Kazuyo Takeda

Wild Mouse Gut Microbiota Promotes Host Fitness and Improves Disease Resistance

Laboratory mice, while paramount for understanding basic biological phenomena, are limited in modeling complex diseases of humans and other free-living mammals. Because the microbiome is a major factor in mammalian physiology, we aimed to identify a naturally evolved reference microbiome to better recapitulate physiological phenomena relevant in the natural world outside the laboratory. Among 21xa0distinct mouse populations worldwide, we identified a closely related wild relative to standard laboratory mouse strains. Its bacterial gut microbiome differed significantly from its laboratory mouse counterpart and was transferred to and maintained in laboratory mice over several generations. Laboratory mice reconstituted with natural microbiota exhibited reduced inflammation and increased survival following influenza virus infection and improved resistance against mutagen/inflammation-induced colorectal tumorigenesis. By demonstrating the host fitness-promoting traits of natural microbiota, our findings should enable the discovery of protective mechanisms relevant in the natural world and improve the modeling of complex diseases of free-living mammals. VIDEO ABSTRACT.

Protein-protein interactions among West Nile non-structural proteins and transmembrane complex formation in mammalian cells.

To study the membrane orientation of flavivirus non-structural proteins (NSPs) in the replication complex, the seven major West Nile (WN) NSPs were separately expressed in monkey cells, and their subcellular localization was investigated by imaging-based techniques. First, we observed by confocal microscopy that four small transmembrane proteins (TP) (NS2A, NS2B, NS4A, and NS4B) were located to the endoplasmic reticulum (ER), whereas the largest NSPs, NS1, NS3, and NS5 were not. We then analyzed the colocalization and the association of WN NSPs using the methods of confocal microscopy, fluorescence resonance energy transfer (FRET), and biologic fluorescence complementation (BiFC). Through these combined imaging techniques, protein-protein interactions (PPI) among WNNSPs were detected. Our data demonstrate that there are interactions between NS2A and NS4A, and interactions of NS2B with three other TPs (NS2A, NS4A, and NS4B) as well as the expected interaction with NS3. PPI between NS2A and NS4B or between NS4A and NS4B were not detected. By the criteria of these techniques, NS5 interacted only with NS3, and NS1 was not shown to be in close proximity with other NSPs. In addition, homo-oligomerization of some NSPs was observed and three-way interactions between NS2A, NS4A, and NA4B with NS2B-NS3 were also observed, respectively. Our results suggest that the four TPs are required for formation of transmembrane complex. NS2B protein seems to play a key role in bringing the TPs together on the ER membrane and in bridging the TPs with non-membrane-associated proteins (NS3 and NS5).

HIV-1 and HIV-2 infections induce autophagy in Jurkat and CD4+ T cells.

Autophagy plays important roles during innate and adaptive immune responses to pathogens, including virus infection. Viruses develop ways to subvert the pathway for their own benefit in order to escape restriction by autophagy, leading to increased viral replication and/or control over apoptosis of their host cells. The effects of HIV infection on the autophagic pathway in host cells have been little documented. Using the susceptible Jurkat cell line and CD4(+) T cells, we studied the relationship of HIV-1 and -2 infections with autophagy. We found that HIV infections significantly increase transcription of ULK1, a member of the autophagy-initiated complex. Two ubiquitin-like conjugation systems, the Atg12 conjugation system and the microtubule-associated protein L chain 3 (LC3) conjugation system that control the elongation of the autophore to form the autophagosome, were activated after HIV infection, with upregulation of Atg12-Atg5 complex and increased transcription of LC3, and formed more autophagosome in infected cells detected using an EM assay. We also found that HIV-1 induced more autophagic death in Jurkat cells relative to HIV-2, and the inhibition of autophagy with 3MA and Beclin-1 knockdown decreased HIV-1 replication significantly. The results indicate that HIV is able to induce the autophagic signaling pathway in HIV-infected host cells, which may be required for HIV infection-mediated apoptotic cell death.

Genetically Modified Live Attenuated Leishmania donovani Parasites Induce Innate Immunity through Classical Activation of Macrophages That Direct the Th1 Response in Mice

ABSTRACT Visceral leishmaniasis (VL) causes significant mortality and there is no effective vaccine. Previously, we have shown that genetically modified Leishmania donovani parasites, here described as live attenuated parasites, induce a host protective adaptive immune response in various animal models. In this study, we demonstrate an innate immune response upon infection with live attenuated parasites in macrophages from BALB/c mice both in vitro and in vivo. In vitro infection of macrophages with live attenuated parasites (compared to that with wild-type [WT] L. donovani parasites) induced significantly higher production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-12 [IL-12], gamma interferon [IFN-γ], and IL-6), chemokines (monocyte chemoattractant protein 1/CCL-2, macrophage inflammatory protein 1α/CCL-3, and IP-10), reactive oxygen species (ROS), and nitric oxide, while concomitantly reducing anti-inflammatory cytokine IL-10 and arginase-1 activities, suggesting a dominant classically activated/M1 macrophage response. The classically activated response in turn helps in presenting antigen to T cells, as observed with robust CD4+ T cell activation in vitro. Similarly, parasitized splenic macrophages from live attenuated parasite-infected mice also demonstrated induction of an M1 macrophage phenotype, indicated by upregulation of IL-1β, TNF-α, IL-12, and inducible nitric oxide synthase 2 and downregulation of genes associated with the M2 phenotype, i.e., the IL-10, YM1, Arg-1, and MRC-1 genes, compared to WT L. donovani-infected mice. Furthermore, an ex vivo antigen presentation assay showed macrophages from live attenuated parasite-infected mice induced higher IFN-γ and IL-2 but significantly less IL-10 production by ovalbumin-specific CD4+ T cells, resulting in proliferation of Th1 cells. These data suggest that infection with live attenuated parasites promotes a state of classical activation (M1 dominant) in macrophages that leads to the generation of protective Th1 responses in BALB/c mice.

Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1

ABSTRACT The intraerythrocytic apicomplexan Babesia microti, the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-length B. microti AMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including other Babesia and Theileria species, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed in Escherichia coli reacted with a mouse anti-BmAMA1 antibody using Western blotting. In vitro binding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation of B. microti parasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.

Inhibition of Hepatitis C Virus by the Cyanobacterial Protein Microcystis viridis Lectin: Mechanistic Differences between the High-Mannose Specific Lectins MVL, CV-N, and GNA

Plant or microbial lectins are known to exhibit potent antiviral activities against viruses with glycosylated surface proteins, yet the mechanism(s) by which these carbohydrate-binding proteins exert their antiviral activities is not fully understood. Hepatitis C virus (HCV) is known to possess glycosylated envelope proteins (gpE1E2) and to be potently inhibited by lectins. Here, we tested in detail the antiviral properties of the newly discovered Microcystis viridis lectin (MVL) along with cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA) against cell culture HCV, as well as their binding properties toward viral particles, target cells, and recombinant HCV glycoproteins. Using infectivity assays, CV-N, MVL, and GNA inhibited HCV with IC50 values of 0.6 nM, 30.4 nM, and 11.1 nM, respectively. Biolayer interferometry analysis demonstrated a higher affinity of GNA to immobilized recombinant HCV glycoproteins compared to CV-N and MVL. Complementary studies, including fluorescence-activated cell sorting (FACS) analysis, confocal microscopy, and pre- and post-virus binding assays, showed a complex mechanism of inhibition for CV-N and MVL that includes both viral and cell association, while GNA functions by binding directly to the viral particle. Combinations of GNA with CV-N or MVL in HCV infection studies revealed synergistic inhibitory effects, which can be explained by different glycan recognition profiles of the mainly high-mannoside specific lectins, and supports the hypothesis that these lectins inhibit through different and complex modes of action. Our findings provide important insights into the mechanisms by which lectins inhibit HCV infection. Overall, the data suggest MVL and CV-N have the potential for toxicity due to interactions with cellular proteins while GNA may be a better therapeutic agent due to specificity for the HCV gpE1E2.

TACI deficiency leads to alternatively activated macrophage phenotype and susceptibility to Leishmania infection

Significance Here, we described a novel role for transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) in determining Mϕ phenotype, a molecule that is previously known to be important in B-cell responses. We found that TACI-deficient mouse Mϕs manifest an M2 phenotype. We also observed that, in WT mouse Mϕs, TACI mediates B-cell activating factor- and a proliferation inducing ligand-induced signals that favor M1 polarization and Leishmania clearance. In TACI-deficient mice, M2-polarized status of Mϕs was responsible for the diminished Th1 response and increased susceptibility to Leishmania infection. These findings have implications in explaining the propensity of infants to develop asthma and weak responses to vaccines because infant Mϕs are likely to be M2-skewed due to severely reduced expression of TACI. The TNF family member, transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), is a key molecule for plasma cell maintenance and is required in infections where protection depends on antibody response. Here, we report that compared with WT mouse, TACI KO Μϕs expressed lower levels of Toll-like receptors (TLRs), CD14, myeloid differentiation primary response protein 88, and adaptor protein Toll/IL-1 receptor domain-containing adapter-inducing IFN-β and responded poorly to TLR agonists. Analysis of Μϕ phenotype revealed that, in the absence of TACI, Μϕs adapt the alternatively activated (M2) phenotype. Steady-state expression levels for M2 markers IL-4Rα, CD206, CCL22, IL-10, Arg1, IL1RN, and FIZZ1 were significantly higher in TACI KO Μϕ than in WT cells. Confirming their M2 phenotype, TACI-KO Mϕs were unable to control Leishmania major infection in vitro, and intradermal inoculation of Leishmania resulted in a more severe manifestation of disease than in the resistant C57BL/6 strain. Transfer of WT Μϕs to TACI KO mice was sufficient to significantly reduce disease severity. TACI is likely to influence Mϕ phenotype by mediating B cell-activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL) signals because both these ligands down-regulated M2 markers in WT but not in TACI-deficient Μϕs. Moreover, treatment of Μϕs with BAFF or APRIL enhanced the clearance of Leishmania from cells only when TACI is expressed. These findings may have implications for understanding the shortcomings of host response in newborns where TACI expression is reduced and in combined variable immunodeficiency patients where TACI signaling is ablated.

Poly(I:C) Induces Human Lung Endothelial Barrier Dysfunction by Disrupting Tight Junction Expression of Claudin-5

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNβ production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelial monolayer permeability with a corresponding dose- and time-dependent decrease in the expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Together, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.

The Use of a Stably Expressed FRET Biosensor for Determining the Potency of Cancer Drugs

Many cancer drugs are intended to kill cancer cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. Here we describe a cell-based fluorescence resonance energy transfer (FRET) biosensor designed to measure the bioactivity of apoptosis inducing cancer drugs. The biosensor contains cyan fluorescent protein (CFP) linked via caspase 3 and caspase 8 specific cleavage recognition sequences to yellow fluorescent protein (YFP). Upon caspase activation, as in the case of apoptosis induction, the linker is cleaved abolishing the cellular FRET signal. This assay closely reflects the mechanism of action of cancer drugs, in killing cancer cells and therefore can function as a potency test for different cancer drugs. We rigorously demonstrate this through characterization of a class of proteins targeting the death receptors. The one-step assay appears to be superior to other apoptosis-based assays because of its simplicity, convenience, and robustness.

Varied Role of Ubiquitylation in Generating MHC Class I Peptide Ligands

CD8+ T cell immunosurveillance is based on recognizing oligopeptides presented by MHC class I molecules. Despite decades of study, the importance of protein ubiquitylation to peptide generation remains uncertain. In this study, we examined the ability of MLN7243, a recently described ubiquitin-activating enzyme E1 inhibitor, to block overall cytosolic peptide generation and generation of specific peptides from vaccinia- and influenza A virus–encoded proteins. We show that MLN7243 rapidly inhibits ubiquitylation in a variety of cell lines and can profoundly reduce the generation of cytosolic peptides. Kinetic analysis of specific peptide generation reveals that ubiquitylation of defective ribosomal products is rate limiting in generating class I peptide complexes. More generally, our findings demonstrate that the requirement for ubiquitylation in MHC class I–restricted Ag processing varies with class I allomorph, cell type, source protein, and peptide context. Thus, ubiquitin-dependent and -independent pathways robustly contribute to MHC class I–based immunosurveillance.