Publications by authors named "Donghong Yan"

33 Publications

NINJ1 mediates plasma membrane rupture during lytic cell death.

Nature 2021 Mar 20;591(7848):131-136. Epub 2021 Jan 20.

Department of Physiological Chemistry, Genentech Inc., South San Francisco, CA, USA.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1 macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1 macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1 mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-021-03218-7DOI Listing
March 2021

Outer membrane vesicles containing OmpA induce mitochondrial fragmentation to promote pathogenesis of Acinetobacter baumannii.

Sci Rep 2021 Jan 12;11(1):618. Epub 2021 Jan 12.

Department of Infectious Diseases, Genentech Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.

Acinetobacter baumannii is a highly antibiotic resistant Gram-negative bacterium that causes life-threatening infections in humans with a very high mortality rate. A. baumannii is an extracellular pathogen with poorly understood virulence mechanisms. Here we report that A. baumannii employs the release of outer membrane vesicles (OMVs) containing the outer membrane protein A (OmpA) to promote bacterial pathogenesis and dissemination. OMVs containing OmpA are taken up by mammalian cells where they activate the host GTPase dynamin-related protein 1 (DRP1). OmpA mediated activation of DRP1 enhances its accumulation on mitochondria that causes mitochondrial fragmentation, elevation in reactive oxygen species (ROS) production and cell death. Loss of DRP1 rescues these phenotypes. Our data show that OmpA is sufficient to induce mitochondrial fragmentation and cytotoxicity since its expression in E. coli transfers its pathogenic properties to E. coli. A. baumannii infection in mice also induces mitochondrial damage in alveolar macrophages in an OmpA dependent manner. We finally show that OmpA is also required for systemic dissemination in the mouse lung infection model. In this study we uncover the mechanism of OmpA as a virulence factor in A. baumannii infections and further establish the host cell factor required for its pathogenic effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-79966-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804284PMC
January 2021

Integration of innate immune signalling by caspase-8 cleavage of N4BP1.

Nature 2020 11 24;587(7833):275-280. Epub 2020 Sep 24.

Department of Physiological Chemistry, Genentech, South San Francisco, CA, USA.

Mutations in the death receptor FAS or its ligand FASL cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2796-5DOI Listing
November 2020

Unstable Mechanisms of Resistance to Inhibitors of Escherichia coli Lipoprotein Signal Peptidase.

mBio 2020 09 8;11(5). Epub 2020 Sep 8.

Department of Infectious Diseases, Genentech, South San Francisco, California, USA

Clinical development of antibiotics with novel mechanisms of action to kill pathogenic bacteria is challenging, in part, due to the inevitable emergence of resistance. A phenomenon of potential clinical importance that is broadly overlooked in preclinical development is heteroresistance, an often-unstable phenotype in which subpopulations of bacterial cells show decreased antibiotic susceptibility relative to the dominant population. Here, we describe a new globomycin analog, G0790, with potent activity against the type II signal peptidase LspA and uncover two novel resistance mechanisms to G0790 in the clinical uropathogenic strain CFT073. Building on the previous finding that complete deletion of Lpp, the major Gram-negative outer membrane lipoprotein, leads to globomycin resistance, we also find that an unexpectedly modest decrease in Lpp levels mediated by insertion-based disruption of regulatory elements is sufficient to confer G0790 resistance and increase sensitivity to serum killing. In addition, we describe a heteroresistance phenotype mediated by genomic amplifications of that result in increased LspA levels sufficient to overcome inhibition by G0790 in culture. These genomic amplifications are highly unstable and are lost after as few as two subcultures in the absence of G0790, which places amplification-containing resistant strains at high risk of being misclassified as susceptible by routine antimicrobial susceptibility testing. In summary, our study uncovers two vastly different mechanisms of resistance to LspA inhibitors in and emphasizes the importance of considering the potential impact of unstable and heterogenous phenotypes when developing antibiotics for clinical use. Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.02018-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7482066PMC
September 2020

Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer.

Front Immunol 2019 4;10:2019. Epub 2019 Sep 4.

Genentech, South San Francisco, CA, United States.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified . By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736990PMC
October 2020

Sustained Circulating Bacterial Deoxyribonucleic Acid Is Associated With Complicated Bacteremia.

Open Forum Infect Dis 2019 Apr 26;6(4):ofz090. Epub 2019 Feb 26.

Genentech, Inc., South San Francisco, California.

Background: (SA) bacteremia often requires a long treatment duration with antibiotics to prevent relapse due to the ability of SA to establish reservoirs of infection in sites such as heart and bone. These metastatic sites of infection cannot be serially sampled to monitor the clearance of SA infection. This study aimed to establish a link between persistence of circulating SA deoxyribonucleic acid (SA-DNA) and tissue reservoirs in patients with SA bacteremia.

Methods: A highly sensitive quantitative polymerase chain reaction was used to measure whole blood SA-DNA and plasma-derived SA cell-free DNA (SA-cfDNA) in a set of longitudinal samples from 73 patients with confirmed SA bacteremia and correlated with clinical features.

Results: Blood SA-DNA was detected for longer than the duration of positive blood cultures. Longer duration of circulating bacterial DNA was observed in complicated SA bacteremia infections, such as endocarditis and osteoarticular infections, compared with uncomplicated bloodstream infections. In contrast, traditional blood cultures demonstrated similar time to clearance regardless of foci of infection. Plasma-derived SA-cfDNA showed concordance with blood SA-DNA levels. Baseline levels of SA-DNA were higher in patients presenting with greater clinical severity and complicated bacteremia.

Conclusions: Prolonged levels of circulating SA-DNA in patients with complicated tissue reservoirs after clearance of blood cultures observed in this single-center study should be validated in additional cohorts to assess the potential utility for monitoring clearance of infection in patients with SA bacteremia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/ofid/ofz090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475589PMC
April 2019

Optimized arylomycins are a new class of Gram-negative antibiotics.

Nature 2018 09 12;561(7722):189-194. Epub 2018 Sep 12.

Department of Infectious Diseases, Genentech, South San Francisco, CA, USA.

Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins-a class of natural products with weak activity and limited spectrum-to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-018-0483-6DOI Listing
September 2018

Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA.

Antimicrob Agents Chemother 2018 11 24;62(11). Epub 2018 Oct 24.

Genentech, Inc., Infectious Diseases, South San Francisco, California, USA

There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01142-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201111PMC
November 2018

Host-derived fatty acids activate type VII secretion in .

Proc Natl Acad Sci U S A 2017 10 2;114(42):11223-11228. Epub 2017 Oct 2.

Department of Infectious Disease, Genentech Inc., South San Francisco, CA 94080;

The type VII secretion system (T7SS) of is a multiprotein complex dedicated to the export of several virulence factors during host infection. This virulence pathway plays a key role in promoting bacterial survival and the long-term persistence of staphylococcal abscess communities. The expression of the T7SS is activated by bacterial interaction with host tissues including blood serum, nasal secretions, and pulmonary surfactant. In this work we identify the major stimulatory factors as host-specific -unsaturated fatty acids. Increased T7SS expression requires host fatty acid incorporation into bacterial biosynthetic pathways by the fatty acid kinase (FAK) complex, and FakA is required for virulence. The incorporated -unsaturated fatty acids decrease membrane fluidity, and these altered membrane dynamics are partially responsible for T7SS activation. These data define a molecular mechanism by which cells sense the host environment and implement appropriate virulence pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1700627114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651732PMC
October 2017

Structural insights into lipoprotein N-acylation by apolipoprotein N-acyltransferase.

Proc Natl Acad Sci U S A 2017 07 11;114(30):E6044-E6053. Epub 2017 Jul 11.

Department of Structural Biology, Genentech, Inc., South San Francisco, CA 94080;

Gram-negative bacteria express a diverse array of lipoproteins that are essential for various aspects of cell growth and virulence, including nutrient uptake, signal transduction, adhesion, conjugation, sporulation, and outer membrane protein folding. Lipoprotein maturation requires the sequential activity of three enzymes that are embedded in the cytoplasmic membrane. First, phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) recognizes a conserved lipobox motif within the prolipoprotein signal sequence and catalyzes the addition of diacylglycerol to an invariant cysteine. The signal sequence is then cleaved by signal peptidase II (LspA) to give an N-terminal S-diacylglyceryl cysteine. Finally, apolipoprotein -acyltransferase (Lnt) catalyzes the transfer of the -1-acyl chain of phosphatidylethanolamine to this N-terminal cysteine, generating a mature, triacylated lipoprotein. Although structural studies of Lgt and LspA have yielded significant mechanistic insights into this essential biosynthetic pathway, the structure of Lnt has remained elusive. Here, we present crystal structures of wild-type and an active-site mutant of Lnt. The structures reveal a monomeric eight-transmembrane helix fold that supports a periplasmic carbon-nitrogen hydrolase domain containing a Cys-Glu-Lys catalytic triad. Two lipids are bound at the active site in the structures, and we propose a putative phosphate recognition site where a chloride ion is coordinated near the active site. Based on these structures and complementary cell-based, biochemical, and molecular dynamics approaches, we propose a mechanism for substrate engagement and catalysis by Lnt.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1707813114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544336PMC
July 2017

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Isolate.

mBio 2017 05 23;8(3). Epub 2017 May 23.

Department of Infectious Diseases, Genentech, South San Francisco, California, USA

Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Their roles in cell-envelope integrity have been documented in laboratory strains, and while Lpp has been linked to serum resistance , the underlying mechanism has not been established. Here, and mutants of uropathogenic strain CFT073 showed reduced survival in a mouse bacteremia model, but only the mutant was sensitive to serum killing The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis and complement-mediated clearance Compared to the wild-type strain, the mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for "group 2" capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic isolates. Uropathogenic (UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistance and for complement-mediated bacterial clearance This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of "group 2" capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for murein lipoprotein in complex interactions between different outer membrane biogenesis pathways and further highlight the importance of lipoprotein assembly and transport in bacterial pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00603-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442458PMC
May 2017

Depletion of major pathogenic cells in asthma by targeting CRTh2.

JCI Insight 2016 05 19;1(7):e86689. Epub 2016 May 19.

Department of Immunology.

Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2 basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2 basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033936PMC
http://dx.doi.org/10.1172/jci.insight.86689DOI Listing
May 2016

A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase.

mBio 2016 09 6;7(5). Epub 2016 Sep 6.

Department of Infectious Diseases, Genentech, Inc., South San Francisco, California, USA

Unlabelled: The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo

Importance: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mBio.00412-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013292PMC
September 2016

Enhanced expression of long non-coding RNA Sox2ot promoted cell proliferation and motility in colorectal cancer.

Minerva Med 2016 Oct 28;107(5):279-86. Epub 2016 Jun 28.

Department of General Surgery, Xinxiang Central Hospital, Xinxiang, China -

Background: Colorectal cancer (CRC) is one of the most common carcinomas throughout the world. Although the diagnostic and therapeutic strategies have made some progression in the treatment of CRC patients, the mortality of CRC remains relatively high. Hence, it is an urgency to find out the detailed mechanisms of how CRC occurs.

Methods: LncRNA Sox2ot expression was explored in CRC tissues and cell lines by using quantitative real-time PCR (qRT-PCR). Cell proliferation, migration and invasion ability was measured following downregulated expression of lncRNA Sox2ot by siRNA in CRC cells. Furthermore, western blot was used to detected the expression of Cyclin B1, Cdc 25C, N-cadherin, and E-cadherin in si-Sox2ot transfected CRC cells.

Results: We found that lncRNA Sox2ot was increased in CRC tissues and cell lines. High expression of Sox2ot was associated with the progression of CRC patients. Decreased Sox2ot expression inhibited the proliferation capacity and caused cell cycle arrested in G0/G1 phase in CRC cells. The key cell cycle regulators Cyclin B1 and Cdc 25C were consistently downregulated by knockdown of Sox2ot. Furthermore, knockdown of Sox2ot suppressed cell migration and invasion and decreased the expression of mesenchymal protein N-cadherin, while it increased the expression of epithelial protein E-cadherin in CRC cells.

Conclusions: LncRNA Sox2ot could promoted CRC cell proliferation and motility and associated with the outcome of CRC patients, suggesting Sox2ot could serve as a potential therapeutic target in the treatment of CRC.
View Article and Find Full Text PDF

Download full-text PDF

Source
October 2016

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo.

J Bacteriol 2016 07 27;198(14):2001-2015. Epub 2016 Jun 27.

Infectious Diseases Department, Genentech Inc., South San Francisco, California, USA

Unlabelled: De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5'-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility.

Importance: De novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.00051-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936099PMC
July 2016

Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

Nature 2015 Nov 4;527(7578):323-8. Epub 2015 Nov 4.

Infectious Diseases Department, Genentech Inc., South San Francisco, California 94080, USA.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature16057DOI Listing
November 2015

Dusp5 negatively regulates IL-33-mediated eosinophil survival and function.

EMBO J 2015 Jan 14;34(2):218-35. Epub 2014 Nov 14.

Department of Immunology, Genentech, Inc., South San Francisco, CA, USA

Mitogen-activated protein kinase (MAPK) activation controls diverse cellular functions including cellular survival, proliferation, and apoptosis. Tuning of MAPK activation is counter-regulated by a family of dual-specificity phosphatases (DUSPs). IL-33 is a recently described cytokine that initiates Th2 immune responses through binding to a heterodimeric IL-33Rα (ST2L)/IL-1α accessory protein (IL-1RAcP) receptor that coordinates activation of ERK and NF-κB pathways. We demonstrate here that DUSP5 is expressed in eosinophils, is upregulated following IL-33 stimulation and regulates IL-33 signaling. Dusp5(-/-) mice have prolonged eosinophil survival and enhanced eosinophil effector functions following infection with the helminth Nippostrongylus brasiliensis. IL-33-activated Dusp5(-/-) eosinophils exhibit increased cellular ERK1/2 activation and BCL-XL expression that results in enhanced eosinophil survival. In addition, Dusp5(-/-) eosinophils demonstrate enhanced IL-33-mediated activation and effector functions. Together, these data support a role for DUSP5 as a novel negative regulator of IL-33-dependent eosinophil function and survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201489456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337062PMC
January 2015

A neutralizing anti-gH/gL monoclonal antibody is protective in the guinea pig model of congenital CMV infection.

PLoS Pathog 2014 Apr 10;10(4):e1004060. Epub 2014 Apr 10.

Department of Infectious Diseases, Genentech, South San Francisco, California, United States of America.

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2-1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1004060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983071PMC
April 2014

Specification of type 2 innate lymphocytes by the transcriptional determinant Gfi1.

Nat Immunol 2013 Dec 20;14(12):1229-36. Epub 2013 Oct 20.

Department of Discovery Immunology, Genentech, South San Francisco, California, USA.

Type 2 innate lymphoid cells (ILC2 cells) participate in host defense against helminth parasites and in allergic inflammation. Given their functional relatedness to type 2 helper T cells (T(H)2 cells), we explored whether Gfi1 acts as a shared transcriptional determinant in ILC2 cells. Gfi1 promoted the development of ILC2 cells and controlled their responsiveness during infection with Nippostrongylus brasiliensis and protease allergen-induced lung inflammation. Gfi1 'preferentially' regulated the responsiveness of ILC2 cells to interleukin 33 (IL-33) by directly activating Il1rl1, which encodes the IL-33 receptor (ST2). Loss of Gfi1 in activated ILC2 cells resulted in impaired expression of the transcription factor GATA-3 and a dysregulated genome-wide effector state characterized by coexpression of IL-13 and IL-17. Our findings establish Gfi1 as a shared determinant that reciprocally regulates the type 2 and IL-17 effector states in cells of the innate and adaptive immune systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ni.2743DOI Listing
December 2013

Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination.

Proc Natl Acad Sci U S A 2013 Sep 9;110(39):15770-5. Epub 2013 Sep 9.

Genentech, Inc., South San Francisco, CA 94080.

Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sμ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sμ. These results suggest that the sufficiency of Sμ to mediate IgH rearrangements may be influenced by context-dependent cues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1221661110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785764PMC
September 2013

An in vivo human-plasmablast enrichment technique allows rapid identification of therapeutic influenza A antibodies.

Cell Host Microbe 2013 Jul;14(1):93-103

Antibody Engineering Department, Genentech, South San Francisco, CA 94080, USA.

Recent advances enabling the cloning of human immunoglobulin G genes have proven effective for discovering monoclonal antibodies with therapeutic potential. However, these antibody-discovery methods are often arduous and identify only a few candidates from numerous antibody-secreting plasma cells or plasmablasts. We describe an in vivo enrichment technique that identifies broadly neutralizing human antibodies with high frequency. For this technique, human peripheral blood mononuclear cells from vaccinated donors are activated and enriched in an antigen-specific manner for the production of numerous antigen-specific plasmablasts. Using this technology, we identified four broadly neutralizing influenza A antibodies by screening only 840 human antibodies. Two of these antibodies neutralize every influenza A human isolate tested and perform better than the current anti-influenza A therapeutic, oseltamivir, in treating severe influenza infection in mice and ferrets. Furthermore, these antibodies elicit robust in vivo synergism when combined with oseltamivir, thus highlighting treatment strategies that could benefit influenza-infected patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2013.06.004DOI Listing
July 2013

Characterization of the guinea pig CMV gH/gL/GP129/GP131/GP133 complex in infection and spread.

Virology 2013 Jun 4;441(1):75-84. Epub 2013 Apr 4.

Department of Infectious Diseases, Genentech, Inc./Hoffmann-LaRoche, Ltd., 1 DNA Way, South San Francisco, CA 94080, United States.

In human cytomegalovirus (HCMV), the UL128-131A locus plays an essential role in cellular tropism and spread. Here, we report the complete annotation of the GP129-133 locus from guinea pig cytomegalovirus (GPCMV) and the discovery of the UL131A homolog, named GP133. We have found that similar to HCMV the GP129-133 proteins form a pentamer complex with the GPCMV glycoproteins gH and gL. In addition, we find that the GP129-133 proteins play a critical role in entry as the GP129-133 deletion mutant shows a defect in both endothelial and fibroblast cell entry. Although the GP129-133 deletion strain can propagate in vitro, we find that the deletion fails to spread in vivo. Interestingly, the wildtype strain can spontaneously give rise to the GP129-133 deletion strain during in vivo spread, suggesting genetic instability at this locus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2013.03.008DOI Listing
June 2013

IgE⁺ memory B cells and plasma cells generated through a germinal-center pathway.

Nat Immunol 2012 Feb 26;13(4):396-404. Epub 2012 Feb 26.

Department of Immunology, Genentech, South San Francisco, California, USA.

Immunoglobulin E (IgE) antibodies are pathogenic in asthma and allergic diseases, but the in vivo biology of IgE-producing (IgE(+)) cells is poorly understood. A model of the differentiation of IgE(+) B cells proposes that IgE(+) cells develop through a germinal-center IgG1(+) intermediate and that IgE memory resides in the compartment of IgG1(+) memory B cells. Here we have used a reporter mouse expressing green fluorescent protein associated with membrane IgE transcripts (IgE-GFP) to assess in vivo IgE responses. In contrast to the IgG1-centered model of IgE switching and memory, we found that IgE(+) cells developed through a germinal-center IgE(+) intermediate to form IgE(+) memory B cells and plasma cells. Our studies delineate a new model for the in vivo biology of IgE switching and memory.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ni.2256DOI Listing
February 2012

Increased targeting of donor switch region and IgE in Sgamma1-deficient B cells.

J Immunol 2010 Jul 28;185(1):166-73. Epub 2010 May 28.

Department of Physiological Chemistry, Genentech, San Francisco, CA 94080, USA.

Ab class switch recombination involves a recombination between two repetitive DNA sequences known as switch (S) regions that vary in length, content, and density of the repeats. Abs expressed by B cells are diversified by somatic hypermutation and class switch recombination. Both class switch recombination and somatic hypermutation are initiated by activation-induced cytidine deaminase (AID), which preferentially recognizes certain hot spots that are far more enriched in the S regions. We found that removal of the largest S region, Sgamma1 (10 kb), in mice can result in the accumulation of mutations and short-range intra-S recombination in the donor Smu region. Furthermore, elevated levels of IgE were detected in trinitrophenol-OVA-immunized mice and in anti-CD40 plus IL-4-stimulated B cells in vitro. We propose that AID availability and targeting in part might be regulated by its DNA substrate. Thus, prominently transcribed S regions, such as Sgamma1, might provide a sufficient sink for AID protein to titrate away AID from other accessible sites within or outside the Ig locus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1000515DOI Listing
July 2010

Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice.

J Clin Invest 2010 Jun 10;120(6):2218-29. Epub 2010 May 10.

Department of Immunology, Genentech Inc., South San Francisco, California 94080, USA.

IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1' domain, and using genetically modified mice that contain the human M1' domain inserted into the mouse IgE locus, we demonstrated that M1'-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1'-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1'-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1'-specific antibodies may be a novel treatment for asthma and allergy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI40141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877936PMC
June 2010

Novel variants in human Aquaporin-4 reduce cellular water permeability.

Hum Mol Genet 2008 Aug 29;17(15):2379-89. Epub 2008 May 29.

Program in Biological and Medical Informatics, University of California, San Francisco, San Francisco, CA 94110, USA.

Cerebral edema contributes significantly to morbidity and mortality after brain injury and stroke. Aquaporin-4 (AQP4), a water channel expressed in astrocytes, plays a key role in brain water homeostasis. Genetic variants in other aquaporin family members have been associated with disease phenotypes. However, in human AQP4, only one non-synonymous single-nucleotide polymorphism (nsSNP) has been reported, with no characterization of protein function or disease phenotype. We analyzed DNA from an ethnically diverse cohort of 188 individuals to identify novel AQP4 variants. AQP4 variants were constructed by site-directed mutagenesis and expressed in cells. Water permeability assays in the cells were used to measure protein function. We identified 24 variants in AQP4 including four novel nsSNPs (I128T, D184E, I205L and M224T). We did not observe the previously documented M278T in our sample. The nsSNPs found were rare ( approximately 1-2% allele frequency) and heterozygous. Computational analysis predicted reduced function mutations. Protein expression and membrane localization were similar for reference AQP4 and the five AQP4 mutants. Cellular assays confirmed that four variant AQP4 channels reduced normalized water permeability to between 26 and 48% of the reference (P < 0.001), while the M278T mutation increased normalized water permeability (P < 0.001). We identified multiple novel AQP4 SNPs and showed that four nsSNPs reduced water permeability. The previously reported M278T mutation resulted in gain of function. Our experiments provide insight into the function of the AQP4 protein. These nsSNPs may have clinical implications for patients with cerebral edema and related disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddn138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2733814PMC
August 2008

Greatly impaired migration of implanted aquaporin-4-deficient astroglial cells in mouse brain toward a site of injury.

FASEB J 2007 Jan 29;21(1):108-16. Epub 2006 Nov 29.

Department of Medicine and Physiology, University of California, San Francisco, San Francisco, CA, USA.

We reported previously that astroglia cultured from aquaporin-4-deficient (AQP4-/-) mice migrate more slowly in vitro than those from wild-type (AQP4+/+) mice (J. Cell Sci. 2005;118, 5691-5698). Here, we investigate the migration of fluorescently labeled AQP4+/+ and AQP4-/- astroglia after implantation into mouse brains in which directional movement was stimulated by a planar stab wound 3 mm away from the axis of the injection needle. Two days after cell injection we determined the location, elongation ratio, and orientation of labeled cells. Migration of AQP4+/+ but not AQP4-/- cells toward the stab was greater than away from the stab. AQP4+/+ astroglia moved on average 1.5 mm toward the stab compared with 0.6 mm for AQP4-/- cells. More than 25% of the migrating AQP4+/+ cells but <3% of AQP4-/- cells appeared elongated (axial ratio>2.5). In transwell assays, AQP4+/+ astroglia migrated faster than AQP4-/- cells in a manner dependent on pore size. At 8 h, approximately 50% of AQP4+/+ cells migrated through 8-microm diameter pores, whereas equivalent migration of AQP4-/- cells was found for 12-microm diameter pores. These results provide in vivo evidence for AQP4-dependent astroglial migration and suggest that modulation of AQP4 expression or function might alter glial scarring.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.06-6848comDOI Listing
January 2007

Controlled cortical impact in swine: pathophysiology and biomechanics.

J Neurotrauma 2006 Feb;23(2):128-39

Department of Neurological Surgery, University of California, San Francisco, California 94110, USA.

Investigations of the basic pathological, cellular, and molecular mechanisms of traumatic brain injury (TBI) over the past two decades have been carried out primarily in rodents. Unfortunately, these studies have not translated into improved outcome in patients with TBI. To better model human TBI, a swine model of controlled cortical impact (CCI) was developed. A CCI device was used to generate a focal lesion in 23 anesthetized male Yorkshire swine. In 10 swine, CCI parameters of velocity and dwell time were varied to achieve a consistent injury (3.5 m/sec, 400 msec, respectively). In 13 swine, depth of depression was varied from 9 to 12 mm. Physiological data, including heart rate (HR), mean arterial blood pressure (MAP), intracranial pressure (ICP), and cerebral perfusion pressure (CPP), were collected for 10 h after injury. Following injury, ICP and HR increased above baseline values in all swine, with a more pronounced elevation in animals impacted to a depth of depression of 12 mm. An 11-mm depth of depression was found to most closely mimic pathological features of human TBI with edema, infiltration of inflammatory cells, pericapillary hemorrhage, and petechial hemorrhages in the white matter. Injury to a depth of depression of 12 mm resulted in cortical laceration obscuring these features. Immunohistological staining with Neu-N, MAP-2, and Fluoro Jade B revealed evidence of degenerating neurons, axonal disruption, and impending cell death. These results indicate that the swine model of CCI results in a defined and reproducible injury with pathological features similar to human TBI. Physiological parameters after injury are readily monitored in a setting mimicking conditions of an intensive care unit, establishing a more clinically relevant experimental model for future investigations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/neu.2006.23.128DOI Listing
February 2006