Intracellular Mycobacterium leprae Utilizes Host Glucose as a Carbon Source in Schwann Cells

ABSTRACT New approaches are needed to control leprosy, but understanding of the biology of the causative agent Mycobacterium leprae remains rudimentary, principally because the pathogen cannot be grown in axenic culture. Here, we applied 13C isotopomer analysis to measure carbon metabolism of M. leprae in its primary host cell, the Schwann cell. We compared the results of this analysis with those of a related pathogen, Mycobacterium tuberculosis, growing in its primary host cell, the macrophage. Using 13C isotopomer analysis with glucose as the tracer, we show that whereas M. tuberculosis imports most of its amino acids directly from the host macrophage, M. leprae utilizes host glucose pools as the carbon source to biosynthesize the majority of its amino acids. Our analysis highlights the anaplerotic enzyme phosphoenolpyruvate carboxylase required for this intracellular diet of M. leprae, identifying this enzyme as a potential antileprosy drug target. IMPORTANCE Leprosy remains a major problem in the world today, particularly affecting the poorest and most disadvantaged sections of society in the least developed countries of the world. The long-term aim of research is to develop new treatments and vaccines, and these aims are currently hampered by our inability to grow the pathogen in axenic culture. In this study, we probed the metabolism of M. leprae while it is surviving and replicating inside its primary host cell, the Schwann cell, and compared it to a related pathogen, M. tuberculosis, replicating in macrophages. Our analysis revealed that unlike M. tuberculosis, M. leprae utilized host glucose as a carbon source and that it biosynthesized its own amino acids, rather than importing them from its host cell. We demonstrated that the enzyme phosphoenolpyruvate carboxylase plays a crucial role in glucose catabolism in M. leprae. Our findings provide the first metabolic signature of M. leprae in the host Schwann cell and identify novel avenues for the development of antileprosy drugs.

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Reprogramming diminishes retention of Mycobacterium leprae in Schwann cells and elevates bacterial transfer property to fibroblasts

F1000Research ◽

10.12688/f1000research.2-198.v1 ◽

2013 ◽

Vol 2 ◽

pp. 198 ◽

Cited By ~ 6

Author(s):

Toshihiro Masaki ◽

Aidan McGlinchey ◽

Simon R. Tomlinson ◽

Jinrong Qu ◽

Anura Rambukkana

Keyword(s):

Schwann Cell ◽

Schwann Cells ◽

Cell Fate ◽

Cell Lineage ◽

Mycobacterium Leprae ◽

Differentiation Markers ◽

Primary Host ◽

Transfer Property ◽

Retention Capacity ◽

Transfer Properties

Background: Bacterial pathogens can manipulate or subvert host tissue cells to their advantage at different stages during infection, from initial colonization in primary host niches to dissemination. Recently, we have shown that Mycobacterium leprae (ML), the causative agent of human leprosy, reprogrammed its preferred host niche de-differentiated adult Schwann cells to progenitor/stem cell-like cells (pSLC) which appear to facilitate bacterial spread. Here, we studied how this cell fate change influences bacterial retention and transfer properties of Schwann cells before and after reprogramming.Results: Using primary fibroblasts as bacterial recipient cells, we showed that non-reprogrammed Schwann cells, which preserve all Schwann cell lineage and differentiation markers, possess high bacterial retention capacity when co-cultured with skin fibroblasts; Schwann cells failed to transfer bacteria to fibroblasts at higher numbers even after co-culture for 5 days. In contrast, pSLCs, which are derived from the same Schwann cells but have lost Schwann cell lineage markers due to reprogramming, efficiently transferred bacteria to fibroblasts within 24 hours.Conclusions: ML-induced reprogramming converts lineage-committed Schwann cells with high bacterial retention capacity to a cell type with pSLC stage with effective bacterial transfer properties. We propose that such changes in cellular properties may be associated with the initial intracellular colonization, which requires long-term bacterial retention within Schwann cells, in order to spread the infection to other tissues, which entails efficient bacterial transfer capacity to cells like fibroblasts which are abundant in many tissues, thereby potentially maximizing bacterial dissemination. These data also suggest how pathogens could take advantage of multiple facets of host cell reprogramming according to their needs during infection.

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Identification and Characterization of Clostridium perfringens Beta Toxin Variants with Differing Trypsin Sensitivity andIn VitroCytotoxicity Activity

Infection and Immunity ◽

10.1128/iai.02864-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1477-1486 ◽

Cited By ~ 3

Author(s):

James R. Theoret ◽

Francisco A. Uzal ◽

Bruce A. McClane

Keyword(s):

Amino Acid ◽

Host Cell ◽

Clostridium Perfringens ◽

Cleavage Site ◽

Cell Binding ◽

Primary Host ◽

Sequence Comparisons ◽

Content Type ◽

Significant Difference ◽

Beta Toxin

By producing toxins,Clostridium perfringenscauses devastating diseases of both humans and animals.C. perfringensbeta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin.

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Rapid Nutritional Remodeling of the Host Cell upon Attachment of Legionella pneumophila

Infection and Immunity ◽

10.1128/iai.01079-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 72-82 ◽

Cited By ~ 15

Author(s):

William M. Bruckert ◽

Christopher T. Price ◽

Yousef Abu Kwaik

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Host Cell ◽

Legionella Pneumophila ◽

Human Monocyte ◽

Bacterial Attachment ◽

Content Type ◽

Cytosolic Side ◽

Novel Strategy ◽

Intracellular Proliferation

ABSTRACTUpon entry ofLegionella pneumophilainto amoebas and macrophages, host-mediated farnesylation of the AnkB effector enables its anchoring to theLegionella-containing vacuole (LCV) membrane. On the LCV, AnkB triggers docking of K48-linked polyubiquitinated proteins that are degraded by the host proteasomes to elevate cellular levels of amino acids needed for intracellular proliferation. Interference with AnkB function triggersL. pneumophilato exhibit a starvation response and differentiate into the nonreplicative phase in response to the basal levels of cellular amino acids that are not sufficient to power intracellular proliferation ofL. pneumophila. Therefore, we have determined whether the biological function of AnkB is temporally and spatially triggered upon bacterial attachment to the host cell to circumvent a counterproductive bacterial differentiation into the nonreplicative phase upon bacterial entry. Here, we show that upon attachment ofL. pneumophilato human monocyte-derived macrophages (hMDMs), the host farnesylation and ubiquitination machineries are recruited by the Dot/Icm system to the plasma membrane exclusively beneath sites of bacterial attachment. Transcription and injection ofankBis triggered by attached extracellular bacteria followed by rapid farnesylation and anchoring of AnkB to the cytosolic side of the plasma membrane beneath bacterial attachment, where K48-linked polyubiquitinated proteins are assembled and degraded by the proteasomes, leading to a rapid rise in the cellular levels of amino acids. Our data represent a novel strategy by an intracellular pathogen that triggers rapid nutritional remodeling of the host cell upon attachment to the plasma membrane, and as a result, a gratuitous surplus of cellular amino acids is generated to support proliferation of the incoming pathogen.

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The Fungal Pathogen Candida albicans Autoinduces Hyphal Morphogenesis by Raising Extracellular pH

mBio ◽

10.1128/mbio.00055-11 ◽

2011 ◽

Vol 2 (3) ◽

Cited By ~ 169

Author(s):

Slavena Vylkova ◽

Aaron J. Carman ◽

Heather A. Danhof ◽

John R. Collette ◽

Huaijin Zhou ◽

...

Keyword(s):

Amino Acids ◽

Candida Albicans ◽

Amino Acid ◽

Carbon Source ◽

Fungal Pathogen ◽

Host Cells ◽

Acid Uptake ◽

Content Type ◽

Virulence Trait ◽

Ph Adaptation

ABSTRACTpH homeostasis is critical for all organisms; in the fungal pathogenCandida albicans, pH adaptation is critical for virulence in distinct host niches. We demonstrate that beyond adaptation,C. albicansactively neutralizes the environment from either acidic or alkaline pHs. Under acidic conditions, this species can raise the pH from 4 to >7 in less than 12 h, resulting in autoinduction of the yeast-hyphal transition, a critical virulence trait. Extracellular alkalinization has been reported to occur in several fungal species, but under the specific conditions that we describe, the phenomenon is more rapid than previously observed. Alkalinization is linked to carbon deprivation, as it occurs in glucose-poor media and requires exogenous amino acids. These conditions are similar to those predicted to exist inside phagocytic cells, and we find a strong correlation between the use of amino acids as a cellular carbon source and the degree of alkalinization. Genetic and genomic approaches indicate an emphasis on amino acid uptake and catabolism in alkalinizing cells. Mutations in four genes,STP2, a transcription factor regulating amino acid permeases,ACH1(acetyl-coenzyme A [acetyl-CoA] hydrolase),DUR1,2(urea amidolyase), andATO5, a putative ammonia transporter, abolish or delay neutralization. The pH changes are the result of the extrusion of ammonia, as observed in other fungi. We propose that nutrient-deprivedC. albicanscells catabolize amino acids as a carbon source, excreting the amino nitrogen as ammonia to raise environmental pH and stimulate morphogenesis, thus directly contributing to pathogenesis.IMPORTANCECandida albicansis the most important fungal pathogen of humans, causing disease at multiple body sites. The ability to switch between multiple morphologies, including a rounded yeast cell and an elongated hyphal cell, is a key virulence trait in this species, as this reversible switch is thought to promote dissemination and tissue invasion in the host. We report here thatC. albicanscan actively alter the pH of its environment and induce its switch to the hyphal form. The change in pH is caused by the release of ammonia from the cells produced during the breakdown of amino acids. This phenomenon is unprecedented in a human pathogen and may substantially impact host physiology by linking morphogenesis, pH adaptation, carbon metabolism, and interactions with host cells, all of which are critical for the ability ofC. albicansto cause disease.

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Role of Host Cell-Derived Amino Acids in Nutrition of Intracellular Salmonella enterica

Infection and Immunity ◽

10.1128/iai.00624-15 ◽

2015 ◽

Vol 83 (12) ◽

pp. 4466-4475 ◽

Cited By ~ 27

Author(s):

Jasmin Popp ◽

Janina Noster ◽

Kim Busch ◽

Alexander Kehl ◽

Gero zur Hellen ◽

...

Keyword(s):

Amino Acids ◽

Host Cell ◽

Cell Line ◽

Hela Cells ◽

Salmonella Enterica ◽

Epithelial Cell Line ◽

Intracellular Replication ◽

Content Type ◽

Raw264.7 Macrophages ◽

Cell Line Hela

The facultative intracellular pathogenSalmonella entericaresides in a specific membrane-bound compartment termed theSalmonella-containing vacuole (SCV). Despite being segregated from access to metabolites in the host cell cytosol,Salmonellais able to efficiently proliferate within the SCV. We set out to unravel the nutritional supply ofSalmonellain the SCV with focus on amino acids. We studied the availability of amino acids by the generation of auxotrophic strains for alanine, asparagine, aspartate, glutamine, and proline in a macrophage cell line (RAW264.7) and an epithelial cell line (HeLa) and examined access to extracellular nutrients for nutrition. Auxotrophies for alanine, asparagine, or proline attenuated intracellular replication in HeLa cells, while aspartate, asparagine, or proline auxotrophies attenuated intracellular replication in RAW264.7 macrophages. The different patterns of intracellular attenuation of alanine- or aspartate-auxotrophic strains support distinct nutritional conditions in HeLa cells and RAW264.7 macrophages. Supplementation of medium with individual amino acids restored the intracellular replication of mutant strains auxotrophic for asparagine, proline, or glutamine. Similarly, a mutant strain deficient in succinate dehydrogenase was complemented by the extracellular addition of succinate. Complementation of the intracellular replication of auxotrophicSalmonellaby external amino acids was possible if bacteria were proficient in the induction ofSalmonella-induced filaments (SIFs) but failed in a SIF-deficient background. We propose that the ability of intracellularSalmonellato redirect host cell vesicular transport provides access of amino acids to auxotrophic strains and, more generally, is essential to continuously supply bacteria within the SCV with nutrients.

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Staphylococcus aureus Tet38 Efflux Pump Structural Modeling and Roles of Essential Residues in Drug Efflux and Host Cell Internalization

Infection and Immunity ◽

10.1128/iai.00811-20 ◽

2021 ◽

Vol 89 (5) ◽

Author(s):

Q. C. Truong-Bolduc ◽

Y. Wang ◽

D. C. Hooper

Keyword(s):

Amino Acids ◽

Staphylococcus Aureus ◽

Amino Acid ◽

Host Cell ◽

Tetracycline Resistance ◽

Drug Efflux ◽

Amino Acid Deletion ◽

Content Type ◽

Cell Internalization ◽

External Loop

ABSTRACT The Staphylococcus aureus Tet38 membrane protein has distinct functions, including drug efflux and host cell attachment and internalization mediated by interaction with host cell CD36. Using structural modeling and site-directed mutagenesis, we identified key amino acids involved in different functions. Tet38, a member of the major facilitator superfamily, is predicted to have 14 transmembrane segments (TMS), 6 cytoplasmic loops, and 7 external loops. Cysteine substitutions of arginine 106 situated at the junction of TMS 4 and external loop L2, and glycine 151 of motif C on TMS 5, resulted in complete or near-complete (8- to 16-fold) reductions in Tet38-mediated resistance to tetracycline, with minimal to no effect on A549 host cell internalization. In contrast, a three-amino-acid deletion, F411P412G413, in external loop L7 situated between TMS 13 and 14 led to a decrease of 4-fold in S. aureus internalization by A549 cells and a partial effect on tetracycline resistance (4-fold reduction). A three-amino-acid deletion, D38D39L40, in external loop L1 situated between TMS-1 and TMS-2, had a similar partial effect on tetracycline resistance but did not affect cell internalization. Using an Ni column retention assay, we showed further that the L7, but not the L1, deletion impaired binding to CD36. Thus, the L7 domain of Tet38 is key for interaction with CD36 and host cell internalization, and amino acids R106 and G151 (TMSs 4 and 5) are particularly important for tetracycline resistance without affecting internalization.

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Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

Applied and Environmental Microbiology ◽

10.1128/aem.00261-16 ◽

2016 ◽

Vol 82 (10) ◽

pp. 3042-3051 ◽

Cited By ~ 28

Author(s):

Kelsi M. Sandoz ◽

Paul A. Beare ◽

Diane C. Cockrell ◽

Robert A. Heinzen

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Transformation ◽

Host Cell ◽

Coxiella Burnetii ◽

Genetic Manipulation ◽

Defined Medium ◽

Gradual Transition ◽

Content Type ◽

Selection Of

ABSTRACTHost cell-free (axenic) culture ofCoxiella burnetiiin acidified citrate cysteine medium-2 (ACCM-2) has provided important opportunities for investigating the biology of this naturally obligate intracellular pathogen and enabled the development of tools for genetic manipulation. However, ACCM-2 has complex nutrient sources that preclude a detailed study of nutritional factors required forC. burnetiigrowth. Metabolic reconstruction ofC. burnetiipredicts that the bacterium cannot synthesize all amino acids and therefore must sequester some from the host. To examineC. burnetiiamino acid auxotrophies, we developed a nutritionally defined medium with known amino acid concentrations, termed ACCM-D. Compared to ACCM-2, ACCM-D supported longer logarithmic growth, a more gradual transition to stationary phase, and approximately 5- to 10-fold greater overall replication. Small-cell-variant morphological forms generated in ACCM-D also showed increased viability relative to that generated in ACCM-2. Lack of growth in amino acid-deficient formulations of ACCM-D revealedC. burnetiiauxotrophy for 11 amino acids, including arginine. Heterologous expression ofLegionella pneumophilaargGHinC. burnetiipermitted growth in ACCM-D missing arginine and supplemented with citrulline, thereby providing a nonantibiotic means of selection ofC. burnetiigenetic transformants. Consistent with bioinformatic predictions, the elimination of glucose did not impairC. burnetiireplication. Together, these results highlight the advantages of a nutritionally defined medium in investigations ofC. burnetiimetabolism and the development of genetic tools.IMPORTANCEHost cell-free growth and genetic manipulation ofCoxiella burnetiihave revolutionized research of this intracellular bacterial pathogen. Nonetheless, undefined components of growth medium have made studies ofC. burnetiiphysiology difficult and have precluded the development of selectable markers for genetic transformation based on nutritional deficiencies. Here, we describe a medium, containing only amino acids as the sole source of carbon and energy, which supports robust growth and improved viability ofC. burnetii. Growth studies confirmed thatC. burnetiicannot replicate in medium lacking arginine. However, genetic transformation of the bacterium with constructs containing the last two genes in theL. pneumophilaarginine biosynthesis pathway (argGH) allowed growth on defined medium missing arginine but supplemented with the arginine precursor citrulline. Our results advance the field by facilitating studies ofC. burnetiimetabolism and allowing non-antibiotic-based selection ofC. burnetiigenetic transformants, an important achievement considering that selectable makers based on antibiotic resistance are limited.

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Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽

10.1128/mbio.00351-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Rajdeep Banerjee ◽

Erin Weisenhorn ◽

Kevin J. Schwartz ◽

Kevin S. Myers ◽

Jeremy D. Glasner ◽

...

Keyword(s):

Amino Acids ◽

Urinary Tract ◽

Amino Acid ◽

Regulatory Network ◽

Iron Homeostasis ◽

Iron Limitation ◽

Content Type ◽

E Coli ◽

General Stress ◽

K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

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How Phillips saw the light

Strategic Direction ◽

10.1108/sd-05-2020-0103 ◽

2020 ◽

Vol 36 (8) ◽

pp. 29-31

Keyword(s):

Design Methodology ◽

Reading Time ◽

Daily Basis ◽

Business World ◽

Content Type ◽

Freak Show ◽

The World ◽

Pertinent Information ◽

The One ◽

Practical Implications

Purpose Reviews the latest management developments across the globe and pinpoints practical implications from cutting-edge research and case studies. Design/methodology/approach This briefing is prepared by an independent writer who adds their own impartial comments and places the articles in context. Findings The problem with developing a reputation of being something of an oracle in the business world is that all of a sudden, everyone expects you to pull off the trick of interpreting the future on a daily basis. Like a freak show circus act or one-hit wonder pop singer, people expect you to perform when they see you, and they expect you to perform the thing that made you famous, even if it is the one thing in the world you don’t want to do. And when you fail to deliver on these heightened expectations, you are dismissed as a one trick pony, however good that trick is in the first place. Originality/value The briefing saves busy executives and researchers hours of reading time by selecting only the very best, most pertinent information and presenting it in a condensed and easy-to-digest format.

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Can housing rights be applied to modern housing systems?

International Journal of Law in the Built Environment ◽

10.1108/17561451011058762 ◽

2010 ◽

Vol 2 (2) ◽

pp. 103-117 ◽

Cited By ~ 1

Author(s):

Padraic Kenna

Keyword(s):

International Law ◽

Case Law ◽

Epistemic Communities ◽

Policy Makers ◽

Content Type ◽

Housing Systems ◽

Effective Implementation ◽

Housing Rights ◽

The World ◽

Effective Translation

PurposeThe purpose of this paper is to outline and examine the growing corpus of housing rights and assess their relevance and applicability to complex contemporary housing systems across the world.Design/methodology/approachThe paper sets out the principal instruments and commentaries on housing rights developed by the United Nations, regional and other bodies. It assesses their relevance in the context of contemporary analysis of housing systems, organized and directed by networks of legal and other professionals within particular domains.FindingsHousing rights instruments are accepted by all States across the world at the level of international law, national constitutions and laws. The findings suggest that there are significant gaps in the international law conception and framework of housing rights, and indeed, human rights generally, which create major obstacles for the effective implementation of these rights. There is a preoccupation with one element of housing systems, that of subsidized or social housing. However, effective housing rights implementation requires application at meso‐, micro‐ and macro‐levels of modern, dynamic housing systems as a whole. Epistemic communities of professionals develop and shape housing law and policy within these domains. The housing rights paradigm must be further fashioned for effective translation into contemporary housing systems.Research limitations/implicationsThe development of housing rights precedents, both within international and national law, is leading to a wide and diffuse corpus of legislation and case law. More research is needed on specific examples of effective coupling between housing rights and elements of housing systems.Originality/valueThis paper offers housing policy makers and lawyers an avenue into the extensive jurisprudence and writings on housing rights, which will inevitably become part of the lexicon of housing law across the world. It also highlights the limitations of housing rights implementation, but offers some new perspectives on more effective application of these rights.

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