Elsevier

Tuberculosis

Volume 88, Issue 5, September 2008, Pages 462-468
Tuberculosis

Resuscitation-promoting factors are expressed in Mycobacterium tuberculosis-infected human tissue

https://doi.org/10.1016/j.tube.2008.01.007Get rights and content

Summary

Resuscitation-promoting factors are small proteins found in many high G+C Gram-positive organisms. In picomolar concentrations in vitro they promote the emergence from dormancy and shorten the lag phase of growth of small inocula of responsive bacteria. Mycobacterium tuberculosis has five rpf gene orthologues all of which are expressed during in vitro growth. Rpfs may play a role in dormancy or reactivation of tubercle bacilli in vivo, and could be a potential target for chemotherapeutic intervention: however it is not known whether they are expressed by M. tuberculosis in vivo in the context of human infection. The work presented here demonstrates that Rpf expression can be detected in human tissues infected with M. tuberculosis and describes the pattern of this expression using immunocytochemistry with anti-Rpf antibodies.

Introduction

Tuberculosis (TB) is recognised as one of the major threats to human health. Approximately 8 million active cases of TB are diagnosed every year and there are over 3 million deaths. Furthermore it was estimated by the 1999 WHO Global Surveillance and Monitoring Project1 that about 1.9 billion people (about one third of the world's population) are latently infected. Conservative estimates suggest that 5–10% of latently infected immunocompetent individuals will develop active TB during their lifetime: but for those co-infected with HIV the figure rises to 5–10% per year of life.2, 3, 4 It is this ‘human reservoir’ which poses the greatest challenge of all to TB control and eradication.

The exact physiological and metabolic state of the tubercle bacillus in latent infection has been the subject of bitter controversy (see Ref. 5 for a review). It is postulated that the anaerobic and acidic conditions within closed lesions in the lung reduce bacterial metabolism. The organisms in this putative state are given the name ‘persisters’.6, 7 The suggestion is that the existence of persisters is one of the reasons for the extended treatment duration required for cure and is fundamental to the problem of treating active TB satisfactorily.

Resuscitation-promoting factors (Rpfs) are small proteins first identified in the supernatant of stationary phase cultures of Micrococcus luteus.8 They are active at picomolar concentrations, increasing the number of culturable M. luteus cells from dormant populations and shortening the lag phase of growth of small inocula. Bioinformatic searches have demonstrated that there are more than 40 examples of rpf-like genes in the high G+C cohort of Gram-positive bacteria, including M. tuberculosis, which contains five rpf gene orthologues. All of these have extensive sequence similarity to the N-terminal domain of M. luteus Rpf.9 The rpf genes have been isolated from M. tuberculosis DNA, cloned and expressed in E. coli.9, 10 Any of these five proteins, used in pM concentrations, permitted the resumption of growth of dormant cells of M. bovis BCG that could not grow in their absence.9

The study of the M. tuberculosis Rpfs may have great clinical relevance. M. tuberculosis cells isolated from murine macrophages, which could not be cultured under normal conditions, could be resuscitated using Rpf in liquid medium.11 Cells in a low state of metabolic activity are less susceptible to drug treatment, so to resuscitate them might be to render them easier to treat with conventional drug treatments. Rpfs might, therefore, be used as an adjunct to conventional treatment and permit shorter courses of chemotherapy to be used. Conversely, where there is a high risk of reactivation of tuberculosis, for example in HIV infection, suppression of Rpf expression or activity might be able to prevent the disease becoming active again.

The five Rpf genes are scattered around the M. tuberculosis genome with no obvious organisation to their distribution. This protein family may show functional redundancy.12 Alternatively, the different molecules may be produced under different conditions or for different specific purposes. All five genes are expressed in M. tuberculosis in vitro.9, 13 One previous study has looked at transcription of mRNA in the homogenised lungs of mice experimentally infected with M. tuberculosis, and found that transcripts were detectable by RT-PCR, suggesting that Rpfs may be expressed in such tissues.13 However, whether or not Rpf proteins are directly detectable in human tissues in the context of M. tuberculosis infection, and if so, whereabouts in the tissues they are to be found, has hitherto not been investigated. This paper reports the expression of Rpfs by M. tuberculosis in tissues obtained from patients with active disease.

Section snippets

Materials and methods

Polyclonal sheep anti-Rpf antibodies were obtained commercially (Micropharm UK) and purified as described previously.9 In brief the sheep was immunized with truncated Rpf (lacking the LysM module) and antibodies were purified using a column packed with Rpf-Sepharose. They were not specific to any one particular Rpf and had been shown to detect recombinant versions of all five Rpf-like proteins of M. tuberculosis.9 Secondary biotinylated rabbit anti-sheep antibodies and peroxidase-conjugated

Staining of M. tuberculosis-infected human tissue with anti-Rpf antibodies

The images are all consecutive sections of the same gut lymph node infected with M. tuberculosis. The lymph node section contained necrotic lesions with caseous centres surrounded by a zone of giant cells and macrophages. Lymphoid follicles were also visible. There was no staining of the negative control section to which anti-Rpf antibody had not been applied. Figure 1a is an image of epithelioid giant cells in the negative control (G0). In sections to which anti-Rpf antibody was applied,

Discussion

This paper reports the localisation of M. tuberculosis Rpf in vivo using an antibody that bound M. tuberculosis anti-Rpf antibodies applied to tissues of patients with tuberculosis.9 The only previously reported work to investigate Rpf expression in vivo used RT-PCR to detect mRNA transcripts in homogenised samples of experimentally infected mouse lung.13 This is the first report of M. tuberculosis Rpf protein expression in human tissue.

Whilst Rpf was present both around the bacilli in the

Acknowledgments

This work was funded by a UK Medical Research Council Clinical Research Training Fellowship (APD) to SHG and BH. We thank Vladimir Koulchin and colleagues (Chemogen) for providing anti-LAM antibodies and Dietrich Mack (Swansea University) for his constructive comments on the manuscript.

References (25)

  • P.A. Selwyn et al.

    A prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection

    N Engl J Med

    (1989)
  • I.M. Orme

    The latent tuberculosis bacillus (I'll let you know if I ever meet one)

    Int J Tuberc Lung Dis

    (2001)
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