scholarly journals Brucella ovis cysteine biosynthesis contributes to peroxide stress survival and fitness in the intracellular niche

2021 ◽  
Author(s):  
Lydia M. Varesio ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Stationary Phase ◽  
Cell Entry ◽  
Host Cells ◽  
Cysteine Biosynthesis ◽  
Brucella Ovis ◽  
Stress Survival ◽  
Host Cell Entry ◽  
Peroxide Stress ◽  
Male Genital

Brucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. These same conditions are often encountered in axenic cultures during stationary phase. Studies of stationary phase may thus inform understanding of Brucella infection biology, yet the genes and pathways that are important in Brucella stationary phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn-himar mutants as a function of growth phase and identified cysE as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine, and we provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis at steps downstream of CysE. Deleting either cysE (ΔcysE) or both cysK1 and cysK2 (ΔcysK1 ΔcysK2) results in premature entry into stationary phase, reduced culture yield and sensitivity to exogenous hydrogen peroxide. These phenotypes can be chemically complemented by cysteine or glutathione. ΔcysE and ΔcysK1 ΔcysK2 strains have no defect in host cell entry in vitro but have significantly diminished intracellular fitness between 2 and 24 hours post infection. Our study has uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in B. ovis, and demonstrates that cysteine anabolism is a determinant of peroxide stress survival and fitness in the intracellular niche.

scholarly journals Cysteine biosynthesis is a determinant of Brucella ovis stress survival and fitness in the intracellular niche

2020 ◽  
Author(s):  
Lydia M. Varesio ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Stationary Phase ◽  
Cell Entry ◽  
Host Cells ◽  
Cysteine Biosynthesis ◽  
Brucella Ovis ◽  
Cysteine Synthesis ◽  
Host Cell Entry ◽  
Phase Entry ◽  
Male Genital

AbstractBrucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. These same conditions are often encountered in stationary phase cultures. Studies of stationary phase may thus inform understanding of Brucella infection biology, yet the genes that are important in Brucella stationary phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn-himar mutants as a function of growth phase and identified cysE as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine. We provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis downstream of CysE. Deleting either cysE or both cysK1 and cysK2 leads to premature entry into stationary phase and reduced culture yield. These phenotypes are rescued by addition of cysteine or glutathione to the medium. We further show that deletion of cysE results in sensitivity to exogenous hydrogen peroxide. Finally, we demonstrate that B. ovis ΔcysE has no defect in host cell entry but is attenuated in macrophage-like cells and in ovine testis epithelial cells at one- and two-days post infection. Our study uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in B. ovis, and demonstrated that cysteine anabolism is an important determinant of stationary phase entry in vitro and fitness in the intracellular niche.

scholarly journals Functional Characterization of a Redundant Plasmodium TRAP Family Invasin, TRAP-Like Protein, by Aldolase Binding and a Genetic Complementation Test

2008 ◽  
Vol 7 (6) ◽  
pp. 1062-1070 ◽  
Author(s):  
Kirsten Heiss ◽  
Hui Nie ◽  
Sumit Kumar ◽  
Thomas M. Daly ◽  
Lawrence W. Bergman ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Cell Entry ◽  
Host Cells ◽  
Complementation Test ◽  
Type I ◽  
Cycle Progression ◽  
Targeted Gene Disruption ◽  
Specific Host ◽  
Host Cell Entry

ABSTRACT Efficient and specific host cell entry is of exquisite importance for intracellular pathogens. Parasites of the phylum Apicomplexa are highly motile and actively enter host cells. These functions are mediated by type I transmembrane invasins of the TRAP family that link an extracellular recognition event to the parasite actin-myosin motor machinery. We systematically tested potential parasite invasins for binding to the actin bridging molecule aldolase and complementation of the vital cytoplasmic domain of the sporozoite invasin TRAP. We show that the ookinete invasin CTRP and a novel, structurally related protein, termed TRAP-like protein (TLP), are functional members of the TRAP family. Although TLP is expressed in invasive stages, targeted gene disruption revealed a nonvital role during life cycle progression. This is the first genetic analysis of TLP, encoding a redundant TRAP family invasin, in the malaria parasite.

scholarly journals A structure-based rationale for sialic acid independent host-cell entry of Sosuga virus

2019 ◽  
Vol 116 (43) ◽  
pp. 21514-21520 ◽  
Author(s):  
Alice J. Stelfox ◽  
Thomas A. Bowden
Keyword(s):  
Sialic Acid ◽  
Host Cell ◽  
Receptor Binding ◽  
Cell Attachment ◽  
Cell Entry ◽  
Head Region ◽  
Close Proximity ◽  
Host Cell Attachment ◽  
Host Cell Entry

The bat-borne paramyxovirus, Sosuga virus (SosV), is one of many paramyxoviruses recently identified and classified within the newly established genus Pararubulavirus, family Paramyxoviridae. The envelope surface of SosV presents a receptor-binding protein (RBP), SosV-RBP, which facilitates host-cell attachment and entry. Unlike closely related hemagglutinin neuraminidase RBPs from other genera of the Paramyxoviridae, SosV-RBP and other pararubulavirus RBPs lack many of the stringently conserved residues required for sialic acid recognition and hydrolysis. We determined the crystal structure of the globular head region of SosV-RBP, revealing that while the glycoprotein presents a classical paramyxoviral six-bladed β-propeller fold and structurally classifies in close proximity to paramyxoviral RBPs with hemagglutinin-neuraminidase (HN) functionality, it presents a receptor-binding face incongruent with sialic acid recognition. Hemadsorption and neuraminidase activity analysis confirms the limited capacity of SosV-RBP to interact with sialic acid in vitro and indicates that SosV-RBP undergoes a nonclassical route of host-cell entry. The close overall structural conservation of SosV-RBP with other classical HN RBPs supports a model by which pararubulaviruses only recently diverged from sialic acid binding functionality.

scholarly journals Shigella applies molecular mimicry to subvert vinculin and invade host cells

2006 ◽  
Vol 175 (3) ◽  
pp. 465-475 ◽  
Author(s):  
Tina Izard ◽  
Guy Tran Van Nhieu ◽  
Philippe R.J. Bois
Keyword(s):  
Host Cell ◽  
Binding Sites ◽  
Type Iii Secretion ◽  
Molecular Mimicry ◽  
Shigella Flexneri ◽  
Cell Entry ◽  
Host Cells ◽  
General Mechanism ◽  
Host Cell Entry ◽  
Cell Spread

Shigella flexneri, the causative agent of bacillary dysentery, injects invasin proteins through a type III secretion apparatus upon contacting the host cell, which triggers pathogen internalization. The invasin IpaA is essential for S. flexneri pathogenesis and binds to the cytoskeletal protein vinculin to facilitate host cell entry. We report that IpaA harbors two vinculin-binding sites (VBSs) within its C-terminal domain that bind to and activate vinculin in a mutually exclusive fashion. Only the highest affinity C-terminal IpaA VBS is necessary for efficient entry and cell–cell spread of S. flexneri, whereas the lower affinity VBS appears to contribute to vinculin recruitment at entry foci of the pathogen. Finally, the crystal structures of vinculin in complex with the VBSs of IpaA reveal the mechanism by which IpaA subverts vinculin's functions, where S. flexneri utilizes a remarkable level of molecular mimicry of the talin–vinculin interaction to activate vinculin. Mimicry of vinculin's interactions may therefore be a general mechanism applied by pathogens to infect the host cell.

scholarly journals Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir

2021 ◽  
Vol 102 (7) ◽  
Author(s):  
Marie O. Pohl ◽  
Idoia Busnadiego ◽  
Francesco Marrafino ◽  
Lars Wiedmer ◽  
Annika Hunziker ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Therapeutic Option ◽  
Cell Entry ◽  
Protease Inhibition ◽  
Antiviral Compounds ◽  
Main Protease ◽  
Anti Cancer ◽  
Synergistic Mechanism ◽  
Host Cell Entry

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC50 value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

scholarly journals Ceftazidime Is a Potential Drug to Inhibit SARS-CoV-2 Infection In Vitro by Blocking Spike Protein-ACE2 Interaction

2020 ◽  
Author(s):  
ChangDong Lin ◽  
Yue Li ◽  
MengYa Yuan ◽  
MengWen Huang ◽  
Cui Liu ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput Screening ◽  
Inhibitory Concentration ◽  
Cell Entry ◽  
Host Cells ◽  
First Line ◽  
Angiotensin Converting Enzyme 2 ◽  
Approved Drugs ◽  
Fda Approved Drugs

SUMMARYCoronavirus Disease 2019 (COVID-19) spreads globally as a sever pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Cell entry of SARS-CoV-2 mainly depends on binding of the viral spike (S) proteins to angiotensin converting enzyme 2 (ACE2) on host cells. Therefore, repurposing of known drugs to inhibit S protein-ACE2 interaction could be a quick way to develop effective therapy for COVID-19. Using a high-throughput screening system to investigate the interaction between spike receptor binding domain (S-RBD) and ACE2 extracellular domain, we screened 3581 FDA-approved drugs and natural small molecules and identified ceftazidime as a potent compound to inhibit S-RBD–ACE2 interaction by binding to S-RBD. In addition to significantly inhibit S-RBD binding to HPAEpiC cells, ceftazidime efficiently prevented SARS-CoV-2 pseudovirus to infect ACE2-expressing 293T cells. The inhibitory concentration (IC50) was 113.2 μM, which is far below the blood concentration (over 300 μM) of ceftazidime in patients when clinically treated with recommended dose. Notably, ceftazidime is a drug clinically used for the treatment of pneumonia with minimal side effects compared with other antiviral drugs. Thus, ceftazidime has both anti-bacterial and anti-SARS-CoV-2 effects, which should be the first-line antibiotics used for the clinical treatment of COVID-19.

scholarly journals Microbe-Independent Entry of Oomycete RxLR Effectors and Fungal RxLR-Like Effectors Into Plant and Animal Cells Is Specific and Reproducible

2013 ◽  
Vol 26 (6) ◽  
pp. 611-616 ◽  
Author(s):  
Brett M. Tyler ◽  
Shiv D. Kale ◽  
Qunqing Wang ◽  
Kai Tao ◽  
Helen R. Clark ◽  
...  
Keyword(s):  
Effector Proteins ◽  
Cell Entry ◽  
Host Cells ◽  
Specific Cell ◽  
Animal Cells ◽  
Major Question ◽  
Root Cells ◽  
Rxlr Effectors ◽  
Nonhost Plant ◽  
Host Cell Entry

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

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