The interaction between flagellin and the glycosphingolipid Gb3 on host cells contributes to Bacillus cereus acute infection

ABSTRACT Viral infection of the central nervous system (CNS) is complicated by the mostly irreplaceable nature of neurons, as the loss of neurons has the potential to result in permanent damage to brain function. However, whether neurons or other cells in the CNS sometimes survive infection and the effects of infection on neuronal function is largely unknown. To address this question, we used the rJHM strain (rJ) of mouse hepatitis virus (MHV), a neurotropic coronavirus that causes acute encephalitis in susceptible strains of mice. To determine whether neurons or other CNS cells survive acute infection with this virulent virus, we developed a recombinant JHMV that expresses Cre recombinase (rJ-Cre) and infected mice that universally expressed a silent (floxed) version of tdTomato. Infection of these mice with rJ-Cre resulted in expression of tdTomato in host cells. The results showed that some cells were able to survive the infection, as demonstrated by continued tdTomato expression after virus antigen could no longer be detected. Most notably, interneurons in the olfactory bulb, which are known to be inhibitory, represented a large fraction of the surviving cells. In conclusion, our results indicated that some neurons are resistant to virus-mediated cell death and provide a framework for studying the effects of prior coronavirus infection on neuron function. IMPORTANCE We developed a novel recombinant virus that allows the study of cells that survive an infection by a central nervous system-specific strain of murine coronavirus. Using this virus, we identified neurons and, to a lesser extent, nonneuronal cells in the brain that were infected during the acute phase of the infection and survived for approximately 2 weeks until the mice succumbed to the infection. We focused on neurons and glial cells within the olfactory bulb because the virus enters the brain at this site. Our results show that interneurons of the olfactory bulb were the primary cell type able to survive infection. Further, these results indicate that this system will be useful for functional and gene expression studies of cells in the brain that survive acute infection.

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Listeria monocytogenes: The Impact of Cell Death on Infection and Immunity

Pathogens ◽

10.3390/pathogens7010008 ◽

2018 ◽

Vol 7 (1) ◽

pp. 8 ◽

Cited By ~ 8

Author(s):

Courtney McDougal ◽

John-Demian Sauer

Keyword(s):

Cell Death ◽

Listeria Monocytogenes ◽

Host Cell ◽

Acute Infection ◽

Host Cells ◽

Cell Mediated Immunity ◽

Cell Death Pathways ◽

Host Cell Death ◽

The Impact

Listeria monocytogenes has evolved exquisite mechanisms for invading host cells and spreading from cell-to-cell to ensure maintenance of its intracellular lifecycle. As such, it is not surprising that loss of the intracellular replication niche through induction of host cell death has significant implications on the development of disease and the subsequent immune response. Although L. monocytogenes can activate multiple pathways of host cell death, including necrosis, apoptosis, and pyroptosis, like most intracellular pathogens L. monocytogenes has evolved a series of adaptations that minimize host cell death to promote its virulence. Understanding how L. monocytogenes modulates cell death during infection could lead to novel therapeutic approaches. In addition, as L. monocytogenes is currently being developed as a tumor immunotherapy platform, understanding how cell death pathways influence the priming and quality of cell-mediated immunity is critical. This review will focus on the mechanisms by which L. monocytogenes modulates cell death, as well as the implications of cell death on acute infection and the generation of adaptive immunity.

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Effect of bovine lactoferrin onChlamydia trachomatisinfection and inflammation

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0049 ◽

2017 ◽

Vol 95 (1) ◽

pp. 34-40 ◽

Cited By ~ 22

Author(s):

Rosa Sessa ◽

Marisa Di Pietro ◽

Simone Filardo ◽

Alessia Bressan ◽

Luigi Rosa ◽

...

Keyword(s):

Acute Infection ◽

Host Cells ◽

Developmental Cycle ◽

Bovine Lactoferrin ◽

Bacterial Disease ◽

Chronic Infections ◽

Sexually Transmitted ◽

Obligate Intracellular Pathogen

Chlamydia trachomatis is an obligate, intracellular pathogen responsible for the most common sexually transmitted bacterial disease worldwide, causing acute and chronic infections. The acute infection is susceptible to antibiotics, whereas the chronic one needs prolonged therapies, thus increasing the risk of developing antibiotic resistance. Novel alternative therapies are needed. The intracellular development of C. trachomatis requires essential nutrients, including iron. Iron-chelating drugs inhibit C. trachomatis developmental cycle. Lactoferrin (Lf), a pleiotropic iron binding glycoprotein, could be a promising candidate against C. trachomatis infection. Similarly to the efficacy against other intracellular pathogens, bovine Lf (bLf) could both interfere with C. trachomatis entry into epithelial cells and exert an anti-inflammatory activity. In vitro and in vivo effects of bLf against C. trachomatis infectious and inflammatory process has been investigated. BLf inhibits C. trachomatis entry into host cells when incubated with cell monolayers before or at the moment of the infection and down-regulates IL-6/IL-8 synthesized by infected cells. Six out of 7 pregnant women asymptomatically infected by C. trachomatis, after 30 days of bLf intravaginal administration, were negative for C. trachomatis and showed a decrease of cervical IL-6 levels. This is the first time that the bLf protective effect against C. trachomatis infection has been demonstrated.

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Molecular Biology of Pseudorabies Virus: Impact on Neurovirology and Veterinary Medicine

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.69.3.462-500.2005 ◽

2005 ◽

Vol 69 (3) ◽

pp. 462-500 ◽

Cited By ~ 370

Author(s):

Lisa E. Pomeranz ◽

Ashley E. Reynolds ◽

Christoph J. Hengartner

Keyword(s):

Molecular Biology ◽

Pseudorabies Virus ◽

Latent Infection ◽

Acute Infection ◽

Model Systems ◽

Host Cells ◽

Viral Dna ◽

Infectious Particle ◽

New Hosts ◽

Regulated Gene Expression

SUMMARY Pseudorabies virus (PRV) is a herpesvirus of swine, a member of the Alphaherpesvirinae subfamily, and the etiological agent of Aujeszky's disease. This review describes the contributions of PRV research to herpesvirus biology, neurobiology, and viral pathogenesis by focusing on (i) the molecular biology of PRV, (ii) model systems to study PRV pathogenesis and neurovirulence, (iii) PRV transsynaptic tracing of neuronal circuits, and (iv) veterinary aspects of pseudorabies disease. The structure of the enveloped infectious particle, the content of the viral DNA genome, and a step-by-step overview of the viral replication cycle are presented. PRV infection is initiated by binding to cellular receptors to allow penetration into the cell. After reaching the nucleus, the viral genome directs a regulated gene expression cascade that culminates with viral DNA replication and production of new virion constituents. Finally, progeny virions self-assemble and exit the host cells. Animal models and neuronal culture systems developed for the study of PRV pathogenesis and neurovirulence are discussed. PRV serves as a self-perpetuating transsynaptic tracer of neuronal circuitry, and we detail the original studies of PRV circuitry mapping, the biology underlying this application, and the development of the next generation of tracer viruses. The basic veterinary aspects of pseudorabies management and disease in swine are discussed. PRV infection progresses from acute infection of the respiratory epithelium to latent infection in the peripheral nervous system. Sporadic reactivation from latency can transmit PRV to new hosts. The successful management of PRV disease has relied on vaccination, prevention, and testing.

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Human Herpesvirus 6B Induces Hypomethylation on Chromosome 17p13.3, Correlating with Increased Gene Expression and Virus Integration

Journal of Virology ◽

10.1128/jvi.02105-16 ◽

2017 ◽

Vol 91 (11) ◽

Cited By ~ 17

Author(s):

Elin Engdahl ◽

Nicky Dunn ◽

Pitt Niehusmann ◽

Sarah Wideman ◽

Peter Wipfler ◽

...

Keyword(s):

Integration Site ◽

Acute Infection ◽

Human Herpesvirus ◽

Epigenetic Modifications ◽

Host Cells ◽

Integration Process ◽

450K Array ◽

Infected Cells ◽

Virus Integration

ABSTRACT Human herpesvirus 6B (HHV-6B) is a neurotropic betaherpesvirus that achieves latency by integrating its genome into host cell chromosomes. Several viruses can induce epigenetic modifications in their host cells, but no study has investigated the epigenetic modifications induced by HHV-6B. This study analyzed methylation with an Illumina 450K array, comparing HHV-6B-infected and uninfected Molt-3 T cells 3 days postinfection. Bisulfite pyrosequencing was used to validate the Illumina results and to investigate methylation over time in vitro. Expression of genes was investigated using quantitative PCR (qPCR), and virus integration was investigated with PCR. A total of 406 CpG sites showed a significant HHV-6B-induced change in methylation in vitro. Remarkably, 86% (351/406) of these CpGs were located <1 Mb from chromosomal ends and were all hypomethylated in virus-infected cells. This was most evident at chromosome 17p13.3, where HHV-6B had induced CpG hypomethylation after 2 days of infection, possibly through TET2, which was found to be upregulated by the virus. In addition, virus-induced cytosine hydroxymethylation was observed. Genes located in the hypomethylated region at 17p13.3 showed significantly upregulated expression in HHV-6B-infected cells. A temporal experiment revealed HHV-6B integration in Molt-3 cell DNA 3 days after infection. The telomere at 17p has repeatedly been described as an integration site for HHV-6B, and we show for the first time that HHV-6B induces hypomethylation in this region during acute infection, which may play a role in the integration process, possibly by making the DNA more accessible. IMPORTANCE The ability to establish latency in the host is a hallmark of herpesviruses, but the mechanisms differ. Human herpesvirus 6B (HHV-6B) is known to establish latency through integration of its genome into the telomeric regions of host cells, with the ability to reactivate. Our study is the first to show that HHV-6B specifically induces hypomethylated regions close to the telomeres and that integrating viruses may use the host methylation machinery to facilitate their integration process. The results from this study contribute to knowledge of HHV-6B biology and virus-host interaction. This in turn will lead to further progress in our understanding of the underlying mechanisms by which HHV-6B contributes to pathological processes and may have important implications in both disease prevention and treatment.

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p47 GTPases Regulate Toxoplasma gondii Survival in Activated Macrophages

Infection and Immunity ◽

10.1128/iai.73.6.3278-3286.2005 ◽

2005 ◽

Vol 73 (6) ◽

pp. 3278-3286 ◽

Cited By ~ 99

Author(s):

Barbara A. Butcher ◽

Robert I. Greene ◽

Stanley C. Henry ◽

Kimberly L. Annecharico ◽

J. Brice Weinberg ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Acute Infection ◽

Host Cells ◽

Intracellular Growth ◽

Latex Beads ◽

Cellular Factors ◽

Ifn Γ ◽

Normal Inhibition

ABSTRACT The cytokine gamma interferon (IFN-γ) is critical for resistance to Toxoplasma gondii. IFN-γ strongly activates macrophages and nonphagocytic host cells to limit intracellular growth of T. gondii; however, the cellular factors that are required for this effect are largely unknown. We have shown previously that IGTP and LRG-47, members of the IFN-γ-regulated family of p47 GTPases, are required for resistance to acute T. gondii infections in vivo. In contrast, IRG-47, another member of this family, is not required. In the present work, we addressed whether these GTPases are required for IFN-γ-induced suppression of T. gondii growth in macrophages in vitro. Bone marrow macrophages that lacked IGTP or LRG-47 displayed greatly attenuated IFN-γ-induced inhibition of T. gondii growth, while macrophages that lacked IRG-47 displayed normal inhibition. Thus, the ability of the p47 GTPases to limit acute infection in vivo correlated with their ability to suppress intracellular growth in macrophages in vitro. Using confocal microscopy and sucrose density fractionation, we demonstrated that IGTP largely colocalizes with endoplasmic reticulum markers, while LRG-47 was mainly restricted to the Golgi. Although both IGTP and LRG-47 localized to vacuoles containing latex beads, neither protein localized to vacuoles containing live T. gondii. These results suggest that IGTP and LRG-47 are able to regulate host resistance to acute T. gondii infections through their ability to inhibit parasite growth within the macrophage.

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Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

REAKTOR ◽

10.14710/reaktor.21.2.59-64 ◽

2021 ◽

Vol 21 (2) ◽

pp. 59-64

Author(s):

Enny Ratnaningsih ◽

Sulistiya Nirta Sunaryo ◽

Idris Idris ◽

Rindia Maharani Putri

Keyword(s):

Bacillus Cereus ◽

Recombinant Expression ◽

Host Cells ◽

Monochloroacetic Acid ◽

Affinity Column ◽

One Pot ◽

Crude Enzyme Extract ◽

Local Strains ◽

Haloacid Dehalogenase ◽

Purification System

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

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The Toxoplasma gondii Rhoptry Kinome Is Essential for Chronic Infection

mBio ◽

10.1128/mbio.00193-16 ◽

2016 ◽

Vol 7 (3) ◽

Cited By ~ 36

Author(s):

Barbara A. Fox ◽

Leah M. Rommereim ◽

Rebekah B. Guevara ◽

Alejandra Falla ◽

Miryam Andrea Hortua Triana ◽

...

Keyword(s):

Innate Immunity ◽

Toxoplasma Gondii ◽

Virulence Factors ◽

Chronic Infection ◽

Parasitophorous Vacuole ◽

Acute Infection ◽

Host Cells ◽

Type Ii ◽

Host Manipulation ◽

Parasite Survival

ABSTRACT Ingestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the Δ rop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5 , ROP17 , ROP18 , ROP35 , or ROP38 / 29 / 19 ( ROP38 , ROP29 , and ROP19 ) severely reduced cyst burdens. Δ rop5 and Δ rop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The Δ rop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the Δ rop35 or Δ rop38 / 29 / 19 strain. Resistance to host IRGs was not affected in the Δ rop17 , Δ rop35 , or Δ rop38 / 29 / 19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondii . IMPORTANCE Reactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections.

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Natural Competence Promotes Helicobacter pylori Chronic Infection

Infection and Immunity ◽

10.1128/iai.01042-12 ◽

2012 ◽

Vol 81 (1) ◽

pp. 209-215 ◽

Cited By ~ 31

Author(s):

Marion S. Dorer ◽

Ilana E. Cohen ◽

Tate H. Sessler ◽

Jutta Fero ◽

Nina R. Salama

Keyword(s):

Helicobacter Pylori ◽

Chronic Infection ◽

Acute Infection ◽

Host Cells ◽

High Rate ◽

Natural Competence ◽

Content Type ◽

Type Iv ◽

Competence Factor ◽

H Pylori

Animal models are important tools for studies of human disease, but developing these models is a particular challenge with regard to organisms with restricted host ranges, such as the human stomach pathogenHelicobacter pylori. In most cases,H. pyloriinfects the stomach for many decades before symptoms appear, distinguishing it from many bacterial pathogens that cause acute infection. To model chronic infection in the mouse, a human clinical isolate was selected for its ability to survive for 2 months in the mouse stomach, and the resulting strain, MSD132, colonized the mouse stomach for at least 28 weeks. During selection, thecagYcomponent of the Cag type IV secretion system was mutated, disrupting a key interaction with host cells. Increases in both bacterial persistence and bacterial burden occurred prior to this mutation, and a mixed population ofcagY+andcagYmutant cells was isolated from a single mouse, suggesting that mutations accumulate during selection and that factors in addition to the Cag apparatus are important for murine adaptation. Diversity in both alleles and genes is common inH. pyloristrains, and natural competence mediates a high rate of interstrain genetic exchange. Mutations of the Com apparatus, a membrane DNA transporter, and DprA, a cytosolic competence factor, resulted in reduced persistence, although initial colonization was normal. Thus, exchange of DNA between genetically heterogeneousH. pyloristrains may improve chronic colonization. The strains and methods described here will be important tools for defining both the spectrum of mutations that promote murine adaptation and the genetic program of chronic infection.

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Direct Neutralization of Type III Effector Translocation by the Variable Region of a Monoclonal Antibody to Yersinia pestis LcrV

Clinical and Vaccine Immunology ◽

10.1128/cvi.00013-14 ◽

2014 ◽

Vol 21 (5) ◽

pp. 667-673 ◽

Cited By ~ 8

Author(s):

Maya I. Ivanov ◽

Jim Hill ◽

James B. Bliska

Keyword(s):

Monoclonal Antibody ◽

Yersinia Pestis ◽

Actin Polymerization ◽

Protective Antigen ◽

Acute Infection ◽

Variable Region ◽

Host Cells ◽

Content Type ◽

Type Iii

ABSTRACTPlague is an acute infection caused by the Gram-negative bacteriumYersinia pestis. Antibodies that are protective against plague target LcrV, an essential virulence protein and component of a type III secretion system ofY. pestis. Secreted LcrV localizes to the tips of type III needles on the bacterial surface, and its function is necessary for the translocation ofYersiniaouter proteins (Yops) into the cytosol of host cells infected byY. pestis. Translocated Yops counteract macrophage functions, for example, by inhibiting phagocytosis (YopE) or inducing cytotoxicity (YopJ). Although LcrV is the best-characterized protective antigen ofY. pestis, the mechanism of protection by anti-LcrV antibodies is not fully understood. Antibodies bind to LcrV at needle tips, neutralize Yop translocation, and promote opsonophagocytosis ofY. pestisby macrophagesin vitro. However, it is not clear if anti-LcrV antibodies neutralize Yop translocation directly or if they do so indirectly, by promoting opsonophagocytosis. To determine if the protective IgG1 monoclonal antibody (MAb) 7.3 is directly neutralizing, an IgG2a subclass variant, a deglycosylated variant, F(ab′)2, and Fab were tested for the ability to inhibit the translocation of Yops intoY. pestis-infected macrophagesin vitro. Macrophage cytotoxicity and cellular fractionation assays show that the Fc of MAb 7.3 is not required for the neutralization of YopJ or YopE translocation. In addition, the use of Fc receptor-deficient macrophages, and the use of cytochalasin D to inhibit actin polymerization, confirmed that opsonophagocytosis is not required for MAb 7.3 to neutralize translocation. These data indicate that the binding of the variable region of MAb 7.3 to LcrV is sufficient to directly neutralize Yop translocation.

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