January 2025
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41 Reads
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January 2025
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41 Reads
September 2024
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19 Reads
The yellow fever virus 17D (YFV-17D) live attenuated vaccine is considered one of the successful vaccines ever generated associated with high antiviral immunity, yet the signaling mechanisms that drive the response in infected cells are not understood. Here, we provide a molecular understanding of how metabolic stress and innate immune responses are linked to drive type I IFN expression in response to YFV-17D infection. Comparison of YFV-17D replication with its parental virus, YFV-Asibi, and a related dengue virus revealed that IFN expression requires RIG-I-like Receptor signaling through MAVS, as expected. However, YFV-17D uniquely induces mitochondrial respiration and major metabolic perturbations, including hyperactivation of electron transport to fuel ATP synthase. Mitochondrial hyperactivity generates reactive oxygen species (mROS) and peroxynitrite, blocking of which abrogated IFN expression in non-immune cells without reducing YFV-17D replication. Scavenging ROS in YFV-17D-infected human dendritic cells increased cell viability yet globally prevented expression of IFN signaling pathways. Thus, adaptation of YFV-17D for high growth uniquely imparts mitochondrial hyperactivity generating mROS and peroxynitrite as the critical messengers that convert a blunted IFN response into maximal activation of innate immunity essential for vaccine effectiveness.
May 2024
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14 Reads
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2 Citations
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection involves an initial viral infection phase followed by a host‐response phase that includes an eicosanoid and cytokine storm, lung inflammation and respiratory failure. While vaccination and early anti‐viral therapies are effective in preventing or limiting the pathogenic host response, this latter phase is poorly understood with no highly effective treatment options. Inhibitors of soluble epoxide hydrolase (sEH) increase levels of anti‐inflammatory molecules called epoxyeicosatrienoic acids (EETs). This study aimed to investigate the impact of sEH inhibition on the host response to SARS‐CoV‐2 infection in a mouse model with human angiotensin‐converting enzyme 2 (ACE2) expression. Mice were infected with SARS‐CoV‐2 and treated with either vehicle or the sEH inhibitor 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) urea (TPPU). At day 5 post‐infection, SARS‐CoV‐2 induced weight loss, clinical signs, a cytokine storm, an eicosanoid storm, and severe lung inflammation with ~50% mortality on days 6–8 post‐infection. SARS‐CoV‐2 infection induced lung expression of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX) pathway genes, while suppressing expression of most cytochrome P450 genes. Treatment with the sEH inhibitor TPPU delayed weight loss but did not alter clinical signs, lung cytokine expression or overall survival of infected mice. Interestingly, TPPU treatment significantly reversed the eicosanoid storm and attenuated viral‐induced elevation of 39 fatty acids and oxylipins from COX, LOX and P450 pathways, which suggests the effects at the level of PLA2 activation. The suppression of the eicosanoid storm by TPPU without corresponding changes in lung cytokines, lung inflammation or mortality reveals a surprising dissociation between systemic oxylipin and cytokine signaling pathways during SARS‐CoV‐2 infection and suggests that the cytokine storm is primarily responsible for morbidity and mortality in this animal model.
May 2024
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1 Read
The Journal of Immunology
Francisella tularensis (Ftt) is a highly virulent bacterial pathogen whose vaccine offers only short-lived immunity. While CD4+ T cells are required for protection, the mechanism behind waning immunity remains unclear. One potential reason is that the local environment induced by the bacteria inhibits T cell function. We therefore carried out a multi-omics study of the infection microenvironment following vaccination and secondary challenge in mice. Flow cytometry and single cell sequencing analysis revealed that during Ftt secondary challenge, the CD4+ T cells in vaccinated animals show a delayed proliferative response and produced high levels of IFN-γ which correlated with an up-regulation of indoleamine 2,3-dioxygenase (IDO1) by endothelial cells. Importantly, increased IDO1 expression corresponded with decreased levels of tryptophan in the lung as measured by LC/MS. Given the requirement for tryptophan in optimal T cell proliferation, we investigated if IDO1 expression was spatially associated with sites of bacterial replication. We performed multiplex immunohistochemistry targeting bacterial antigen, immune cells, and nutrient depleting enzymes and quantified the proximity of CD4+ T cells to IDO1-expressing cells which arose just outside the points of bacterial replication. Overall, this research uncovered a potential systems level of regulation that is inhibiting T cell function. All studies were approved by the IACUC and was supported by the IRP of the NIH/NIAID
May 2024
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1 Read
The Journal of Immunology
Following pulmonary insults there are persistent alterations in the lung that influence responsiveness to subsequent injury. Over 600 million individuals have survived SARS-CoV-2 (SCV2) infection. Thus, the impact of SCV2 on the ability of the newly remodeled lung to handle various pulmonary challenges is of significant interest and therapeutic importance. Using the K18-hACE2 mouse model, we found that SCV2 resulted in an expanded pool of recruited alveolar macrophages along with persistent lymphocytic perivascular cuffs up to 90 days post-infection in the absence of detectable viral antigen. SCV2 resolved lungs had only modest changes following an OVA-specific allergic response compared to mock infected controls. In contrast, despite no differences in bacterial burdens, SCV2 experienced lungs had significantly less inflammation following infection with Bordetella pertussis compared to mock controls. However, this was not consistent for all bacterial driven inflammatory responses. Specifically, we observed an exacerbation of LPS derived inflammation in mice that had recovered from SCV2. The balance of inflammatory responses in these models was associated with specific changes in lipid mediators (LMs). Together, our data reveal the impact of SCV2 infection on the ability of the lung to optimally respond to a variety of inflammation provoking stimuli and underscore the potential role of LMs in the trained innate immune response.
March 2024
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12 Reads
Journal of Biological Chemistry
February 2024
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19 Reads
The PINK1/Parkin pathway of mitophagy has been implicated in the pathogenesis of Parkinson’s disease. In prion diseases, a transmissible neurodegenerative disease caused by the misfolded and infectious prion protein (PrPSc), expression of both PINK1 and Parkin are elevated, suggesting that PINK1/Parkin mediated mitophagy may also play a role in prion pathogenesis. Using mice in which expression of either PINK1 (PINK1KO) or Parkin (ParkinKO) has been ablated, we analyzed the potential role of PINK1 and Parkin in prion pathogenesis. Prion infected PINK1KO and ParkinKO mice succumbed to disease more rapidly (153 and 150 days, respectively) than wild-type control C57Bl/6 mice (161 days). Faster incubation times in PINK1KO and ParkinKO mice did not correlate with altered prion pathology in the brain, altered expression of proteins associated with mitochondrial dynamics, or prion-related changes in mitochondrial respiration. However, the expression level of mitochondrial respiration Complex I, a major site for the formation of reactive oxygen species (ROS), was higher in prion infected PINK1KO and ParkinKO mice when compared to prion infected control mice. Our results demonstrate a protective role for PINK1/Parkin mitophagy during prion disease, likely by helping to minimize ROS formation via Complex I, leading to slower prion disease progression.
February 2024
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74 Reads
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2 Citations
Frontiers in Bacteriology
Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires' disease. As a prototypical vacuolar pathogen L. pneumophila establishes a unique endoplasmic reticulum (ER)-derived organelle within which bacterial replication takes place. Bacteria-derived proteins are deposited in the host cytosol and in the lumen of the pathogen-occupied vacuole via a type IVb (T4bSS) and a type II (T2SS) secretion system respectively. These secretion system effector proteins manipulate multiple host functions to facilitate intracellular survival of the bacteria. Subversion of host membrane glycerophospholipids (GPLs) by the internalized bacteria via distinct mechanisms feature prominently in trafficking and biogenesis of the Legionella-containing vacuole (LCV). Conventional GPLs composed of a glycerol backbone linked to a polar headgroup and esterified with two fatty acids constitute the bulk of membrane lipids in eukaryotic cells. The acyl chain composition of GPLs dictates phase separation of the lipid bilayer and therefore determines the physiochemical properties of biological membranes-such as membrane disorder, fluidity and permeability. In mammalian cells, fatty acids esterified in membrane GPLs are sourced endogenously from de novo synthesis or via internalization from the exogenous pool of lipids present in serum and other interstitial fluids. Here, we exploited the preferential utilization of exogenous fatty acids for GPL synthesis by macrophages to reprogram the acyl chain composition of host membranes and investigated its impact on LCV homeostasis and L. pneumophila intracellular replication. Using saturated fatty acids as well as cis-and trans-isomers of monounsaturated fatty acids we discovered that under conditions promoting lipid packing and membrane rigidification L. pneumophila intracellular replication was significantly reduced. Palmitoleic acid-a C16:1 monounsaturated fatty acid-that promotes Frontiers in Bacteriology
February 2024
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97 Reads
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3 Citations
The intestinal microbiome plays an important role in mammalian health, disease, and immune function. In light of this function, recent studies have aimed to characterize the microbiomes of various bat species, which are noteworthy for their roles as reservoir hosts for several viruses known to be highly pathogenic in other mammals. Despite ongoing bat microbiome research, its role in immune function and disease, especially the effects of changes in the microbiome on host health, remains nebulous. Here, we describe a novel methodology to investigate the intestinal microbiome of captive Jamaican fruit bats (Artibeus jamaicensis). We observed a high degree of individual variation in addition to sex- and cohort-linked differences. The intestinal microbiome was correlated with intestinal metabolite composition, possibly contributing to differences in immune status. This work provides a basis for future infection and field studies to examine in detail the role of the intestinal microbiome in antiviral immunity.
November 2023
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23 Reads
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1 Citation
Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires disease. As a prototypical vacuolar pathogen L. pneumophila establishes a unique endoplasmic reticulum (ER)—derived organelle within which bacterial replication takes place. Bacteria—derived proteins are deposited in the host cytosol and in the lumen of the pathogen—occupied vacuole via a type IVb (T4bSS) and a type II (T2SS) secretion system respectively. These secretion system effector proteins manipulate multiple host functions to facilitate intracellular survival of the bacteria. Subversion of host membrane glycerophospholipids (GPLs) by the internalized bacteria via distinct mechanisms feature prominently in trafficking and biogenesis of the Legionella —containing vacuole (LCV). Conventional GPLs composed of a glycerol backbone linked to a polar headgroup and esterified with two fatty acids constitute the bulk of membrane lipids in eukaryotic cells. The acyl chain composition of GPLs dictates phase separation of the lipid bilayer and therefore determines the physiochemical properties of biological membranes — such as membrane disorder, fluidity and permeability. In mammalian cells, fatty acids esterified in membrane GPLs are sourced endogenously from de novo synthesis or via internalization from the exogenous pool of lipids present in serum and other interstitial fluids. Here, we exploited the preferential utilization of exogenous fatty acids for GPL synthesis by macrophages to reprogram the acyl chain composition of host membranes and investigated its impact on LCV homeostasis and L. pneumophila intracellular replication. Using saturated fatty acids as well as cis— and trans— isomers of monounsaturated fatty acids we discovered that under conditions promoting lipid packing and membrane rigidification L. pneumophila intracellular replication was significantly reduced. Palmitoleic acid — a C16:1 monounsaturated fatty acid — that promotes membrane disorder when enriched in GPLs significantly increased bacterial replication within human and murine macrophages but not in axenic growth assays. Lipidome analysis of infected macrophages showed that treatment with exogenous palmitoleic acid resulted in membrane acyl chain reprogramming in a manner that promotes membrane disorder and live—cell imaging revealed that the consequences of increasing membrane disorder impinge on several LCV homeostasis parameters. Collectively, we provide experimental evidence that L. pneumophila replication within its intracellular niche is a function of the lipid bilayer disorder and hydrophobic thickness.
... To avoid a pyroptotic cell death response triggered by cytosolic delivery of flagellin 78 , which can convolute data interpretation in infection-based growth assays, we used a flagellin clean deletion mutant. We engineered two distinct L. pneumophila Philadelphia-1 derived strains-Lp01 and JR32-to bioluminescence by inserting the LuxR operon (luxC luxD luxA luxB luxE) from Photorhabdus luminescens on the bacterial chromosome under the constitutive IcmR promoter using two different genetic approaches 79,80 (Supplementary Fig. 1a). For both strains the bioluminescence output increased exponentially during logarithmic growth in axenic cultures and peaked as the bacteria entered stationary phase ( Supplementary Fig. 1b, c) 79,80 . ...
February 2024
Frontiers in Bacteriology
... Alpha diversity analyses revealed significant differences between sexes and among male reproductive stages (active and inactive). Previous analyses of the microbiota of phyllostomid bats, comparing sexes and reproductive conditions within sexes, have generally shown that both reproductive males and females harbor the most diverse bacterial communities [8,48]. However, in our study, reproductively active males exhibited a more diverse fecal microbiota compared to both females and inactive males. ...
February 2024
... While these results seem controversial, given that BHB has been demonstrated to cause hyperacetylation in some contexts by inhibiting HDACs, recent work has identified a non-canonical function for HDACs. This research demonstrated that Class I HDACs unexpectedly catalyze protein lysine modification with BHB [42]. Thus, the reduction in transcription may be a regulatory mechanism to avoid excessive Kbhb accumulation. ...
November 2023
... This could be due to the failure of newer vaccine formulations in engaging an immune response that is like infection, or it could be their inability to engage both the innate and adaptive arms of the immune system. A recent study showed that depending on the route of infection, F. tularensis has different, specific metabolic niches [98]. This could mean that there is no one answer to vaccinating against F. tularensis and that having multiple types of vaccines available is ideal for combatting different routes of infection. ...
October 2023
... This hypothesis would for instance accommodate the observation that PRNP transcripts are induced upon long-term ( Supplementary Fig. S3) but not short-term (Supplementary Fig. S2F,G) TGFβ exposure. Our data further highlight a PrP C -dependent regulation of EGFR expression and signalling, recalling that previously described in neuroblastoma cells [50] and neural and dental pulp stem cells [26,51,52]. Because PrP C and EGFR co-localize in LUAD cells (Fig. 5I), as well as in other cancerous and non-cancerous cells [40,50], we speculate that PrP C modulates the dynamics of EGFR signalling, as already suggested over a decade ago in the study by Solis et al. [53]. ...
October 2023
Journal of Biological Chemistry
... In silico screening for treatment candidates with activity against CoV-2 followed by in vitro testing for activity against OC43 has resulted in the identification of several drugs that may be useful in treating COVID-19, including the antipsychotic lurasidone, the antimicrobial atovaquone, and the antivirals elbasvir and pibrentasvir (Milani et al., 2021;von Beck et al., 2023). In some cases, the drug candidate was further tested in vivo against CoV-2 using mouse models to confirm its efficacy (von Beck et al., 2023;Ojha et al., 2023;Ordonez et al., 2022). For example, the phytochemical sulforaphane was shown to inhibit both CoV-2 and OC43 in vitro, and oral treatment ameliorated disease in K18-hACE2 mice infected with CoV-2 (Ordonez et al., 2022). ...
June 2023
International Journal of Antimicrobial Agents
... Neuronal bursts and raster plots were analyzed and generated by MEAnalyzer [23]. Oscillatory powers and peak frequency of delta (0.5-4 Hz) and gamma oscillations were analyzed by MAT-LAB (R2021a) as described previously [24]. ...
January 2023
... In contrast, the mutagenic nucleoside N 4hydroxycytidine (NHC) is far more potent in vitro where it is possible to show a clear relationship between antiviral activity and mutation load in the viral genome, and its antiviral activity has been shown against a diverse group of RNA viruses [18][19][20][21][22]. The 5'-isopropyl ester prodrug of NHC, molnupiravir (MOV), is active in in vivo models for treating respiratory virus infections and is being used to treat SARS-CoV-2 in humans [23][24][25][26][27][28][29][30]. ...
December 2022
JCI Insight
... [1][2][3][4] Small molecule therapeutics that can inhibit the RNA dependent polymerase (RdRp) and Main protease (Mpro) are clinically approved and had a big impact on reducing COVID-19 mortality. [5][6][7][8] The success of these small molecule drugs has created a tremendous interest in developing inhibitors against other proteins from SCoV-2 that are also essential for viral replication. 9,10 The papain-like protease (PLpro) from SCoV-2 is an essential protein for viral replication and an attractive target for developing small-molecule drugs. ...
September 2022
... The arginine deiminase system produces ornithine (BB_0842) (49), which is the peptidoglycan crosslinking diamino acid (50,51). Again, matching recent experimental data, two glycerol-3-phos phate dehydrogenases were predicted: one using NADH as a cofactor (BB_0368) (52) and one using FADH (BB_0243) (53). ...
September 2022
Molecular Microbiology