stop latent tuberculosis

NUMBER THREE / 1 Sep 2010

HEALTH-F3-2008-200999

StopLATENT-TB

WHO WE ARE

The consortitum is composed by seven laboratories. Five laboratories are from four European Countries (France[Beneficiary 8], Italy [Beneficiary 2], Spain [Beneficiaries 1 and 4] and United Kingdom [Beneficiary 5]) and two laboratories are from two Latinamerican countries (Colombia [Beneficiary 6] and Mexico [Beneficiary 7]).

StopLATENTTB is a Collaborative Project funded by the European Union under the 7th Framework Programme. The main aim of the project is the analysis of the basic acitvity of M. tuberculosis during dormancy, and the changes in that activity with drug treatment.

Could the study of ribosomes contribute to our understanding of mycobacterial dormancy?

Ribosomes are essential for the metabolism of bacteria, including mycobacteria, because they are central to the cell's factory for polypeptide synthesis. They are also one of the more abundant components of the bacteria.

A ribosome's structure is complex; its components include three different molecules of ribosomal RNA (rRNA) and more than 50 different proteins, distributed into two (small and large) subunits.

Despite their complexity, ribosomes can form crystals and their structure has been unravelled by x-ray crystallography. The scientists responsible (A. Yonath [Weizman], T. Steitz [Yale] and V. Ramakrishnan [Cambridge]) deserved their Nobel Prize in Chemistry (2009).

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Path of 50S subunit at high resolution (see reference: The Royal Swedish Academy of Sciences)

The importance of the ribosomes in the mycobacterial field goes further than their main physiological role. Their study has also made important contribution to our understanding of mycobacteria, including the main pathogen Mycobacterium tuberculosis.

Scientists studying mycobacterial species have made use of the ribosomes at least once in their research. As an example, the gene coding for the 16S rRNA was the first mycobacterial gene to be sequenced (Suzuki, et al. 1988). In addition, the analysis of the synthesis of the rRNA operon in mycobacteria has helped our understanding of the cell machinery that controls the gene expression (Cook, et al. 2009; Arnvig, et al. 2005).

The basic results were applied to two are main activities thought to be relevant for the control of the disease of Tuberculosis: diagnostic and drugs development. The ribosomes have participated as a main target in both of them:

- Ribosomes are a principal target for drugs active against mycobacteria, including Streptomycin, a first-line drug active against M. tuberculosis. This drug was one of the earliest medications discovered in the treatment of the disease (Iseman, 2002).

- Within the field of diagnostics, the sequence of the 16SrRNA is being frequently used in the identification of mycobacteria isolated from clinical or environmental sources (Tortoli, 2003). Moreover, commercial probes available for the identification of M. tuberculosis and other mycobacteria of clinical interest (Musial, et al. 1988) are aimed at the rrn operon.

Ribosomes have also being used in the implementation of novel molecular technologies, such as microarrays and quantitative PCR. Thus, the sequence coding for the 23SrRNA is considered the positive control to determine if the results derived from microarray analysis have good quality (http://bugs.sgul.ac.uk/). On the other hand, the promoter region of the rrn operon was shown to be a good reference product in qRT-PCR because it was synthesized in a low and constitutive level under several environmental conditions, such as hydrogen peroxide (Nuñez, et al. 2008)

Furthermore, it is expected that the study of ribosomes could also be of help to gain insights into latency, the most prevalent state of the main pathogen M. tuberculosis. Could we use ribosomes in any way to characterize this mycobacterium during latent infection?.

The situation of the tubercle bacilli during latency usually has several names: dormancy, persistence, long-lasting infection, etc... All these terms overlap one with the other so that these states are not always clearly differentiated. However, they all concern a biological condition of the bacilli: bacilli that are viable but not cultivable. How this can be analyzed?.

In microbiology, viable means organisms capable of reproducing (or replicating in the case of viruses) under appropriate conditions, and non cultivable refer to the inability of growth a particular organism under controlled conditions (Singleton & Sainsbury, 1987).

The ability to culture M. tuberculosis is usually tested in vitro, by counting the number of colonies visible on plates utilizing a specific culture media; the lack of colonies after an appropriate time is interpreted as the presence of "non cultivable" bacilli.

How can viability be determined?. This is the place where we hope the ribosomes could be of help. If one bacterium is able to reproduce, it would certainly need ribosomes; also, it must need to synthesize ribosomal components, such as rRNA. Suppose we use the ability of bacilli to synthesize rRNA as a marker of their viability?.

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Mycobacteria need to control and to couple the synthesis of ribosomal RNA and ribosomal proteins, in order to built ribosomes according to the cell's needs.(see: Garcia et al, 2010)

Previous studies of members of the StopLATENT-TB consortium characterized the promoter region of the rrn operons of several mycobacteria, including M. tuberculosis (Gonzalez-y-Merchand, et al. 1997; Menendez, et al. 2002). It was also shown that the pre-rRNA (the promoter region of the rrn operon) is detected in a similar level than other mRNAs, contrary to the detection of mature 16SrRNA which is usually detected near 103 times compared to other mRNA molecules (Garcia, et al. 2010). Recently, the detection of pre-rRNA (Menendez, et al. 2005) has been used in the analysis of another mycobacterium, namely M. avium subsp. paratuberculosis (MAP). In this work, authors used the detection of the pre-16SrRNA gene ratio to determine the MAP viability in the presence of a drug (Bull, et al. 2009).

Perhaps all these previous studies show a way to detect live bacilli during latent infection. Again, the ribosomes may lend us a hand to understand this cryptic phase that is frequently found when M. tuberculosis infects humans.

REFERENCES

Arnvig, K.B. et al. The mechanism of upstream activation in the rrnB operon of Mycobacterium smegmatis is different from the Escherichia coli paradigm. Microbiology. 2005. 151:467-473.

Bull, T.J. et al. A rhodamine agent active against non-replicating intracellular Mycobacterium avium subspecies paratuberculosis. Gut Pathogens. 2009, 1:25 doi:10.1186/1757-4749-1-25

Cook, G.M. et al. Physiology of Mycobacteria. Advances in Microbial Physiology. 2009. 55:81-182.

Garcia, M.J. et al. Measurement of the rates of synthesis of three components of ribosomes of Mycobacterium fortuitum: a theoretical approach to qRT-PCR experimentation. PLoS ONE. 2010. 5(7): e11575. doi:10.1371/journal.pone.0011575

Gonzalez-y-Merchand, J.A., et al. Strategies used by pathogenic and non-pathogenic Mycobacteria to synthesize rRNA. Journal of Bacteriology. 1997. 179:6949-6958.

Iseman M.D. Tuberculosis therapy: past, present and future. Eur.resp. J. 2002. 20(36)87s-94s.

Menendez, M.C. et al. Characterization of an rRNA operon (rrnB) of Mycobacterium fortuitum and other mycobacterial species; implications for the classification of mycobacteria. Journal of Bacteriology. 2002. 184: 1078-1088.

Menendez, M.C. et al. Analysis of the precursor-rRNA fractions of rapidly growing mycobacteria: quantification by methods that include the use of a promoter (rrnA P1) as a novel standard. Journal of Bacteriology. 2005. 187:534-543-

Musial, C.E. et al. Identification of mycobacteria from culture by using the Gen-Probe Rapid Diagnostic System for Mycobacterium avium complex and Mycobacterium tuberculosis complex. J Clin Microbiol. 1988. 26:2120-3.

Nuñez, M.C. et al. Transcriptional analysis of Mycobacterium fortuitum upon hydrogen peroxide treatment using the novel standard rrnA-P1. BMC Microbiology. 2008. 8:100 doi:10.1186/1471-2180-8-100.

Singleton, P. & D. Sainsbury. Dictionary of Microbiology and Molecular Biology. 2nd Ed. J Wiley & sons. 1987.

Suzuki, Y. et al. Complete nucleotide sequence of the 16S rRNA gene of Mycobacterium bovis BCG. J Bacteriol. 1988. 170(6): 2886-2889.

The Royal Swedish Academy of Sciences. Structure and function of the ribosome. Scientific background of the Nobel Prize in Chemistry 2009. 7 October 2009.

Tortoli E. Impact of Genotypic studies on Mycobacterial Taxonomy: The New Mycobacteria of the 1990s. Clinical Microbiology Reviews. 2003. 16(2):319-354.

NUMBER THREE / 1 September 2010