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

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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Intracellular localisation of Mycobacterium tuberculosis affects efficacy of the antibiotic pyrazinamide

Nature Communications ◽

10.1038/s41467-021-24127-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Pierre Santucci ◽

Daniel J. Greenwood ◽

Antony Fearns ◽

Kai Chen ◽

Haibo Jiang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Host Cell ◽

Combination Chemotherapy ◽

In Vitro Activity ◽

In Vivo Efficacy ◽

Human Macrophages ◽

Ion Microscopy ◽

Intracellular Localisation

AbstractTo be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis. However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. Here, we use correlative light, electron, and ion microscopy to investigate how various microenvironments within human macrophages affect the activity of pyrazinamide (PZA), a key antibiotic against TB. We show that PZA accumulates heterogeneously among individual bacteria in multiple host cell environments. Crucially, PZA accumulation and efficacy is maximal within acidified phagosomes. Bedaquiline, another antibiotic commonly used in combined TB therapy, enhances PZA accumulation via a host cell-mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy. Our results may explain the potent in vivo efficacy of PZA, compared to its modest in vitro activity, and its critical contribution to TB combination chemotherapy.

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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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Perturbation of host autophagy by the Irish potato famine pathogen

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314091736 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C826-C826

Author(s):

Abbas Maqbool ◽

Richard Richard ◽

Tolga Bozkurt ◽

Yasin Dagdas ◽

Khaoula Belhai ◽

...

Keyword(s):

Host Cell ◽

Plant Cells ◽

Irish Potato ◽

Effector Proteins ◽

Host Cells ◽

Cell Physiology ◽

Biochemical Basis ◽

The Impact ◽

Potato Famine ◽

Irish Potato Famine

Autophagy is a catabolic process involving degradation of dysfunctional cytoplasmic components to ensure cellular survival under starvation conditions. The process involves formation of double-membrane vesicles called autophagosomes and delivery of the inner constituents to lytic compartments. It can also target invading pathogens, such as intracellular bacteria, for destruction and is thus implicated in innate immune pathways [1]. In response, certain mammalian pathogens deliver effector proteins into host cells that inhibit autophagy and contribute to enabling parasitic infection [2]. Pyhtophthora infestans, the Irish potato famine pathogen, is a causative agent of late blight disease in potato and tomato crops. It delivers a plethora of modular effector proteins into plant cells to promote infection. Once inside the cell, RXLR-type effector proteins engage with host cell proteins, to manipulate host cell physiology for the benefit of the pathogen. As plants lack an adaptive immune system, this provides a robust mechanism for pathogens to circumvent host defense. PexRD54 is an intracellular RXLR-type effector protein produced by P. infestans. PexRD54 interacts with potato homologues of autophagy protein ATG8 in plant cells. We have been investigating the structural and biochemical basis of the PexRD54/ATG8 interaction in vitro. We have purified PexRD54 and ATG8 independently and in complex from E. coli. Using protein/protein interaction studies we have shown that PexRD54 binds ATG8 with sub-micromolar affinity. We have also determined the structure of PexRD54 in the presence of ATG8. This crystal structure provides key insights into how the previously reported WY-fold of oomycete RXLR-type effectors [3] can be organized in multiple repeats. The structural data also provides insights into the interaction between PexRD54 and ATG8, suggesting further experiments to understand the impact of this interaction on host cell physiology and how this benefits the pathogen.

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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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Suppression of influenza A virus replication in human lung epithelial cells by noncytotoxic concentrations bafilomycin A1

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00011.2014 ◽

2015 ◽

Vol 308 (3) ◽

pp. L270-L286 ◽

Cited By ~ 46

Author(s):

Behzad Yeganeh ◽

Saeid Ghavami ◽

Andrea L. Kroeker ◽

Thomas H. Mahood ◽

Gerald L. Stelmack ◽

...

Keyword(s):

Epithelial Cells ◽

Host Cell ◽

Influenza A Virus ◽

Influenza A ◽

Life Cycles ◽

Host Cells ◽

Nuclear Accumulation ◽

Low Concentration ◽

High Concentrations ◽

The Impact

Subcellular trafficking within host cells plays a critical role in viral life cycles, including influenza A virus (IAV). Thus targeting relevant subcellular compartments holds promise for effective intervention to control the impact of influenza infection. Bafilomycin A1(Baf-A1), when used at relative high concentrations (≥10 nM), inhibits vacuolar ATPase (V-ATPase) and reduces endosome acidification and lysosome number, thus inhibiting IAV replication but promoting host cell cytotoxicity. We tested the hypothesis that much lower doses of Baf-A1also have anti-IAV activity, but without toxic effects. Thus we assessed the antiviral activity of Baf-A1at different concentrations (0.1–100 nM) in human alveolar epithelial cells (A549) infected with IAV strain A/PR/8/34 virus (H1N1). Infected and mock-infected cells pre- and cotreated with Baf-A1were harvested 0–24 h postinfection and analyzed by immunoblotting, immunofluorescence, and confocal and electron microscopy. We found that Baf-A1had disparate concentration-dependent effects on subcellular organelles and suppressed affected IAV replication. At concentrations ≥10 nM Baf-A1inhibited acid lysosome formation, which resulted in greatly reduced IAV replication and release. Notably, at a very low concentration of 0.1 nM that is insufficient to reduce lysosome number, Baf-A1retained the capacity to significantly impair IAV nuclear accumulation as well as IAV replication and release. In contrast to the effects of high concentrations of Baf-A1, very low concentrations did not exhibit cytotoxic effects or induce apoptotic cell death, based on morphological and FACS analyses. In conclusion, our results reveal that low-concentration Baf-A1is an effective inhibitor of IAV replication, without impacting host cell viability.

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Intracellular localisation of Mycobacterium tuberculosis affects antibiotic efficacy

10.1101/2020.11.25.398636 ◽

2020 ◽

Author(s):

Pierre Santucci ◽

Daniel J. Greenwood ◽

Antony Fearns ◽

Kai Chen ◽

Haibo Jiang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Host Cell ◽

Combination Chemotherapy ◽

Human Macrophage ◽

Ion Microscopy ◽

Antibiotic Efficacy ◽

Subcellular Level ◽

Intracellular Localisation

AbstractTo be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of Mycobacterium tuberculosis (Mtb). However, how host cell environments affect antibiotic accumulation and efficacy remains elusive. Pyrazinamide (PZA) is a key antibiotic against TB, yet its behaviour is not fully understood. Here, by using correlative light, electron, and ion microscopy to image PZA at the subcellular level, we investigated how human macrophage environments affect PZA activity. We discovered that PZA accumulates heterogeneously between individual bacteria in multiple host cell environments. Crucially, Mtb phagosomal localisation and acidification increase PZA accumulation and efficacy. By imaging two antibiotics commonly used in combined TB therapy, we showed that bedaquiline (BDQ) significantly enhances PZA accumulation by a host cell mediated mechanism. Thus, intracellular localisation and specific microenvironments affect PZA accumulation and efficacy; explaining the potent in vivo efficacy compared to its modest in vitro activity and the critical contribution to TB combination chemotherapy.

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Mycobacterium tuberculosis infection of host cells in space and time

FEMS Microbiology Reviews ◽

10.1093/femsre/fuz006 ◽

2019 ◽

Vol 43 (4) ◽

pp. 341-361 ◽

Cited By ~ 31

Author(s):

Claudio Bussi ◽

Maximiliano G Gutierrez

Keyword(s):

Mycobacterium Tuberculosis ◽

Host Cell ◽

Cell Biology ◽

Current Knowledge ◽

Bacterial Pathogen ◽

Infectious Agent ◽

Host Cells ◽

Cellular Mechanisms ◽

Mycobacterium Tuberculosis Infection ◽

Human Host

ABSTRACTTuberculosis (TB) caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb) remains one of the deadliest infectious diseases with over a billion deaths in the past 200 years (Paulson 2013). TB causes more deaths worldwide than any other single infectious agent, with 10.4 million new cases and close to 1.7 million deaths in 2017. The obstacles that make TB hard to treat and eradicate are intrinsically linked to the intracellular lifestyle of Mtb. Mtb needs to replicate within human cells to disseminate to other individuals and cause disease. However, we still do not completely understand how Mtb manages to survive within eukaryotic cells and why some cells are able to eradicate this lethal pathogen. Here, we summarise the current knowledge of the complex host cell-pathogen interactions in TB and review the cellular mechanisms operating at the interface between Mtb and the human host cell, highlighting the technical and methodological challenges to investigating the cell biology of human host cell-Mtb interactions.

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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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