Gabriel L. Hendricks

Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus

Background: Better treatments are needed for combating influenza. Results: LSTc-sialoside-bearing decoy liposomes competitively bind to influenza A virus, as assessed by hemagglutination inhibition, flow cytometry, and growth inhibition studies. Decoy liposomes co-localize with influenza virus, as assessed by confocal imaging. Conclusion: LSTc-sialoside-bearing decoy liposomes are highly effective in capturing influenza virus. Significance: Decoy liposomes may serve as an effective platform for presenting anti-pathogen receptors. Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.

TRIM13 Is a Negative Regulator of MDA5-Mediated Type I Interferon Production

ABSTRACT Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential intracellular detectors of viral RNA. They contribute to the type I interferon (IFN) response that is crucial for host defense against viral infections. Given the potent antiviral and proinflammatory activities elicited by the type I IFNs, induction of the type I IFN response is tightly regulated. Members of the tripartite motif (TRIM) family of proteins have recently emerged as key regulators of antiviral immunity. We show that TRIM13, an E3 ubiquitin ligase, is expressed in immune cells and is upregulated in bone marrow-derived macrophages upon stimulation with inducers of type I IFN. TRIM13 interacts with MDA5 and negatively regulates MDA5-mediated type I IFN production in vitro, acting upstream of IFN regulatory factor 3. We generated Trim13 −/− mice and show that upon lethal challenge with encephalomyocarditis virus (EMCV), which is sensed by MDA5, Trim13 −/− mice produce increased amounts of type I IFNs and survive longer than wild-type mice. Trim13 −/− murine embryonic fibroblasts (MEFs) challenged with EMCV or poly(I·C) also show a significant increase in beta IFN (IFN-β) levels, but, in contrast, IFN-β responses to the RIG-I-detected Sendai virus were diminished, suggesting that TRIM13 may play a role in positively regulating RIG-I function. Together, these results demonstrate that TRIM13 regulates the type I IFN response through inhibition of MDA5 activity and that it functions nonredundantly to modulate MDA5 during EMCV infection. IMPORTANCE The type I interferon (IFN) response is crucial for host defense against viral infections, and proper regulation of this pathway contributes to maintaining immune homeostasis. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are intracellular detectors of viral RNA that induce the type I IFN response. In this study, we show that expression of the gene tripartite motif 13 (Trim13) is upregulated in response to inducers of type I IFN and that TRIM13 interacts with both MDA5 and RIG-I in vitro. Through the use of multiple in vitro and in vivo model systems, we show that TRIM13 is a negative regulator of MDA5-mediated type I IFN production and may also impact RIG-I-mediated type I IFN production by enhancing RIG-I activity. This places TRIM13 at a key junction within the viral response pathway and identifies it as one of the few known modulators of MDA5 activity.

Dopamine Inhibits N-Type Channels in Visceral Afferents to Reduce Synaptic Transmitter Release Under Normoxic and Chronic Intermittent Hypoxic Conditions

Glutamatergic synaptic currents elicited in second-order neurons in the nucleus of the solitary tract (nTS) by activation of chemosensory and other visceral afferent fibers are severely reduced following 10 days of chronic intermittent hypoxia (CIH). The mechanism by which this occurs is unknown. A strong candidate for producing the inhibition is dopamine, which is also released from the presynaptic terminals and which we have shown exerts a tonic presynaptic inhibition on glutamate release. We postulated that tonic activation of the D2 receptors inhibits presynaptic calcium currents to reduce transmitter release and that in CIH this occurs in conjunction with an increase in the dopamine inhibitory response due to the increase in presynaptic D2 receptors or an increase in dopamine release further suppressing the evoked excitatory postsynaptic current (eEPSC). Thus we predicted that blockade of the D2 receptors would return the EPSC to values of animals maintained under normoxic conditions. We found that dopamine and quinpirole, the selective D2-like agonist, inhibit calcium currents via the D2 receptors by acting on the N-type calcium channel in presynaptic neurons and their nTS central terminals. However, in brain slice studies from CIH animals, although the D2 antagonist sulpiride increased the CIH-reduced amplitude of synaptic currents, EPSCs were not restored to normal levels. This indicates that while the dopamine inhibitory effect remains intact in CIH, most of the reduction in the eEPSC amplitude occurs via alternative mechanisms.

An update on the use of C. elegans for preclinical drug discovery: screening and identifying anti-infective drugs

ABSTRACT Introduction: The emergence of antibiotic-resistant and -tolerant bacteria is a major threat to human health. Although efforts for drug discovery are ongoing, conventional bacteria-centered screening strategies have thus far failed to yield new classes of effective antibiotics. Therefore, new paradigms for discovering novel antibiotics are of critical importance. Caenorhabditis elegans, a model organism used for in vivo, offers a promising solution for identification of anti-infective compounds. Areas covered: This review examines the advantages of C. elegans-based high-throughput screening over conventional, bacteria-centered in vitro screens. It discusses major anti-infective compounds identified from large-scale C. elegans-based screens and presents the first clinically-approved drugs, then known bioactive compounds, and finally novel small molecules. Expert opinion: There are clear advantages of using a C. elegans-infection based screening method. A C. elegans-based screen produces an enriched pool of non-toxic, efficacious, potential anti-infectives, covering: conventional antimicrobial agents, immunomodulators, and anti-virulence agents. Although C. elegans-based screens do not denote the mode of action of hit compounds, this can be elucidated in secondary studies by comparing the results to target-based screens, or conducting subsequent target-based screens, including the genetic knock-down of host or bacterial genes.

A new class of synthetic retinoid antibiotics effective against bacterial persisters.

A challenge in the treatment of Staphylococcus aureus infections is the high prevalence of methicillin-resistant S. aureus (MRSA) strains and the formation of non-growing, dormant ‘persister’ subpopulations that exhibit high levels of tolerance to antibiotics and have a role in chronic or recurrent infections. As conventional antibiotics are not effective in the treatment of infections caused by such bacteria, novel antibacterial therapeutics are urgently required. Here we used a Caenorhabditis elegans–MRSA infection screen to identify two synthetic retinoids, CD437 and CD1530, which kill both growing and persister MRSA cells by disrupting lipid bilayers. CD437 and CD1530 exhibit high killing rates, synergism with gentamicin, and a low probability of resistance selection. All-atom molecular dynamics simulations demonstrated that the ability of retinoids to penetrate and embed in lipid bilayers correlates with their bactericidal ability. An analogue of CD437 was found to retain anti-persister activity and show an improved cytotoxicity profile. Both CD437 and this analogue, alone or in combination with gentamicin, exhibit considerable efficacy in a mouse model of chronic MRSA infection. With further development and optimization, synthetic retinoids have the potential to become a new class of antimicrobials for the treatment of Gram-positive bacterial infections that are currently difficult to cure.

Expanding the nematode model system: The molecular basis of inflammation and infection recovery in C. elegans

Inflammation is an acute response to invading pathogens that temporarily reduces fitness, but allows the host to survive infection. Dysregulated inflammation is a chronic state that is now recognized as the central pathological process in a number of diverse diseases. In the past two decades, the initiation of inflammation has been extensively studied. The molecular basis of initiation of each of the four hallmarks of inflammation—heat, redness, swelling, and pain, has been revealed. Recently a number of anti-inflammatory drugs have come onto the market to treat or alleviate chronic inflammatory diseases, including rheumatoid arthritis, multiple sclerosis, vitiligo, and lupus. However, studies have also shown that chronic inflammatory diseases result more from dysregulated resolution than dysregulated initiation. Pro-resolution drugs may prove superior to or synergistic with antiinflammatory drugs, by short-circuiting the inflammatory process. However, the resolution of inflammation remains poorly understood. Resolution pathways are heterogeneous, and may be tissueand pathogen-specific. The progression from the inflammatory to the resolved state is no longer thought to occur when the pro-inflammatory cascade simply fizzles out due to a lack of stimuli. Instead, it is a complex interplay between many concurring pathways. First, once the source of the pathogenor danger-associated molecular patterns (PAMPs and DAMPS) are eliminated, anti-inflammatory cytokines and regulatory T-cells inhibit pro-inflammatory immune responses. The remaining cytokines, chemokines, and reactive oxygen species are catabolized, stopping further immune cell recruitment. The remaining leukocytes and lymphocytes either reenter circulation or are cleared by alternatively activated (M2) macrophages by efferocytosis. M2 macrophages are activated by specific efferocytic receptors, engulf dying leukocytes, and produce additional anti-inflammatory cytokines. Underpinning each of these steps are highly conserved cell-to-cell receptorligand interactions, humoral-mediated signaling and intracellular kinase cascades. Recent studies have relied on in vitro assays to elucidate the genetic pathways of inflammation resolution. However, to fully understand the interplay between all the resolution and repair mechanisms, a whole-animal model system would be the best approach. In this issue of Virulence, Head et al., used the wholeorganism Caenorhabditis elegans-pathogen model to study the host recovery response from acute Pseudomonas aeruginosa infection. They utilized whole genome expression profiling to characterize the resolution process from the moment of pathogen exposure to early and late phases of recovery. The authors found 1,323 genes, or more than 6% of the genome, whose expression increased or decreased more than 2-fold during the four phases of recovery. The authors categorized the altered genes into four major groups with different responses during exposure and recovery. Furthermore, they compared the worm recovery responses to P. aeruginosa and Salmonella enterica to elucidate shared and pathogen-specific pathways. Clustering the shared and unique genes into gene ontology (GO) groups, the authors were able to see shared pathways, like the downregulation of the antimicrobial C-type lectins and aging genes, and the up-regulation of the UGT/Transferase gene cluster. Taking advantage of the genetically tractable C. elegans system, they found that recovery was dependent on the GATA transcription factor elt-2 and the p38-mitogen-activated protein kinase (MAPK) pmk1. Both of these pathways are highly conserved, and are

Strategies against methicillin-resistant Staphylococcus aureus persisters

Chronic Staphylococcus aureus infections are complicated by frequent relapses not only from the development of drug resistance to conventional antibiotics, but also through the formation of persister bacterial cells. Bacterial persisters are in a transient, metabolically inactive state, making conventional antibiotics that target essential cellular growth processes ineffective, resulting in high clinical failure rates of antibiotic chemotherapy. The development of new antibiotics against persistent S. aureus is an urgent issue. Over the last decade, new strategies to identify S. aureus persister-active compounds have been proposed. This review summarizes the proposed targets, antipersister compounds and innovative methods that may augment conventional antibiotics against S. aureus persisters. The reviewed antipersister strategies can be summarized as two broad categories; directly targeting growth-independent targets and potentiating existing, ineffective antibiotics by aiding uptake or accessibility.