Orientia tsutsugamushi modulates cellular levels of NF-κB inhibitor p105

Background Scrub typhus is a neglected tropical disease that threatens more than one billion people. If antibiotic therapy is delayed, often due to mis- or late diagnosis, the case fatality rate can increase considerably. Scrub typhus is caused by the obligate intracellular bacterium, Orientia tsutsugamushi, which invades phagocytes and endothelial cells in vivo and diverse tissue culture cell types in vitro. The ability of O. tsutsugamushi to replicate in the cytoplasm indicates that it has evolved to counter eukaryotic host cell immune defense mechanisms. The transcription factor, NF-κB, is a tightly regulated initiator of proinflammatory and antimicrobial responses. Typically, the inhibitory proteins p105 and IκBα sequester the NF-κB p50:p65 heterodimer in the cytoplasm. Canonical activation of NF-κB via TNFα involves IKKβ-mediated serine phosphorylation of IκBα and p105, which leads to their degradation and enables NF-κB nuclear translocation. A portion of p105 is also processed into p50. O. tsutsugamushi impairs NF-κB translocation into the nucleus, but how it does so is incompletely defined. Principal findings Western blot, densitometry, and quantitative RT-PCR analyses of O. tsutsugamushi infected host cells were used to determine if the pathogen’s ability to inhibit NF-κB is linked to modulation of p105. Results demonstrate that p105 levels are elevated several-fold in O. tsutsugamushi infected HeLa and RF/6A cells with only a nominal increase in p50. The O. tsutsugamushi-stimulated increase in p105 is bacterial dose- and protein synthesis-dependent, but does not occur at the level of host cell transcription. While TNFα-induced phosphorylation of p105 serine 932 proceeds unhindered in infected cells, p105 levels remain elevated and NF-κB p65 is retained in the cytoplasm. Conclusions O. tsutsugamushi specifically stabilizes p105 to inhibit the canonical NF-κB pathway, which advances understanding of how it counters host immunity to establish infection.

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Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs

Journal of Bacteriology ◽

10.1128/jb.00276-15 ◽

2015 ◽

Vol 197 (19) ◽

pp. 3097-3109 ◽

Cited By ~ 13

Author(s):

Andrea R. Beyer ◽

Lauren VieBrock ◽

Kyle G. Rodino ◽

Daniel P. Miller ◽

Brittney K. Tegels ◽

...

Keyword(s):

Host Cell ◽

Ubiquitin Ligase ◽

Scrub Typhus ◽

Ankyrin Repeat ◽

Intracellular Bacterium ◽

Host Cells ◽

Orientia Tsutsugamushi ◽

Ligand Interaction ◽

Content Type ◽

Cell Functions

ABSTRACTA rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions.Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here,in silicoand manual sequence analyses revealed that a large proportion ofO. tsutsugamushistrain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAG-Ank4_01 or FLAG-Ank9, a glutathioneS-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitatedO. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. AligningO. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate thatO. tsutsugamushistrain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that anO. tsutsugamushiAnk does so during infection, and advance overall understanding of microbial F-box proteins.IMPORTANCEAnkyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets.Orientia tsutsugamushi, which causes a debilitating infection called scrub typhus in one of the most densely populated regions of the world, encodes one of the largest Ank armamentariums of any sequenced bacterium. This study demonstrates thatO. tsutsugamushistrain Ikeda Anks also bear F-box motifs that interact with host cell polyubiquitination machinery. By proving that anOrientia-derived Ank interacts with SKP1 in infected cells, this evidences the first bona fideOrientiaeffector and the first example of an endogenous F-box-containing Ank–mammalian-host ligand interaction for any intracellular bacterium. Also, importantly, this work identifies key residues that are essential for microbial F-box function.

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Revisiting Ehrlichia ruminantium Replication Cycle Using Proteomics: The Host and the Bacterium Perspectives

Microorganisms ◽

10.3390/microorganisms9061144 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1144

Author(s):

Isabel Marcelino ◽

Philippe Holzmuller ◽

Ana Coelho ◽

Gabriel Mazzucchelli ◽

Bernard Fernandez ◽

...

Keyword(s):

Endothelial Cells ◽

Host Cell ◽

Cell Metabolism ◽

Replication Cycle ◽

Host Cells ◽

Ehrlichia Ruminantium ◽

Host Interaction ◽

Infected Cells ◽

Related Proteins

The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.

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Cellular and immunological basis of the host-parasite relationship during infection withNeospora caninum

Parasitology ◽

10.1017/s0031182006000485 ◽

2006 ◽

Vol 133 (3) ◽

pp. 261-278 ◽

Cited By ~ 76

Author(s):

A. HEMPHILL ◽

N. VONLAUFEN ◽

A. NAGULESWARAN

Keyword(s):

Host Cell ◽

Cell Biology ◽

Neospora Caninum ◽

Host Cells ◽

Term Survival ◽

Prevention Of Infection ◽

Parasite Relationship ◽

Potential Applications

Neospora caninumis an apicomplexan parasite that is closely related toToxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast toT. gondii, N. caninumrepresents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused byN. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cellsin vitroandin vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite orvice versa). Thirdly, by analogy withT. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features ofN. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

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1,3,4-Thiadiazoles Effectively Inhibit Proliferation of Toxoplasma gondii

Cells ◽

10.3390/cells10051053 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1053

Author(s):

Lidia Węglińska ◽

Adrian Bekier ◽

Katarzyna Dzitko ◽

Barbara Pacholczyk-Sienicka ◽

Łukasz Albrecht ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cell Invasion ◽

Direct Action ◽

Human Fibroblasts ◽

Imidazole Ring ◽

Host Cells ◽

In Vitro Assays ◽

Host Cell Invasion

Congenital and acquired toxoplasmosis caused by the food- and water-born parasite Toxoplasma gondii (T. gondii) is one of the most prevalent zoonotic infection of global importance. T. gondii is an obligate intracellular parasite with limited capacity for extracellular survival, thus a successful, efficient and robust host cell invasion process is crucial for its survival, proliferation and transmission. In this study, we screened a series of novel 1,3,4-thiadiazole-2-halophenylamines functionalized at the C5 position with the imidazole ring (1b–12b) for their effects on T. gondii host cell invasion and proliferation. To achieve this goal, these compounds were initially subjected to in vitro assays to assess their cytotoxicity on human fibroblasts and then antiparasitic efficacy. Results showed that all of them compare favorably to control drugs sulfadiazine and trimethoprim in terms of T. gondii growth inhibition (IC50) and selectivity toward the parasite, expressed as selectivity index (SI). Subsequently, the most potent of them with meta-fluoro 2b, meta-chloro 5b, meta-bromo 8b, meta-iodo 11b and para-iodo 12b substitution were tested for their efficacy in inhibition of tachyzoites invasion and subsequent proliferation by direct action on established intracellular infection. All the compounds significantly inhibited the parasite invasion and intracellular proliferation via direct action on both tachyzoites and parasitophorous vacuoles formation. The most effective was para-iodo derivative 12b that caused reduction in the percentage of infected host cells by 44% and number of tachyzoites per vacuole by 93% compared to non-treated host cells. Collectively, these studies indicate that 1,3,4-thiadiazoles 1b–12b, especially 12b with IC50 of 4.70 µg/mL and SI of 20.89, could be considered as early hit compounds for future design and synthesis of anti-Toxoplasma agents that effectively and selectively block the invasion and subsequent proliferation of T. gondii into host cells.

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Abstract 1772: Cardiomyocyte Damage-Released S100A1 Protein Evokes A Proinflammatory Phenotype In Cardiac Fibroblasts

Circulation ◽

10.1161/circ.118.suppl_18.s_386-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

David Rohde ◽

Melanie Boerries ◽

Herzog Nicole ◽

Gang Qiu ◽

Philipp Ehlermann ◽

...

Keyword(s):

Western Blotting ◽

Immune Cells ◽

Nuclear Translocation ◽

Cardiac Fibroblasts ◽

Innate Immune ◽

Host Cells ◽

Boyden Chamber ◽

Innate Immune Cells ◽

Danger Signal

Background: S100A1, a cardiomyocyte specific inotropic calcium sensor protein, is released from infarcted human myocardium in the extracellular environment and circulation, reaching peak serum levels (1–2 μM) 8–9 hours after clinical onset. As growing evidence indicates that S100 proteins can act as pre-existing danger signals triggering the innate immune system into action upon release from injured host cells, we hypothesized that damage-released S100A1 can act as a cardiac danger signal alerting innate immune cells. Methods and Results: Here we report for the first time that necrotic cardiomyocytes release S100A1 protein in vitro, which is exclusively internalized by cardiac fibroblasts (CFs) in a clathrin- and caveolin-independent manner as shown by IF. Internalized S100A1 specifically activated MAPKs/SAPKs (p38, ERK1/2 and JNK) resulting in nuclear translocation of p65 (NF-kB) as assessed by Western blotting, EMSA and IF. In turn, S100A1 triggered an inflammatory gene program in CFs including enhanced expression of adhesion molecules, integrins, chemokines and cytokines including I-CAM, V-CAM, CD11b/18, IL1-alpha, MCP-1, TNF-alpha, SDF-1 among others as obtained by RT-PCR, Western blotting and ELISA. This resulted in enhanced chemoattraction and adhesion of monocytotic and stem cells to S100A1-activated CF as shown by Boyden-chamber and adhesion assays. In line with their proinflammatory transition, S100A1-activated CFs exhibited decreased collagen-1/-3 expression and de-novo collagen production, enhanced collagenolytic MMP-9 abundance and activity and increased levels of the antiangiogenic matricellular factor thrombospondin-2 reflecting extracellular matrix net degradation. Importantly, the immun-modulatory and antifibrotic actions of S100A1 protein in vitro were restricted to CFs, RAGE independent and occurred at concentrations (0.1–1 μM) that were found in patients after AMI. Conclusion: Our in vitro results indicate that S100A1 has the properties of a pre-exisiting endogenous cardiomyocyte danger signal transforming cardiac fibroblasts into immunmodulatory cells that might recruit innate immune cells to the site of cardiac injury and link cardiomyocyte damage to post-MI inflammation.

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Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

Journal of Experimental Medicine ◽

10.1084/jem.20151248 ◽

2016 ◽

Vol 213 (5) ◽

pp. 809-825 ◽

Cited By ~ 75

Author(s):

Yancheng Liu ◽

Shumin Tan ◽

Lu Huang ◽

Robert B. Abramovitch ◽

Kyle H. Rohde ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Host Cell ◽

Stress Responses ◽

Drug Action ◽

Host Cells ◽

Transcriptional Analysis ◽

Drug Pressure ◽

Cytokine Activation ◽

The Impact

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs.

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Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein

mBio ◽

10.1128/mbio.01712-17 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 16

Author(s):

Javeed Ahmad ◽

Aisha Farhana ◽

Rita Pancsa ◽

Simran Kaur Arora ◽

Alagiri Srinivasan ◽

...

Keyword(s):

Dendritic Cells ◽

Mycobacterium Tuberculosis ◽

Host Cell ◽

Terminal Segment ◽

Host Protein ◽

Host Cells ◽

Productive Infection ◽

Disordered Proteins ◽

Intrinsically Disordered

ABSTRACT Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion—an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis. IMPORTANCE To hijack the human host cell machinery to enable survival inside macrophages, the pathogen Mycobacterium tuberculosis requires a repertoire of proteins that can mimic host protein function and modulate host cell machinery. Here, we have shown how a single protein can play multiple functions and hijack the host cell for the benefit of the pathogen. Full-length membrane-anchored PPE37 protein is cleaved into N- and C-terminal domains under iron-depleted conditions. The N-terminal domain facilitates the propathogen semimature tolerogenic state of dendritic cells, whereas the C-terminal segment is localized into host cell nucleus and induces apoptosis. The immune implications of these in vitro observations were assessed and validated in mice and also human TB patients. This study presents novel mechanistic insight adopted by M. tuberculosis to survive inside host cells.

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Complementation of the fadA Mutation in Fusobacterium nucleatum Demonstrates that the Surface-Exposed Adhesin Promotes Cellular Invasion and Placental Colonization

Infection and Immunity ◽

10.1128/iai.00209-09 ◽

2009 ◽

Vol 77 (7) ◽

pp. 3075-3079 ◽

Cited By ~ 48

Author(s):

Akihiko Ikegami ◽

Peter Chung ◽

Yiping W. Han

Keyword(s):

Host Cell ◽

Cell Attachment ◽

Adverse Pregnancy Outcome ◽

Fusobacterium Nucleatum ◽

Host Cells ◽

Mouse Placenta ◽

Host Cell Attachment ◽

Adverse Pregnancy

ABSTRACT Fusobacterium nucleatum is a gram-negative oral anaerobe implicated in periodontal disease and adverse pregnancy outcome. The organism colonizes the mouse placenta, causing localized infection and inflammation. The mechanism of placental colonization has not been elucidated. Previous studies identified a novel adhesin from F. nucleatum, FadA, as being involved in the attachment and invasion of host cells. The fadA deletion mutant F. nucleatum 12230 US1 was defective in host cell attachment and invasion in vitro, but it also exhibited pleiotropic effects with altered cell morphology and growth rate. In this study, a fadA-complementing clone, F. nucleatum 12230 USF81, was constructed. The expression of FadA on USF81 was confirmed by Western blotting and immunofluorescent labeling. USF81 restored host cell attachment and invasion activities. The ability of F. nucleatum 12230, US1, and USF81 to colonize the mouse placenta was examined. US1 was severely defective in placental colonization compared to the wild type and USF81. Thus, FadA plays an important role in F. nucleatum colonization in vivo. These results also represent the first complementation studies for F. nucleatum. FadA may be a therapeutic target for preventing F. nucleatum colonization of the host.

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Effects of Mycoplasmas on the Host Cell Signaling Pathways

Pathogens ◽

10.3390/pathogens9040308 ◽

2020 ◽

Vol 9 (4) ◽

pp. 308

Author(s):

Sergei N. Borchsenius ◽

Innokentii E. Vishnyakov ◽

Olga A. Chernova ◽

Vladislav M. Chernov ◽

Nikolai A. Barlev

Keyword(s):

Host Cell ◽

Intracellular Signaling ◽

Nuclear Translocation ◽

Inflammatory Responses ◽

Cell Interaction ◽

Host Cells ◽

Nuclear Factor ◽

New Therapeutic Approaches ◽

Anti Inflammatory ◽

Living Organisms

Mycoplasmas are the smallest free-living organisms. Reduced sizes of their genomes put constraints on the ability of these bacteria to live autonomously and make them highly dependent on the nutrients produced by host cells. Importantly, at the organism level, mycoplasmal infections may cause pathological changes to the host, including cancer and severe immunological reactions. At the molecular level, mycoplasmas often activate the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) inflammatory response and concomitantly inhibit the p53-mediated response, which normally triggers the cell cycle and apoptosis. Thus, mycoplasmal infections may be considered as cancer-associated factors. At the same time, mycoplasmas through their membrane lipoproteins (LAMPs) along with lipoprotein derivatives (lipopeptide MALP-2, macrophage-activating lipopeptide-2) are able to modulate anti-inflammatory responses via nuclear translocation and activation of Nrf2 (the nuclear factor-E2-related anti-inflammatory transcription factor 2). Thus, interactions between mycoplasmas and host cells are multifaceted and depend on the cellular context. In this review, we summarize the current information on the role of mycoplasmas in affecting the host’s intracellular signaling mediated by the interactions between transcriptional factors p53, Nrf2, and NF-κB. A better understanding of the mechanisms underlying pathologic processes associated with reprogramming eukaryotic cells that arise during the mycoplasma-host cell interaction should facilitate the development of new therapeutic approaches to treat oncogenic and inflammatory processes.

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Sequence, expression and localization of calmodulin-domain protein kinases inEimeria tenellaandEimeria maxima

Parasitology ◽

10.1017/s0031182000081506 ◽

1996 ◽

Vol 113 (5) ◽

pp. 439-448 ◽

Cited By ~ 21

Author(s):

P. P. J. Dunn ◽

J. M. Bumstead ◽

F. M. Tomley

Keyword(s):

Host Cell ◽

Protein Kinases ◽

Catalytic Domain ◽

Sequence Data ◽

Host Cells ◽

Cdna Clones ◽

Host Cell Membrane ◽

Amino Terminal ◽

Domain Protein

SUMMARYWe have isolated and sequenced cDNA clones fromEimeria tenellaandEimeria maximawhich encode proteins that share homology with a recently described family of calmodulin-domain protein kinases. The primary sequence data show that each of the protein kinases can be divided into 2 main functional domains – an amino-terminal catalytic domain typical of serine/threonine protein kinases and a carboxy-terminal domain homologous to calmodulin, which is capable of binding calcium ions at 4 ‘EF-hand’ motifs. Expression of theE. tenellacalmodulin-domain protein kinase (EtCDPK) increased towards the end of oocyst sporulation, as judged by Northern and Western blotting, and indirect immunofluorescent antibody labelling showed that within a few minutes of adding sporozoites to target host cells inin vitroculture EtCDPK was found to be specifically associated with a filament-like structure that converges at the apical end of the parasite. Once the parasite entered the host cell EtCDPK appeared to be left on the host cell membrane at the point of entry, indicating a brief yet specific role for this molecule in the invasion of host cells byE. tenella.

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