scholarly journals Enteric pathogens deploy cell cycle inhibiting factors to block the bactericidal activity of Perforin-2

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Ryan M McCormack ◽  
Kirill Lyapichev ◽  
Melissa L Olsson ◽  
Eckhard R Podack ◽  
George P Munson
Keyword(s):  
Cell Cycle ◽  
Bactericidal Activity ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Biological Significance ◽  
Enteric Pathogens ◽  
Host Cells ◽  
Inhibiting Factors ◽  
Molecular Events ◽  
Molecular Patterns

Perforin-2 (MPEG1) is an effector of the innate immune system that limits the proliferation and spread of medically relevant Gram-negative, -positive, and acid fast bacteria. We show here that a cullin-RING E3 ubiquitin ligase (CRL) complex containing cullin-1 and βTrCP monoubiquitylates Perforin-2 in response to pathogen associated molecular patterns such as LPS. Ubiquitylation triggers a rapid redistribution of Perforin-2 and is essential for its bactericidal activity. Enteric pathogens such as Yersinia pseudotuberculosis and enteropathogenic Escherichia coli disarm host cells by injecting cell cycle inhibiting factors (Cifs) into mammalian cells to deamidate the ubiquitin-like protein NEDD8. Because CRL activity is dependent upon NEDD8, Cif blocks ubiquitin dependent trafficking of Perforin-2 and thus, its bactericidal activity. Collectively, these studies further underscore the biological significance of Perforin-2 and elucidate critical molecular events that culminate in Perforin-2-dependent killing of both intracellular and extracellular, cell-adherent bacteria.

scholarly journals Author response: Enteric pathogens deploy cell cycle inhibiting factors to block the bactericidal activity of Perforin-2

2015 ◽  
Author(s):  
Ryan M McCormack ◽  
Kirill Lyapichev ◽  
Melissa L Olsson ◽  
Eckhard R Podack ◽  
George P Munson
Keyword(s):  
Cell Cycle ◽  
Bactericidal Activity ◽  
Enteric Pathogens ◽  
Author Response ◽  
Inhibiting Factors

scholarly journals Arf6 and Phosphoinositol-4-Phosphate-5-Kinase Activities Permit Bypass of the Rac1 Requirement for β1 Integrin–mediated Bacterial Uptake

2003 ◽  
Vol 198 (4) ◽  
pp. 603-614 ◽  
Author(s):  
Ka-Wing Wong ◽  
Ralph R. Isberg
Keyword(s):  
Target Cell ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Small Gtpase ◽  
Host Cells ◽  
Β1 Integrin ◽  
Integrin Receptors ◽  
Bacterial Binding ◽  
Bacterial Uptake ◽  
Dependent Pathway

Efficient entry of the bacterium Yersinia pseudotuberculosis into mammalian cells requires the binding of the bacterial invasin protein to β1 integrin receptors and the activation of the small GTPase Rac1. We report here that this Rac1-dependent pathway involves recruitment of phosphoinositol-4-phosphate-5-kinase (PIP5K) to form phosphoinositol-4,5-bisphosphate (PIP2) at the phagocytic cup. Reducing the concentration of PIP2 in the target cell by using a membrane-targeted PIP2-specific phosphatase lowered bacterial uptake proportionately. PIP2 formation is regulated by Arf6. An Arf6 derivative defective for nucleotide binding (Arf6N122I) interfered with uptake and decreased the level of PIP2 around extracellular bacteria bound to host cells. This reduction in PIP2 occurred in spite of fact that PIP5K appeared to be recruited efficiently to the site of bacterial binding, indicating a role for Arf6 in activation of the kinase. The elimination of the Rac1-GTP–bound form from the cell by the introduction of the Y. pseudotuberculosis YopE RhoGAP protein could be bypassed by the overproduction of either PIP5K or Arf6, although the degree of bypass was greater for Arf6 transfectants. These results indicate that both Arf6 and PIP5K are involved in integrin-dependent uptake, and that Arf6 participates in both activation of PIP5K as well as in other events associated with bacterial uptake.

scholarly journals A Solvent-Exposed Patch in Chaperone-Bound YopE Is Required for Translocation by the Type III Secretion System

2010 ◽  
Vol 192 (12) ◽  
pp. 3114-3122 ◽  
Author(s):  
Loren Rodgers ◽  
Romila Mukerjea ◽  
Sara Birtalan ◽  
Devorah Friedberg ◽  
Partho Ghosh
Keyword(s):  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Effector Proteins ◽  
Host Cells ◽  
Type Iii ◽  
Potential Association ◽  
Chaperone Binding ◽  
Bacterial Type ◽  
Characteristic Manner

ABSTRACT Most effector proteins of bacterial type III secretion (T3S) systems require chaperone proteins for translocation into host cells. Such effectors are bound by chaperones in a conserved and characteristic manner, with the chaperone-binding (Cb) region of the effector wound around the chaperone in a highly extended conformation. This conformation has been suggested to serve as a translocation signal in promoting the association between the chaperone-effector complex and a bacterial component required for translocation. We sought to test a prediction of this model by identifying a potential association site for the Yersinia pseudotuberculosis chaperone-effector pair SycE-YopE. We identified a set of residues in the YopE Cb region that are required for translocation but are dispensable for expression, SycE binding, secretion into the extrabacterial milieu, and stability in mammalian cells. These residues form a solvent-exposed patch on the surface of the chaperone-bound Cb region, and thus their effect on translocation is consistent with the structure of the chaperone-bound Cb region serving as a signal for translocation.

scholarly journals c-Myb Contributes to G2/M Cell Cycle Transition in Human Hematopoietic Cells by Direct Regulation of Cyclin B1 Expression

2007 ◽  
Vol 27 (6) ◽  
pp. 2048-2058 ◽  
Author(s):  
Yuji Nakata ◽  
Susan Shetzline ◽  
Chizuko Sakashita ◽  
Anna Kalota ◽  
Ravikumar Rallapalli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Biological Significance ◽  
Cyclin B1 ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
M Cell ◽  
Cell Cycle Transition

ABSTRACTMyb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G1/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells. As a result, it contributes directly to G2/M cell cycle progression. In cell lines and primary cells, cyclin B1 levels varied directly with c-Myb expression. Chromatin immunoprecipitation assays, mutation analysis, and luciferase reporter assays revealed that c-Myb bound the cyclin B1 promoter preferentially at a site just downstream of the transcriptional start site. The biological significance of c-Myb, versus B-Myb, binding the cyclin B1 promoter was demonstrated by the fact that expression of inducible dominant negative c-Myb in K562 cells accelerated their exit from M phase. In addition, expression of c-Myb in HCT116 cells rescued cyclin B1 expression after B-mybexpression was silenced with small interfering RNA. These results suggest that c-Myb protein plays a previously unappreciated role in the G2/M cell cycle transition of normal and malignant human hematopoietic cells and expands the known repertoire of c-mybfunctions in regulating human hematopoiesis.

scholarly journals Post-Glycosylation Modification of Sialic Acid and Its Role in Virus Pathogenesis

Vaccines ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 171 ◽  
Author(s):  
Park
Keyword(s):  
Sialic Acid ◽  
Mammalian Cells ◽  
Biological Significance ◽  
Extraction Methods ◽  
Sialic Acids ◽  
Neuraminic Acid ◽  
Host Cells ◽  
Viral Receptor ◽  
Site Specific ◽  
Viral Binding

Sialic acids are a family of nine carbon keto-aldononulosonic acids presented at the terminal ends of glycans on cellular membranes. α-Linked sialoglycoconjugates often undergo post-glycosylation modifications, among which O-acetylation of N-acetyl neuraminic acid (Neu5Ac) is the most common in mammalian cells. Isoforms of sialic acid are critical determinants of virus pathogenesis. To date, the focus of viral receptor-mediated attachment has been on Neu5Ac. O-Acetylated Neu5Acs have been largely ignored as receptor determinants of virus pathogenesis, although it is ubiquitous across species. Significantly, the array of structures resulting from site-specific O-acetylation by sialic acid O-acetyltransferases (SOATs) provides a means to examine specificity of viral binding to host cells. Specifically, C4 O-acetylated Neu5Ac can influence virus pathogenicity. However, the biological implications of only O-acetylated Neu5Ac at C7–9 have been explored extensively. This review will highlight the biological significance, extraction methods, and synthetic modifications of C4 O-acetylated Neu5Ac that may provide value in therapeutic developments and targets to prevent virus related diseases.

scholarly journals Impact of Host Membrane Pore Formation by the Yersinia pseudotuberculosis Type III Secretion System on the Macrophage Innate Immune Response

2013 ◽  
Vol 81 (3) ◽  
pp. 905-914 ◽  
Author(s):  
Laura Kwuan ◽  
Walter Adams ◽  
Victoria Auerbuch
Keyword(s):  
Cell Death ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Effector Proteins ◽  
Innate Immune ◽  
Host Cells ◽  
Content Type ◽  
Host Cell Death ◽  
Tnf Α

ABSTRACTType III secretion systems (T3SSs) are used by Gram-negative pathogens to form pores in host membranes and deliver virulence-associated effector proteins inside host cells. In pathogenicYersinia, the T3SS pore-forming proteins are YopB and YopD. Mammalian cells recognize theYersiniaT3SS, leading to a host response that includes secretion of the inflammatory cytokine interleukin-1β (IL-1β), Toll-like receptor (TLR)-independent expression of the stress-associated transcription factor Egr1 and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), and host cell death. The knownYersiniaT3SS effector proteins are dispensable for eliciting these responses, but YopB is essential. Three models describe how theYersiniaT3SS might trigger inflammation: (i) mammalian cells sense YopBD-mediated pore formation, (ii) innate immune stimuli gain access to the host cytoplasm through the YopBD pore, and/or (iii) the YopB-YopD translocon itself or its membrane insertion is proinflammatory. To test these models, we constructed aYersinia pseudotuberculosismutant expressing YopD devoid of its predicted transmembrane domain (YopDΔTM) and lacking the T3SS cargo proteins YopHEMOJTN. This mutant formed pores in macrophages, but it could not mediate translocation of effector proteins inside host cells. Importantly, this mutant did not elicit rapid host cell death, IL-1β secretion, or TLR-independent Egr1 and TNF-α expression. These data suggest that YopBD-mediated translocation of unknown T3SS cargo leads to activation of host pathways influencing inflammation, cell death, and response to stress. As the YopDΔTMY. pseudotuberculosismutant formed somewhat smaller pores with delayed kinetics, an alternative model is that the wild-type YopB-YopD translocon is specifically sensed by host cells.

scholarly journals Structural and Biological Basis of Alphacoronavirus nsp1 Associated with Host Proliferation and Immune Evasion

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 812
Author(s):  
Zhou Shen ◽  
Yiling Yang ◽  
Siqi Yang ◽  
Guangxu Zhang ◽  
Shaobo Xiao ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Cycle ◽  
Immune Evasion ◽  
Mammalian Cells ◽  
Full Length ◽  
Host Protein ◽  
Host Cells ◽  
Renilla Luciferase ◽  
Sequencing Analysis ◽  
Viral Virulence

Non-structural protein 1 (nsp1) is only characterized in alphacoronaviruses (α-CoVs) and betacoronaviruses (β-CoVs). There have been extensive researches on how the β-CoVs nsp1 regulates viral virulence by inhibiting host protein synthesis, but the regulatory mechanism of the α-CoVs nsp1 is still unclear. Here, we report the 2.1-Å full-length crystal structure of nsp1 in emerging porcine SADS-CoV and the 1.8-Å full-length crystal structure of nsp1 in the highly lethal cat FIPV. Although they belong to different subtypes of α-CoVs, these viruses all have a bucket-shaped fold composed of six β-sheets, similar to the crystal structure of PEDV and TGEV nsp1. Comparing the above four structures, we found that the structure of α-CoVs nsp1 in the same subtype was more conserved. We then selected mammalian cells that were treated with SADS-CoV and FIPV nsp1 for RNA sequencing analysis and found that nsp1 had a specific inhibitory effect on interferon (IFN) and cell cycle genes. Using the Renilla luciferase (Rluc) assay and Western blotting, we confirmed that seven representative α-CoVs nsp1s could significantly inhibit the phosphorylation of STAT1-S727 and interfere with the effect of IFN-I. Moreover, the cell cycle experiment confirmed that α-CoVs nsp1 could encourage host cells to stay in the G0/G1 phase. Based on these findings, we not only greatly improved the crystal structure data on α-CoVs nsp1, but we also speculated that α-CoVs nsp1 regulated host proliferation and immune evasion-related biological functions by inhibiting the synthesis of host proteins, thus creating an environment conducive to the virus.

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