Symbiotic Gene Sym31 Controls the Presence of a Lectinlike Glycoprotein in the Symbiosome Compartment of Nitrogen-Fixing Pea Nodules

The distribution of a lectinlike glycoprotein, PsNLEC-1, was examined with a specific antiserum in nodule tissue from a symbiotically defective pea mutant, Sprint2Fix¯(sym31), and the parent line, Sprint2. Immunostaining of Western blots (immunoblots) revealed that, whereas wild-type nodules contained three antigenic isoforms of PsNLEC-1, nodule homogenates of mutant sym31 contained only one isoform, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was not associated with symbiosomes in either the mutant or the wild-type parent (unlike the other two isoforms from wild-type nodules). Light microscopy revealed that PsNLEC-1 antigen was more abundant in the infected tissues of wild-type nodules than in nodules of sym31. By contrast, in situ hybridization indicated that the PsNlec1 gene transcript was strongly expressed in infected cells of both Sprint2 and sym31 nodule tissues. At the ultrastructural level, most of the PsNLEC-1 antigen in sym31 nodule tissue was visualized as inclusion bodies in the vacuolar compartment of infected host cells but it was apparently absent from the symbiosome compartment. The results suggest an aberrant vesicle targeting pathway during symbiosome development in this mutant.

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Analysis of mouse polyomavirus mutants with lesions in the minor capsid proteins

Journal of General Virology ◽

10.1099/0022-1317-83-9-2309 ◽

2002 ◽

Vol 83 (9) ◽

pp. 2309-2319 ◽

Cited By ~ 28

Author(s):

Petra Mannová ◽

David Liebl ◽

Nina Krauzewicz ◽

Anna Fejtová ◽

Jitka Štokrová ◽

...

Keyword(s):

Host Cells ◽

Cell Fractionation ◽

Wild Type ◽

Terminal Amino Acid ◽

Viral Antigens ◽

Cell Structures ◽

Viral Particles ◽

Infected Cells ◽

Terminal Amino

Polyomavirus mutants E, Q and H, expressing non-myristylated VP2, were generated by replacing the N-terminal glycine residue with glutamic acid, glutamine or histidine, respectively. Viruses mutated in either VP2 or VP3 translation initiation codons were also prepared. All mutated genomes, when transfected into murine host cells, gave rise to viral particles. Infectivity of VP2− and VP3− viruses, as measured by the number of cells expressing viral antigens, was dramatically diminished, indicative of defects in the early stages of infection. In contrast, the absence of a myristyl moiety on VP2 did not substantially affect the early steps of virus infection. No differences in numbers of cells expressing early or late viral antigens were observed between wild-type (wt) and E or Q myr− viruses during the course of a life cycle. Furthermore, no delay in virus DNA replication was detected. However, when cells were left for longer in culture, the number of infected cells, measured by typical virus bursts, was much lower when mutant rather than wt genomes were used. In situ, cell fractionation studies revealed differences in the interaction of viral particles with host cell structures. The infectivity of mutants was affected not only by loss of the myristyl group on VP2, but also, and to a greater extent, by alterations of the N-terminal amino acid composition.

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Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

Journal of Virology ◽

10.1128/jvi.80.1.360-371.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 360-371 ◽

Cited By ~ 42

Author(s):

Dorothea L. Sawicki ◽

Silvia Perri ◽

John M. Polo ◽

Stanley G. Sawicki

Keyword(s):

Host Response ◽

Rna Synthesis ◽

Late Phase ◽

Host Cells ◽

Minus Strand ◽

Wild Type ◽

Persistent Infections ◽

Infected Cells ◽

Replicative Intermediates ◽

Transcription Complexes

ABSTRACT In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and ∼10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC+) that are active in plus-strand synthesis. Minus-strand synthesis and incorporation of [3H]uridine into replicative intermediates differed among PI replicons, depending on the location of the mutation in nsP2. Minus-strand synthesis by PI cells appeared normal; it was dependent on continuous P123 and P1234 polyprotein synthesis and ceased when protein synthesis was inhibited. The failure by the PI replicons to shut off minus-strand synthesis was not due to some defect in the PI cells but rather was due to the loss of some function in the mutated nsP2. This was demonstrated by showing that superinfection of PI cells with wt SFV triggered the shutdown of minus-strand synthesis, which we believe is a host response to infection with alphaviruses. Together, the results indicate alphavirus nsP2 functions to engage the host response to infection and activate a switch from the early-to-late phase. The loss of this function leads to continuous viral minus-strand synthesis and the production of unstable RC+.

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Direct intracellular visualization of Ebola virus-receptor interaction by in situ proximity ligation

10.1101/2020.05.05.080085 ◽

2020 ◽

Author(s):

Eva Mittler ◽

Tanwee P Alkutkar ◽

Rohit K Jangra ◽

Kartik Chandran

Keyword(s):

Receptor Binding ◽

Ebola Virus ◽

Host Cells ◽

Proximity Ligation Assay ◽

Viral Glycoprotein ◽

Virus Receptor ◽

Proximity Ligation ◽

Infected Cells ◽

Viral Surface

ABSTRACTEbola virus (EBOV) entry into host cells comprises stepwise and extensive interactions of the sole viral surface glycoprotein GP with multiple host factors. During the intricate process, following virus uptake and trafficking to late endosomal/lysosomal compartments, GP is proteolytically processed to GPCL by the endosomal proteases cathepsin B and L unmasking GP’s receptor-binding site. Engagement of GPCL with the universal filoviral intracellular receptor Niemann-Pick C1 (NPC1) eventually culminates in fusion between viral and cellular membranes, cytoplasmic escape of the viral nucleocapsid and subsequent infection. Mechanistic delineation of the indispensable GPCL:NPC1 binding step has been severely hampered by the unavailability of a robust cell-based assay assessing interaction of GPCL with full-length endosomal NPC1.Here, we describe a novel in situ assay to monitor GPCL:NPC1 engagement in intact, infected cells. Visualization of the subcellular localization of binding complexes is based on the principle of DNA-assisted, antibody-mediated proximity ligation. Virus-receptor binding monitored by proximity ligation was contingent on GP’s proteolytic cleavage, and was sensitive to perturbations in the GPCL:NPC1 interface. Our assay also specifically decoupled detection of virus-receptor binding from steps post-receptor binding, such as membrane fusion and infection. Testing of multiple FDA-approved small molecule inhibitors revealed that drug treatments inhibited virus entry and GPCL:NPC1 recognition by distinctive mechanisms. Together, here we present a newly established proximity ligation assay, which will allow us to dissect cellular and viral requirements for filovirus-receptor binding, and to delineate the mechanisms of action of inhibitors on filovirus entry in a cell-based system.IMPORTANCEEbola virus causes episodic but increasingly frequent outbreaks of severe disease in Middle Africa, as shown by a currently ongoing outbreak in the Democratic Republic of Congo. Despite considerable effort, FDA-approved anti-filoviral therapeutics or targeted interventions are not available yet. Virus host-cell invasion represents an attractive target for antivirals; however our understanding of the inhibitory mechanisms of novel therapeutics is often hampered by fragmented knowledge of the filovirus-host molecular interactions required for viral infection. To help close this critical knowledge gap, here, we report an in situ assay to monitor binding of the EBOV glycoprotein to its receptor NPC1 in intact, infected cells. We demonstrate that our in situ assay based on proximity ligation represents a powerful tool to delineate receptor-viral glycoprotein interactions. Similar assays can be utilized to examine receptor interactions of diverse viral surface proteins whose studies have been hampered until now by the lack of robust in situ assays.

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Loss of Virulence of the Phytopathogen Ralstonia solanacearum Through Infection by φRSM Filamentous Phages

Phytopathology ◽

10.1094/phyto-11-11-0319-r ◽

2012 ◽

Vol 102 (5) ◽

pp. 469-477 ◽

Cited By ~ 47

Author(s):

Hardian S. Addy ◽

Ahmed Askora ◽

Takeru Kawasaki ◽

Makoto Fujie ◽

Takashi Yamada

Keyword(s):

Ralstonia Solanacearum ◽

Virulence Genes ◽

Host Cells ◽

Extracellular Polysaccharides ◽

Twitching Motility ◽

Wild Type ◽

Tomato Plants ◽

Type Iv ◽

Infected Cells ◽

Filamentous Phages

φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of β-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.

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Leishmania-infected macrophages release extracellular vesicles that can promote lesion development

Life Science Alliance ◽

10.26508/lsa.202000742 ◽

2020 ◽

Vol 3 (12) ◽

pp. e202000742

Author(s):

Anna Gioseffi ◽

Tim Hamerly ◽

Kha Van ◽

Naixin Zhang ◽

Rhoel R Dinglasan ◽

...

Keyword(s):

Endothelial Cells ◽

Extracellular Vesicles ◽

Leishmania Donovani ◽

Protein Profile ◽

Tube Formation ◽

Host Cells ◽

Western Blots ◽

Host Cell Proteins ◽

Transgenic Parasite ◽

Infected Cells

Leishmania donovani infection of macrophages results in quantitative and qualitative changes in the protein profile of extracellular vesicles (EVs) released by the infected host cells. We confirmed mass spectrometry results orthogonally by performing Western blots for several Leishmania-infected macrophage-enriched EVs (LieEVs) molecules. Several host cell proteins in LieEVs have been implicated in promoting vascular changes in other systems. We also identified 59 parasite-derived proteins in LieEVs, including a putative L. donovani homolog of mammalian vasohibins (LdVash), which in mammals promotes angiogenesis. We developed a transgenic parasite that expressed an endogenously tagged LdVash/mNeonGreen (mNG) and confirmed that LdVash/mNG is indeed expressed in infected macrophages and in LieEVs. We further observed that LieEVs induce endothelial cells to release angiogenesis promoting mediators including IL-8, G-CSF/CSF-3, and VEGF-A. In addition, LieEVs induce epithelial cell migration and tube formation by endothelial cells in surrogate angiogenesis assays. Taken together, these studies show that Leishmania infection alters the composition of EVs from infected cells and suggest that LieEVs may play a role in the promotion of vascularization of Leishmania infections.

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phrR-Like Gene praR of Azorhizobium caulinodans ORS571 Is Essential for Symbiosis with Sesbania rostrata and Is Involved in Expression of reb Genes

Applied and Environmental Microbiology ◽

10.1128/aem.00238-10 ◽

2010 ◽

Vol 76 (11) ◽

pp. 3475-3485 ◽

Cited By ~ 19

Author(s):

Noriko Akiba ◽

Toshihiro Aono ◽

Hiroki Toyazaki ◽

Satoru Sato ◽

Hiroshi Oyaizu

Keyword(s):

Nitrogen Fixation ◽

Host Cells ◽

Sesbania Rostrata ◽

Azorhizobium Caulinodans ◽

Wild Type ◽

Nitrogen Fixation Activity ◽

Homologous Genes ◽

Fixation Activity ◽

Putative Transcription Factor ◽

Infected Cells

ABSTRACT This study focuses on the function of the gene praR that encodes a putative transcription factor in Azorhizobium caulinodans ORS571, a microsymbiont of Sesbania rostrata. The praR gene is a homolog of the phrR gene of Sinorhizobium medicae WSM419, and the praR and phrR homologs are distributed throughout the class Alphaproteobacteria. The growth and nitrogen fixation activity of an A. caulinodans praR deletion mutant in the free-living state were not significantly different from those of the wild-type strain. However, the stem nodules formed by the praR mutant showed lower nitrogen fixation activity than the wild-type stem nodules. Microscopy revealed that infected host cells with an oval or elongated shape were observed at early stages in the nodules formed by the praR mutant, but these infected cells gradually fell into two types. One maintained an oval or elongated shape, but the vacuoles in these cells gradually enlarged and the bacteria gradually disappeared. The other cells were shrunken with bacteria remaining inside. Microarrays revealed that genes homologous to the reb genes of Caedibacter taeniospiralis were highly expressed in the praR mutant. Furthermore, the stem nodules formed by an A. caulinodans mutant with a deletion of praR and reb-homologous genes showed high nitrogen fixation activity, comparable to that of the wild-type stem nodules, and were filled with oval or elongated host cells. These results suggest that PraR controls the expression of the reb-homologous genes and that high expression of reb-homologous genes causes aberrance in A. caulinodans-S. rostrata symbiosis.

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Murine Cytomegalovirus M25 Proteins Sequester the Tumor Suppressor Protein p53 in Nuclear Accumulations

Journal of Virology ◽

10.1128/jvi.00574-20 ◽

2020 ◽

Vol 94 (20) ◽

Author(s):

Ivana Kutle ◽

Katarzyna M. Szymańska-de Wijs ◽

Boris Bogdanow ◽

Berislav Cuvalo ◽

Lars Steinbrück ◽

...

Keyword(s):

Tumor Suppressor ◽

Defense Mechanisms ◽

Target Genes ◽

Host Cells ◽

Tumor Suppressor Protein ◽

Wild Type ◽

Tumor Suppressor Protein P53 ◽

Protein P53 ◽

Infected Cells ◽

Infection Interaction

ABSTRACT To ensure productive infection, herpesviruses utilize tegument proteins and nonstructural regulatory proteins to counteract cellular defense mechanisms and to reprogram cellular pathways. The M25 proteins of mouse cytomegalovirus (MCMV) belong to the betaherpesvirus UL25 gene family that encodes viral proteins implicated with regulatory functions. Through affinity purification and mass spectrometric analysis, we discovered the tumor suppressor protein p53 as a host factor interacting with the M25 proteins. M25-p53 interaction in infected and transfected cells was confirmed by coimmunoprecipitation. Moreover, the proteins colocalized in nuclear dot-like structures upon both infection and inducible expression of the two M25 isoforms. p53 accumulated in wild-type MCMV-infected cells, while this did not occur upon infection with a mutant lacking the M25 gene. Both M25 proteins were able to mediate the effect, identifying them as the first CMV proteins responsible for p53 accumulation during infection. Interaction with M25 proteins led to substantial prolongation of the half-life of p53. In contrast to the higher abundance of the p53 protein in wild-type MCMV-infected cells, the transcript levels of the prominent p53 target genes Cdkn1a and Mdm2 were diminished compared to cells infected with the ΔM25 mutant, and this was associated with reduced binding of p53 to responsive elements within the respective promoters. Notably, the productivity of the M25 deletion mutant was partially rescued on p53-negative fibroblasts. We propose that the MCMV M25 proteins sequester p53 molecules in the nucleus of infected cells, reducing their availability for activating a subset of p53-regulated genes, thereby dampening the antiviral role of p53. IMPORTANCE Host cells use a number of factors to defend against viral infection. Viruses are, however, in an arms race with their host cells to overcome these defense mechanisms. The tumor suppressor protein p53 is an important sensor of cell stress induced by oncogenic insults or viral infections, which upon activation induces various pathways to ensure the integrity of cells. Viruses have to counteract many functions of p53, but complex DNA viruses such as cytomegaloviruses may also utilize some p53 functions for their own benefit. In this study, we discovered that the M25 proteins of mouse cytomegalovirus interact with p53 and mediate its accumulation during infection. Interaction with the M25 proteins sequesters p53 molecules in nuclear dot-like structures, limiting their availability for activation of a subset of p53-regulated target genes. Understanding the interaction between viral proteins and p53 may allow to develop new therapeutic strategies against cytomegalovirus and other viruses.

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The EspD Protein of EnterohemorrhagicEscherichia coli Is Required for the Formation of Bacterial Surface Appendages and Is Incorporated in the Cytoplasmic Membranes of Target Cells

Infection and Immunity ◽

10.1128/iai.67.9.4834-4842.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4834-4842 ◽

Cited By ~ 61

Author(s):

Andreas U. Kresse ◽

Manfred Rohde ◽

Carlos A. Guzmán

Keyword(s):

Shiga Toxin ◽

Host Cells ◽

Target Cells ◽

Wild Type ◽

Bacterial Surface ◽

E Coli ◽

Molecular Events ◽

Cytoplasmic Membranes ◽

Infected Cells

ABSTRACT The formation of EspA-containing surface appendages in pathogenicEscherichia coli strains, both enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. colistrains, is essential for critical events in the infective process, e.g., localized bacterial adherence to host cells with formation of microcolonies and induction of attaching and effacing lesions. It has been reported that EPEC mutants deficient in the production of EspD, which is encoded by the esp operon, are unable to accumulate actin underneath adherent bacteria but exhibit an attachment similar to that of the wild type. Here, we report the construction and characterization of an in-frame espD deletion mutant of the enterohemorrhagic E. coli (EHEC) strain EDL933. In contrast to what was observed in EPEC mutants, the EDL933 espDmutant not only lacked the capacity to accumulate actin but also exhibited an impaired attachment to HeLa cells. The synthesis of the EspD protein was also essential for the formation of EspA-containing filaments. Finally, localization studies demonstrated that the EspD protein is transferred to the cytoplasm and integrated into the cytoplasmic membranes of infected cells. These results help to elucidate the underlying molecular events in infections caused by EHEC.

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Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051657 ◽

1974 ◽

Vol 32 ◽

pp. 328-329

Author(s):

Joseph E. Mazurkiewicz

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Ultrastructural Level ◽

Electron Microscopic ◽

Biological Interest ◽

Investigative Approach ◽

Immunologic Activity ◽

Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

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Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

International Journal of Molecular Sciences ◽

10.3390/ijms22094823 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4823

Author(s):

María Fernanda González ◽

Paula Díaz ◽

Alejandra Sandoval-Bórquez ◽

Daniela Herrera ◽

Andrew F. G. Quest

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Outer Membrane ◽

Extracellular Vesicles ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Gastric Epithelium ◽

Infected Cells ◽

H Pylori

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.

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