST infection. Our comparative analysis showed that HIO inflammatory responses are the dominant response in all infections, but ST infection induces the weakest upregulation of inflammatory mediators relative to the other serovars. In addition, we identified several cellular processes, including cell cycle and mitochondrial functions, that were inversely regulated between STM and SE infection despite these serovars causing similar localized intestinal infection in humans. Our findings reinforce HIOs as an emerging model system to study Salmonella serovar infection, and provide global host transcriptional response profiles as a foundation for understanding human infection outcomes.

microbiology↗

bioRxiv2020

Salmonella enterica serovar Typhimurium SPI-1 and SPI-2 shape the transcriptional landscape of epithelial cells in a human intestinal organoid model system

The intestinal epithelium is a primary interface for engagement of the host response by foodborne pathogens, like Salmonella enterica serovar Typhimurium (STm). While interaction of STm with the mammalian host has been well studied in vitro in transformed epithelial cell lines or in the complex intestinal environment in vivo, few tractable models recapitulate key features of the intestinal epithelium. Human intestinal organoids (HIOs) contain a polarized epithelium with functionally differentiated cell subtypes, including enterocytes and goblet cells. HIOs contain luminal space that supports bacterial replication and are more amenable to experimental manipulation than animals while more reflective of physiological epithelial responses. Here we use the HIO model to define transcriptional responses of the host epithelium to STm infection, also determining host pathways dependent on Salmonella Pathogenicity Island-1 (SPI-1) and -2 (SPI-2) encoded Type 3 secretion systems (T3SS). Consistent with prior findings, we find that STm strongly stimulates pro-inflammatory gene expression. Infection-induced cytokine gene expression was rapid, transient and largely independent of SPI-1 T3SS-mediated invasion, likely due to continued luminal stimulation. Notably, STm infection led to significant down-regulation of host genes associated with cell cycle and DNA repair, an effect that required SPI-1 and SPI-2 T3SS. The transcriptional profile of cell cycle-associated target genes implicates multiple miRNAs as likely mediators of STm-dependent cell cycle suppression. These findings from Salmonella-infected HIOs delineate common and distinct contributions of SPI-1 and SPI-2 T3SSs in inducing early host responses during enteric infection and reveal host cell cycle as a potential target during STm intracellular infection. ImportanceSalmonella enterica serovar Typhimurium (STm) causes a significant health burden worldwide, yet host responses to initial stages of intestinal infection remain poorly understood. Due to differences in infection outcome between mice and humans, evaluating physiological host responses driven by major virulence determinants of Salmonella have been difficult to date. Here we use the 3D human intestinal organoid model to define early responses to infection with wildtype STm and mutants defective in the SPI-1 or SPI-2 Type 3 secretion systems. Both secretion system mutants show defects in a mouse model of oral Salmonella infection but the specific contributions of each secretion system are less well understood. We show that STm upregulates pro-inflammatory pathways independently of either secretion system while downregulation of host cell cycle pathways is dependent on both SPI-1 and SPI-2. These findings lay the groundwork for future studies investigating how SPI-1- and SPI-2-driven host responses affect infection outcome and show the potential of this model to study host-pathogen interactions with other serovars to understand how initial interactions with the intestinal epithelium may affect pathogenesis.

microbiology↗

bioRxiv2020

The branched chain aminotransferase IlvE promotes growth, stress resistance, and pathogenesis of Listeria monocytogenes

The bacterial plasma membrane is a key interface during pathogen-host interactions, and membrane composition enhances resistance against host antimicrobial defenses. Branched chain fatty acids (BCFAs) are the major plasma membrane component in the intracellular Gram-positive pathogen Listeria monocytogenes (Lm) and BCFA metabolism is essential for Lm growth and virulence. BCFA synthesis requires branched chain amino acids (BCAAs), and the BCAA Isoleucine (Ile) is a necessary substrate for the predominant membrane anteiso-BCFAs (ai-BCFAs) as well as an environmental signal for virulence regulation in Lm. In this study, we explored how two proteins that metabolize or sense Ile contribute to Lm growth, BCFA metabolism, and virulence. The IlvE aminotransferase incorporates Ile into ai-BCFAs, while CodY is an Ile-sensing regulator that coordinates BCAA synthesis and virulence gene expression. Analysis of deletion mutants lacking IlvE ({Delta}ilvE) or CodY ({Delta}codY) revealed a major role for IlvE under nutrient restriction and stress conditions. Cultures of the {Delta}ilvE mutant contained proportionally less ai-BCFAs relative to wild type, while of the {Delta}codY mutant had a lower proportion of ai-BCFAs in stationary phase, despite containing more cell-associated Ile. Both {Delta}ilvE and {Delta}codY mutants required exogenous Ile for optimal growth, but the {Delta}ilvE mutant had an absolute requirement for Valine and Leucine when Ile was absent. IlvE was also necessary for resistance to membrane stress, cell-to-cell spread, infection of primary macrophages, and virulence in mice. Our findings implicate IlvE as an integral aspect of Lm stress resistance and emphasize the central importance of Ile in Lm growth and virulence.

microbiology↗

bioRxiv2018

Central carbon metabolism is an intrinsic factor for optimal replication of a norovirus

The metabolic pathways of central carbon metabolism, glycolysis and oxidative phosphorylation (OXPHOS), are important host factors that determine the outcome of viral infections and can therefore be manipulated by some viruses to favor infection. However, mechanisms of metabolic modulation and their effects on viral replication vary widely. Herein, we present the first metabolomics profile of norovirus-infected cells, which revealed increases in glycolysis, OXPHOS, and the pentose phosphate pathway (PPP) during murine norovirus infection. Inhibiting glycolysis with 2-deoxyglucose (2DG) in transformed and primary macrophages revealed that host cell metabolism is an important factor for optimal murine norovirus (MNV) infection. 2DG affected an early stage in the viral life cycle after viral uptake and capsid uncoating, leading to decreased levels of viral protein translation and viral RNA replication. The requirement of central carbon metabolism was specific for MNV (but not astrovirus) infection, independent of the Type I interferon antiviral response, and unlikely to be due to a lack of host cell nucleotide synthesis. MNV infection increased activation of the protein kinase Akt, but not AMPK, two master regulators of cellular metabolism, suggesting Akt signaling may play a role in upregulating central carbon metabolism during norovirus infection. In conclusion, our findings suggest that the metabolic state of target cells is an intrinsic host factor that determines the extent of norovirus replication and implicates metabolism as a virulence determinant. They further implicate cellular metabolism as a novel therapeutic target for norovirus infections and improvements of current human norovirus culture systems.\n\nIMPORTANCEViruses depend on the host cells they infect to provide the machinery and substrates for replication. Host cells are highly dynamic systems that can alter their intracellular environment and metabolic behavior, which may be helpful or inhibitory for an infecting virus. In this study, we show that macrophages, a target cell of murine norovirus (MNV), increase central carbon metabolism upon viral infection, which is important for early steps in MNV infection. Human noroviruses (hNoV) are a major cause of gastroenteritis globally, causing enormous morbidity and economic burden. Currently, no effective antivirals or vaccines exist for hNoV, mainly due to the lack of high efficiency in vitro culture models for their study. Thus, insights gained from the MNV model may reveal aspects of host cell metabolism that can be targeted for improving hNoV cell culture systems and for developing effective antiviral therapies.

microbiology↗

bioRxiv2018

Perturbation of ubiquitin homeostasis promotes macrophage oxidative defenses

Innate immune responses rely on specific pattern recognition receptors that induce downstream signaling cascades and promote inflammatory responses. Emerging evidence suggests that cells may also recognize alterations in cellular processes induced by infection. Protein ubiquitination is a post-translational modification essential for maintaining cellular homeostasis, and infection can cause global alterations in the host ubiquitin proteome. Here we used a chemical biology approach to perturb the cellular ubiquitin proteome as a simplified model to study the direct effect of ubiquitin homeostasis on macrophage responses. We show that perturbation of ubiquitin homeostasis results in a rapid and transient burst of reactive oxygen species (ROS) that promotes macrophage anti-infective capacity. ROS production was dependent on the activity of the phagocyte NADPH oxidase NOX2 and was associated with an increase in intracellular calcium. Our findings suggest that major changes in the host ubiquitin landscape may be a potent signal to rapidly deploy innate immune defenses.

immunology↗

bioRxiv2018

Mitochondria-derived vesicles deliver antimicrobial payload to control phagosomal bacteria

Pathogenic bacteria taken up into the macrophage phagosome are the target of many anti-microbial effector molecules. Although mitochondria-derived antimicrobial effectors such as reactive oxygen species (mROS) are reported to aid in bacterial killing, it is unclear how these effectors reach bacteria within the phagosomal lumen. To examine the crosstalk between mitochondria and phagosomes, we monitored the production and the spatial localization of mROS during methicillin-resistant Staphylococcus aureus (MRSA) infection. We showed here mROS, specifically hydrogen peroxide (mH2O2) can be delivered into phagosomes via infection-induced mitochondria-derived vesicles, which are generated in a Parkin-dependent manner. Accumulation of mH2O2 in phagosomes required TLR signaling and the mitochondrial superoxide dismutase, Sod2, which converts superoxide into mH2O2. These data highlight a novel mechanism by which the mitochondrial redox capacity enhances macrophage antimicrobial function by delivering mitochondria-derived effector molecules into bacteria-containing phagosomes.

immunology↗