Pareek M, Hingley-Wilson S, Sridhar S, Lalvani A, Evans J, Smith G, Hawkey P, Innes J, Dedicoat M, Lougheed KE (2012) Ethnicity and mycobacterial lineage as determinants of tuberculosis disease phenotype, Thorax
Background: Emerging evidence suggests that Mycobacterium tuberculosis (Mtb) lineage and host ethnicity can determine tuberculosis (TB) clinical disease patterns but their relative importance and interaction are unknown. Methods: We evaluated prospectively collected TB surveillance and Mtb strain typing data in an ethnically heterogeneous UK population. Lineage assignment was denoted using 15-loci mycobacterial interspersed repetitive units containing variable numbers of tandem repeats (MIRU-VNTR) and MIRU-VNTRplus. Geographical and ethnic associations of the six global Mtb lineages were identified and the influence of lineage and demographic factors on clinical phenotype were assessed using multivariate logistic regression. Results: Data were available for 1070 individuals with active TB which was pulmonary only, extrapulmonary only and concurrent pulmonary-extrapulmonary in 52.1%, 36.9% and 11.0% respectively. The most prevalent lineages were Euro-American (43.7%), East African Indian (30.2%), Indo-Oceanic (13.6%) and East Asian (12.2%) and were geo-ethnically restricted with, for example, Indian subcontinent ethnicity inversely associated with Euro-American lineage (OR 0.23; 95% CI 0.14 to 0.39) and positively associated with the East African-Indian lineage (OR 4.04; 95% CI 2.19 to 7.45). Disease phenotype was most strongly associated with ethnicity (OR for extrathoracic disease 21.14 (95% CI 6.08 to 73.48) for Indian subcontinent and 14.05 (3.97 to 49.65) for Afro-Caribbean), after adjusting for lineage. With East Asian lineage as the reference category, the Euro-American (OR 0.54; 95% CI 0.32 to 0.91) and East-African Indian (OR 0.50; 95% CI 0.29 to 0.86) lineages were negatively associated with extrathoracic disease, compared with pulmonary disease, after adjusting for ethnicity. Conclusions: Ethnicity is a powerful determinant of clinical TB phenotype independently of mycobacterial lineage and the role of ethnicity-associated factors in pathogenesis warrants investigation. Copyright Article author (or their employer) 2012.
Hingley-Wilson SM, Casey R, Connell D, Bremang S, Evans JT, Hawkey PM, Smith GE, Jepson A, Philip S, Kon OM, Lalvani A (2013) Undetected Multidrug-Resistant Tuberculosis Amplified by First-line Therapy in Mixed Infection, EMERGING INFECTIOUS DISEASES 19 (7) pp. 1138-1141
CENTERS DISEASE CONTROL
Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance.
Trauner A, Lougheed KEA, Bennett MH, Hingley-Wilson SM, Williams HD (2012) The dormancy regulator DosR controls ribosome stability in hypoxic mycobacteria, Journal of Biological Chemistry 287 (28) pp. 24053-24063
It is thought that during latent infection, Mycobacterium tuberculosis bacilli are retained within granulomas in a lowoxygen environment. The dormancy survival (Dos) regulon, regulated by the response regulator DosR, appears to be essential for hypoxic survival in M. tuberculosis, but it is not known how the regulon promotes survival. Here we report that mycobacteria, in contrast to enteric bacteria, do not form higher-order structures (e.g. ribosomal dimers) upon entry into stasis. Instead, ribosomes are stabilized in the associated form (70S). Using a strategy incorporating microfluidic, proteomic, and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during hypoxic stasis, we show that the dormancy regulator DosR is required for optimal ribosome stabilization. We present evidence that the majority of this effect is mediated by the DosR-regulated protein MSMEG-3935 (a S30AE domain protein), which is associated with the ribosome under hypoxic conditions. A 3935 mutant phenocopies the dosR mutant during hypoxia, and complementation of dosR with the MSMEG-3935 gene leads to complete recovery of dosR mutant phenotypes during hypoxia. We suggest that this protein is named ribosome-associated factor under hypoxia (RafH) and that it is the major factor responsible for DosR-mediated hypoxic survival in mycobacteria. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
Hingley-Wilson SM (2013) Metagenomic analysis of tuberculosis - Current limitations, New England Journal of Medicine 369 (16)
Montamat-Sicotte DJ, Millington KA, Willcox CR, Hingley-Wilson S, Hackforth S, Innes J, Kon OM, Lammas DA, Minnikin DE, Besra GS, Willcox BE, Lalvani A (2011) A mycolic acid-specific CD1-restricted T cell population contributes to acute and memory immune responses in human tuberculosis infection., J Clin Invest 121 (6) pp. 2493-2503
Current tuberculosis (TB) vaccine strategies are largely aimed at activating conventional T cell responses to mycobacterial protein antigens. However, the lipid-rich cell wall of Mycobacterium tuberculosis (M. tuberculosis) is essential for pathogenicity and provides targets for unconventional T cell recognition. Group 1 CD1-restricted T cells recognize mycobacterial lipids, but their function in human TB is unclear and their ability to establish memory is unknown. Here, we characterized T cells specific for mycolic acid (MA), the predominant mycobacterial cell wall lipid and key virulence factor, in patients with active TB infection. MA-specific T cells were predominant in TB patients at diagnosis, but were absent in uninfected bacillus Calmette-Guérin-vaccinated (BCG-vaccinated) controls. These T cells were CD1b restricted, detectable in blood and disease sites, produced both IFN-³ and IL-2, and exhibited effector and central memory phenotypes. MA-specific responses contracted markedly with declining pathogen burden and, in patients followed longitudinally, exhibited recall expansion upon antigen reencounter in vitro long after successful treatment, indicative of lipid-specific immunological memory. T cell recognition of MA is therefore a significant component of the acute adaptive and memory immune response in TB, suggesting that mycobacterial lipids may be promising targets for improved TB vaccines.
Terry Alli OA, Hingley-Wilson S, Spreadbury Claire L, Terry Alli OA, Ogbolu DO, Hingley-Wilson S (2008) The Mycobacterium tuberculosis homologue of the Mycobacterium avium mig gene is not specifically expressed in the macrophage, African Journal Biomedical Research 11 (2) pp. 173-181
With the completion of genome sequencing of Mycobacterium tuberculosis and upsurge in the incidence of M. tuberculosis infection worldwide partly as a result of HIV pandemic, there is need for rationale approach to vaccine and chemotherapy discoveries for M. tuberculosis. The homologue of mig gene of Mycobacterium avium was searched for in the M. tuberculosis database at The Institute of Genomic Research (TIGR), USA and The Sanger Institute, UK. Homologue of the gene was found and comprehensively analysed. Reverse transcription PCR (RT-PCR) was carried out on the mig (fadD19) gene homologue and echA19 gene. The result of the RT-PCR showed that the mig gene was at least 2-fold upregulated during intracellular infection of macrophage compared to the broth grown bacilli as opposed to the demonstrated specific expression of mig gene in M. avium infected macrophage. The echA19 gene was also found to be upregulated. . © Ibadan Biomedical Communications Group.
Hingley-Wilson S, Lalvani A (2008) An exit strategy for the tubercle bacillus?, Nat Rev Microbiol 6 (12)
Allmendinger A, Spavieri J, Kaiser M, Casey R, Hingley-Wilson S, Lalvani A, Guiry M, Blunden G, Tasdemir D (2010) Antiprotozoal, antimycobacterial and cytotoxic potential of twenty-three British and Irish red algae, Phytotherapy Research 24 (7) pp. 1099-1103
As part of our continuing research on seaweeds, we have screened the crude extracts of 23 red marine algae collected from England and Ireland. The clinically important blood-stage life forms of Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani and Mycobacterium tuberculosis were used as test organisms in the in vitro assays. The selectivity of the extracts was determined by using mammalian skeletal myoblast (L6) cells. All algal extracts showed activity against T. brucei rhodesiense, with Corallina officinalis and Ceramium virgatum being the most potent (IC50 values 4.8 and 5.4 ¼g/ml), whilst none of the algal extracts inhibited the growth of T. cruzi. Except for Porphyra leucosticta, extracts from all seaweeds also showed leishmanicidal activity with IC50 values ranging from 16.5 to 85.6 ¼g/ml. Only the crude extract of Calliblepharis jubata showed some weak activity against Mycobacterium tuberculosis (MIC value 256 ¼g/ml), while the others were inactive at this concentration. Corallina officinalis was the only seaweed that displayed some marginal cytotoxicity (IC50 value 88.6 ¼g/ml), and all remaining extracts were non-toxic towards L6 cells at 90 ¼g/ml concentration. To our knowledge, this is the first study reporting antiprotozoal and antimycobacterial activity of British and Irish red algae. Copyright © 2010 John Wiley & Sons, Ltd.
Süzgeaç-Selaçuk S, Meriaçli AH, Güven KC, Kaiser M, Casey R, Hingley-Wilson S, Lalvani A, Tasdemir D (2011) Evaluation of Turkish seaweeds for antiprotozoal, antimycobacterial and cytotoxic activities, Phytotherapy Research 25 (5) pp. 778-783
As part of our continuing research on seaweeds, crude MeOH extracts of two green, three brown and six red algae collected from Marmara, Black, Aegean and Mediterranean Seas were screened. Four parasitic protozoa, i.e. Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani and the tubercle bacillus Mycobacterium tuberculosis were used as test organisms for the in vitro assays. The selective toxicity of the extracts was also determined against mammalian L6 cells. All seaweed extracts were active against T. brucei rhodesiense; the Dasya pedicellata extract was the most potent (IC 50 value 0.37 ¼g/mL). The same extract also weakly inhibited the growth of T. cruzi (IC 50 62.02 ¼g/mL). All seaweed extracts also showed leishmanicidal activity (IC 50 values 16.76-69.98 ¼g/mL). The majority of the extracts also exhibited antiplasmodial potential and the most potent extracts were those from D. pedicellata (IC 50 0.38 ¼g/mL), Codium bursa (IC 50 1.38 ¼g/mL) and Caulerpa rasemosa (IC 50 3.12 ¼g/mL). One brown and two red algal extracts showed some weak activity against Mycobacterium tuberculosis (MIC values 125-256 ¼g/mL). Except for the extract of Dasya pedicellata, none of the extracts displayed any cytotoxicity. This is the second study investigating the antiprotozoal activities of Turkish marine algae and identifies Dasya pedicellata, an understudied algal species, as a candidate for further studies. © 2010 John Wiley & Sons, Ltd.
Hingley-Wilson S, Kon OM, Hsu T, Jr JW, Lalvani A (2010) Mycobacterium tuberculosis RD1 is a requisite for virulence-associated cellular recruitment, IMMUNOLOGY 131 pp. 173-173
Lalvani A, Hingley-Wilson S (2009) Editorial: Live or let die--does HIV exacerbate tuberculosis by attenuating M. tuberculosis-induced apoptosis?, J Leukoc Biol 86 (1) pp. 9-11
Millington KA, Fortune SM, Low J, Garces A, Hingley-Wilson SM, Wickremasinghe M, Kon OM, Lalvani A (2011) Rv3615c is a highly immunodominant RD1 (Region of difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection, Proceedings of the National Academy of Sciences of the United States of America 108 (14) pp. 5730-5735
The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 10-kDa culture filtrate antigen (CFP-10), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv3615c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-10 and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospot- and flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus Calmette- Guérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-10 in both active and latent TB infection. EspC contained broadly recognized CD4+ and CD8+ epitopes, inducing a predominantly CD4+ T-cell response that comprised functional T-cell subsets secreting both IFN-³ and IL-2 as well as functional T-cell subsets secreting only IFN-³. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-10, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-10, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis.
Broniatowska B, Allmendinger A, Tasdemir D, Kaiser M, Montamat-Sicotte D, Hingley-Wilson S, Lalvani A, Guiry M, Blunden G (2011) Antiprotozoal, antitubercular and cytotoxic potential of cyanobacterial (blue-green algal) extracts from Ireland, Natural Product Communications 6 (5) pp. 689-694
Cyanobacteria (= blue-green algae) are prolific producers of structurally distinct and biologically active metabolites. In the continuation of our search for new sources of anti-infective natural products, we have assessed the in vitro antiprotozoal (Plasmodium falciparum, Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani) and antitubercular (Mycobacterium tuberculosis) potential of samples of two terrestrial cyanobacteria, Nostoc commune (collected when desiccated and wet) and Rivularia biasolettiana. The cytotoxic potential of the extracts was also evaluated against primary L6 cells. Except for T. cruzi and M. tuberculosis, the crude extracts were active against all the organisms tested and showed no toxicity. The crude extracts were then partitioned between n-hexane, chloroform and aqueous methanol and retested against the same panel of pathogens. The chloroform sub-extracts of both N. commune samples showed significant activity against T. b. rhodesiense (IC
values 2.0 and 3.5 ¼g/mL) and P. falciparum (IC
s 7.4 and 5.8 ¼g/mL), with low toxicity. This trend was also true for R. biasolettiana extracts, and its chloroform sub-extract showed notable activity against all parasitic protozoa. There were differences in the biological activity profiles of extracts derived from desiccated and hydrated forms of N. commune. To our knowledge, this is the first study assessing the anti-infective activity of desiccated and hydrated forms of N. commune, as well as R. biasolettiana. Furthermore, the present work reports such biological activity in terrestrial cyanobacteria from Ireland for the first time. These results warrant the further study of Irish terrestrial cyanobacteria as a valuable source of new natural product leads for the treatment of parasitic protozoal infections.
Spavieri J, Kaiser M, Casey R, Hingley-Wilson S, Lalvani A, Blunden G, Tasdemir D (2010) Antiprotozoal, antimycobacterial and cytotoxic potential of some British green algae, Phytotherapy Research 24 (7) pp. 1095-1098
In the continuation of our search for natural sources for antiprotozoal and antitubercular molecules, we have screened the crude extracts of four green marine algae (Cladophora rupestris, Codium fragile ssp. tomentosoides, Ulva intestinalis and Ulva lactuca) collected from the Dorset area of England. Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani and Mycobacterium tuberculosis were used as test organisms in the in vitro assays. The selective toxicity of the extracts was also determined toward mammalian skeletal myoblast (L6) cells. The crude seaweed extracts had no activity against M. tuberculosis, but showed antiprotozoal activity against at least two protozoan species. All algal extracts were active against T. brucei rhodesiense, with C. rupestris being the most potent one (IC50 value 3.7 ¼g/ml), whilst only C. rupestris and U. lactuca had moderate trypanocidal activity against T. cruzi (IC50 values 80.8 and 34.9 ¼g/ml). Again, all four extracts showed leishmanicidal activity with IC50 values ranging between 12.0 and 20.2 ¼g/ml. None of the extracts showed cytotoxicity toward L6 cells, indicating that their antiprotozoal activity is specific. This is the first study reporting antiprotozoal and antimycobacterial activity of British marine algae. Copyright © 2009 John Wiley & Sons, Ltd.
Spavieri J, Allmendinger A, Kaiser M, Casey R, Hingley-Wilson S, Lalvani A, Guiry MD, Blunden G, Tasdemir D (2010) Antimycobacterial, antiprotozoal and cytotoxic potential of twenty-one brown algae (phaeophyceae) from British and Irish waters, Phytotherapy Research 24 (11) pp. 1724-1729
In the continuation of our research on seaweeds, crude extracts of 21 brown algae collected from the south coast of England and the west coast of Ireland were screened for in vitro trypanocidal, leishmanicidal and antimycobacterial activities. Mammalian stages of a small set of parasitic protozoa; i.e. Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani, and the tubercle bacillus Mycobacterium tuberculosis were used as test organisms. The extracts were also evaluated for selectivity by testing on a mammalian cell line (L6 cells). Only four extracts were moderately active against T. cruzi, whereas all algal extracts showed significant activity against T. brucei rhodesiense, with Halidrys siliquosa and Bifurcaria bifurcata (Sargassaceae) being the most potent (IC50 values 1.2 and 1.9 ¼g/mL). All algal extracts also displayed leishmanicidal activity, with H. siliquosa and B. bifurcata again being the most active (IC50s 6.4 and 8.6 ¼g/mL). When tested against M. tuberculosis, only the B. bifurcata extract was found to have some antitubercular potential (MIC value 64.0 ¼g/mL). Only three seaweed extracts, i.e. H. siliquosa, B. bifurcata and Cystoseira tamariscifolia showed some cytotoxicity. To our knowledge, this is the first study on the antiprotozoal and antimycobacterial activity of brown algae from British and Irish waters. © 2010 John Wiley & Sons, Ltd.
Pareek M, Evans J, Innes J, Smith G, Hingley-Wilson S, Lougheed KE, Sridhar S, Dedicoat M, Hawkey P, Lalvani A (2013) Ethnicity and mycobacterial lineage as determinants of tuberculosis disease phenotype, Thorax 68 (3) pp. 221-229
Background: Emerging evidence suggests that Mycobacterium tuberculosis (Mtb) lineage and host ethnicity can determine tuberculosis (TB) clinical disease patterns but their relative importance and interaction are unknown. Methods: We evaluated prospectively collected TB surveillance and Mtb strain typing data in an ethnically heterogeneous UK population. Lineage assignment was denoted using 15-loci mycobacterial interspersed repetitive units containing variable numbers of tandem repeats (MIRU-VNTR) and MIRU-VNTRplus. Geographical and ethnic associations of the six global Mtb lineages were identified and the influence of lineage and demographic factors on clinical phenotype were assessed using multivariate logistic regression. Results: Data were available for 1070 individuals with active TB which was pulmonary only, extrapulmonary only and concurrent pulmonary-extrapulmonary in 52.1%, 36.9% and 11.0% respectively. The most prevalent lineages were Euro-American (43.7%), East African Indian (30.2%), Indo-Oceanic (13.6%) and East Asian (12.2%) and were geo-ethnically restricted with, for example, Indian subcontinent ethnicity inversely associated with Euro-American lineage (OR 0.23; 95% CI 0.14 to 0.39) and positively associated with the East African-Indian lineage (OR 4.04; 95% CI 2.19 to 7.45). Disease phenotype was most strongly associated with ethnicity (OR for extrathoracic disease 21.14 (95% CI 6.08 to 73.48) for Indian subcontinent and 14.05 (3.97 to 49.65) for Afro-Caribbean), after adjusting for lineage. With East Asian lineage as the reference category, the Euro-American (OR 0.54; 95% CI 0.32 to 0.91) and East-African Indian (OR 0.50; 95% CI 0.29 to 0.86) lineages were negatively associated with extrathoracic disease, compared with pulmonary disease, after adjusting for ethnicity. Conclusions: Ethnicity is a powerful determinant of clinical TB phenotype independently of mycobacterial lineage and the role of ethnicity-associated factors in pathogenesis warrants investigation.
Montamat-Sicotte D, Millington K, Willcox CR, Hingley-Wilson S, Hackforth S, Innes J, Kon OM, Minnikin DE, Besra GS, Willcox BE, Lalvani A (2010) Mycolic acid-specific T-cells in human tuberculosis are dynamically related to antigen load and exhibit memory expansion after cure, IMMUNOLOGY 131 pp. 171-171
Hingley-Wilson SM, Lougheed KEA, Ferguson K, Leiva S, Williams HD (2010) Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro, Tuberculosis 90 (4) pp. 236-244
Mycobacterium tuberculosis has 10 universal stress proteins, whose function is unknown. However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines. Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial species, suggests a potential role in the persistence and/or intracellular survival of Mtb. Knock-out mutants of individual usp genes encoding the USPs Rv1996, Rv2005c, Rv2026c and Rv2028c were generated and their growth and survival under hypoxic and other stress conditions examined. Although the majority of usp genes are highly induced in hypoxic conditions, mutation did not affect the long term survival of Mtb under these conditions, or in response to a range of stress conditions chosen to represent the environmental onslaughts experienced by the bacillus during an infection, nor during infection of mouse and human - derived macrophage cell lines. The possibility remains that these USPs are functionally redundant in Mtb. © 2010 Elsevier Ltd. All rights reserved.
Hingley-Wilson S, Connell D, Pollock K, Min KO, Jr JW, Lalvani A (2011) Fractalkine production mediates virulence-associated monocyte recruitment and Mycobacterium tuberculosis infection, IMMUNOLOGY 135 pp. 167-167
Thillai M, Eberhardt C, Lewin AM, Potiphar L, Hingley-Wilson S, Sridhar S, Macintyre J, Kon OM, Wickremasinghe M, Wells A, Weeks ME, Mitchell D, Lalvani A (2012) Sarcoidosis and tuberculosis cytokine profiles: indistinguishable in bronchoalveolar lavage but different in blood., PLoS One 7 (7)
The clinical, radiological and pathological similarities between sarcoidosis and tuberculosis can make disease differentiation challenging. A complicating factor is that some cases of sarcoidosis may be initiated by mycobacteria. We hypothesised that immunological profiling might provide insight into a possible relationship between the diseases or allow us to distinguish between them.
Cell growth experiments with a microfluidic device produce large scale time-lapse image data, which contain important information on cell growth and patterns in their genealogy. To extract such information, we propose a scheme to segment and track bacterial cells automatically. In contrast to most published approaches, which often split segmentation and tracking into two independent procedures, we focus on designing an algorithm that describes cell properties evolving between consecutive frames by feeding segmentation and tracking results from one frame to the next one. The cell boundaries are extracted by minimising the Distance Regularised Level Set Evolution model. Each individual cell was identified and tracked by identifying cell septum and membrane as well as developing a trajectory energy minimisation function along time-lapse series. Experiments show that by applying this scheme, cell growth and division can be measured automatically. The results show the efficiency of the approach when testing on different datasets while comparing with other existing algorithms. The proposed approach demonstrates great potential for large scale bacterial cell growth analysis.
Hsu T, Hingley-Wilson S, Chen B, Chen M, Dai A, Morin P, Marks C, Padiyar J, Goulding C, Gingery M, Eisenberg D, Russell R, Derrick S, Collins F, Morris S, King C, Jacobs W (2003) The primary mechanism of attenuation of bacillus
Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue, Proceedings of the National Academy of Sciences of the United States of America 100 (21) pp. 12420-12425
National Academy of Sciences
Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette?Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting RD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis RD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the RD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette?Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.
Studying defined mutants of Mycobacterium tuberculosis in the mouse model of infection has led to the discovery of attenuated mutants that fall into several phenotypic classes. These mutants are categorized by their growth characteristics compared with those of wild-type M. tuberculosis, and include severe growth in vivo mutants, growth in vivo mutants, persistence mutants, pathology mutants and dissemination mutants. Here, examples of each of these mutant phenotypes are described and classified accordingly. Defining the importance of mycobacterial gene products responsible for in vivo growth, persistence and the induction of immunopathology will lead to a greater understanding of the host-pathogen interaction and potentially to new antimycobacterial treatment options.
Upton N, Jackson D, Nikonova A, Hingley-Wilson S, Khaitov M, Del Rosario A, Traub S, Trujillo-Torralbo M, Habibi M, Elkin S, Kon O, Edwards M, Mallia P, Footitt J, Macintyre J, Stanciu L, Johnston S, Sykes A (2017) Rhinovirus induction of fractalkine (CX3CL1) in airway and peripheral blood mononuclear cells in asthma, PLoS One 12 (8) e0183864 pp. 1-13
Public Library of Science
Rhinovirus infection is associated with the majority of asthma exacerbations. The role of fractalkine in anti-viral (type 1) and pathogenic (type 2) responses to rhinovirus infection in allergic asthma is unknown. To determine whether (1) fractalkine is produced in airway cells and in peripheral blood leucocytes, (2) rhinovirus infection increases production of fractalkine and (3) levels of fractalkine differ in asthmatic compared to non-asthmatic subjects. Fractalkine protein and mRNA levels were measured in bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) from non-asthmatic controls (n = 15) and mild allergic asthmatic (n = 15) subjects. Protein levels of fractalkine were also measured in macrophages polarised ex vivo to give M1 (type 1) and M2 (type 2) macrophages and in BAL fluid obtained from mild (n = 11) and moderate (n = 14) allergic asthmatic and non-asthmatic control (n = 10) subjects pre and post in vivo rhinovirus infection. BAL cells produced significantly greater levels of fractalkine than PBMCs. Rhinovirus infection increased production of fractalkine by BAL cells from non-asthmatic controls (P
Mcl-1 protein expression was found to be up-regulated during infection with virulent Mycobacterium tuberculosis strain H37Rv. Mcl-1 induction in THP-1 cells was optimal at a multiplicity of infection of 0.8?1.2 bacilli per macrophage and was independent of opsonin coating of the bacteria. Mcl-1 expression was elevated as early as 4 h, peaked at 5.8-fold above control cells at 24 h, and remained elevated at 48 h after infection. In THP-1 cells, mMcl-1 mRNA was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. In THP-1 cells and monocyte-derived macrophages (MDMs), Mcl-1 protein was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. Treatment of uninfected, H37Ra-infected, and H37Rv-infected THP-1 cells and MDMs with antisense oligonucleotides to mcl-1 reduced Mcl-1 expression by >84%. This resulted in an increase in apoptosis of both MDMs and THP-1 cells that were infected with H37Rv, but not cells that were uninfected or infected with H37Ra. Increased apoptosis correlated with a decrease in M. tuberculosis CFUs recovered from antisense-treated, H37Rv-infected cells at 4 and 7 days after infection. In contrast, CFU recoveries from sense-treated, H37Rv-infected cells or from antisense- or sense-treated, H37Ra-infected cells were unchanged from controls. Thus, the antiapoptotic effect of the induction of Mcl-1 expression in H37Rv-infected macrophages promotes the survival of virulent M. tuberculosis.
Hingley-Wilson S, Connell D, Pollock K, Grass L, Potiphar L, Bremang S, Lalvani A, Hsu T, Jacobs Jr W, Tchilian E, Sykes A, Kon O (2014) ESX1-dependent fractalkine mediates chemotaxis and Mycobacterium tuberculosis infection in humans, Tuberculosis 94 (3) pp. 262-270
Mycobacterium tuberculosis-induced cellular aggregation is essential for granuloma formation and may assist establishment and early spread of M. tuberculosis infection. The M. tuberculosis ESX1 mutant, which has a non-functional type VII secretion system, induced significantly less production of the host macrophage-derived chemokine fractalkine (CX3CL1). Upon infection of human macrophages ESX1-dependent fractalkine production mediated selective recruitment of CD11b+ monocytic cells and increased infection of neighbouring cells consistent with early local spread of infection. Fractalkine levels were raised in vivo at tuberculous disease sites in humans and were significantly associated with increased CD11b+ monocytic cellular recruitment and extent of granulomatous disease. These findings suggest a novel fractalkine-dependent ESX1-mediated mechanism in early tuberculous disease pathogenesis in humans. Modulation of M. tuberculosis-mediated fractalkine induction may represent a potential treatment option in the future, perhaps allowing us to switch off a key mechanism required by the pathogen to spread between cells. © 2014 The Authors.