Margarita O. Shleeva

Mutants of Mycobacterium tuberculosis lacking three of the five rpf-like genes are defective for growth in vivo and for resuscitation in vitro

ABSTRACT Mycobacterium tuberculosis contains five genes, rpfA through rpfE, that bear significant homology to the resuscitation-promoting factor (rpf) gene of Micrococcus luteus, whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf-like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf-like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the three strains tested (the ΔrpfB, ΔrpfD, and ΔrpfE strains). In contrast, two multiple strains, KDT8 (ΔrpfA-mutation ΔrpfC ΔrpfB) and KDT9 (ΔrpfA ΔrpfC ΔrpfD), which lack three of the five rpf-like genes, were significantly yet differentially attenuated in a mouse infection model. These mutants were also unable to resuscitate spontaneously in vitro, demonstrating the importance of the Rpf-like proteins of M. tuberculosis in resuscitation from the nonculturable state. These results strongly suggest that the biological functions of the five rpf-like genes of M. tuberculosis are not wholly redundant and underscore the potential utility of these proteins as targets for therapeutic intervention.

Dormant ovoid cells of Mycobacterium tuberculosis are formed in response to gradual external acidification

It is believed that latent tuberculosis is associated with the persistence of Mycobacterium tuberculosis (MTB) in a dormant-like state. Dormant cells of MTB with coccoid morphology were produced in some in vivo studies, but similar forms were not produced in the known in vitro models in sufficient amounts to permit their characterization. This work demonstrates the efficient formation of phase-dark ovoid cells in MTB cultures within 150 days after the onset of stationary phase. During this time the medium underwent gradual acidification (pH 8.5 → 4.7) as a result of cellular metabolism. A rapid change in the external pH resulted in cell degradation and death. In common with the dormant forms found in other organisms, the ovoid cells had thickened cell walls, a low metabolic activity and elevated resistance to antibiotics and heating. The ovoid cells had lost the ability to form colonies on solid medium and were thus regarded as operationally «non-culturable». At an early stage in the acidification process (about 40 days post inoculation), the ovoid cells self-resuscitated when placed in fresh liquid medium. However, ovoid cells, stored for a prolonged time, required supernatant from active MTB cells, or externally added recombinant form of resuscitation promoting factor (Rpf) for successful resuscitation. It is suggested that the adaptation of cellular metabolism leading to gradual acidification of the external medium results in the formation of morphologically distinct dormant MTB cells in vitro. The model of MTB dormancy developed here could be a useful tool for the development of new drugs against latent TB.

Finding of the low molecular weight inhibitors of resuscitation promoting factor enzymatic and resuscitation activity.

Background Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs. Methodology/Principal Findings Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC50 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC50. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations. Conclusions/Significance NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins.

Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids

One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant (“non-culturable”) cells of Mycobacterium smegmatis (mc2155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6–10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3–10 mM) or dibutyryl-cAMP (0.5–1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.

A product of RpfB and RipA joint enzymatic action promotes the resuscitation of dormant mycobacteria

Resuscitation‐promoting factor proteins (Rpfs) are known to participate in reactivating the dormant forms of actinobacteria. Structural analysis of the Rpf catalytic domain demonstrates its similarity to lysozyme and to lytic transglycosylases – the groups of enzymes that cleave the β‐1,4‐glycosidic bond between N‐acetylmuramic acid (MurNAc) and GlcNAc, and concomitantly form a 1,6‐anhydro ring at the MurNAc residue. Analysis of the products formed from mycobacterial peptidoglycan hydrolysis reactions containing a mixture of RpfB and resuscitation‐promoting factor interacting protein (RipA) allowed us to identify the suggested product of their action – N‐acetylglucosaminyl‐β(1→4)‐N‐glycolyl‐1,6‐anhydromuramyl‐l‐alanyl‐d‐isoglutamate. To identify the role of this resulting product in resuscitation, we used a synthetic 1,6‐anhydrodisaccharide‐dipeptide, and tested its ability to stimulate resuscitation by using the dormant Mycobacterium smegmatis model. It was found that the disaccharide‐dipeptide was the minimal structure capable of resuscitating the dormant mycobacterial cells over the concentration range of 9–100 ng·mL−1. The current study therefore provides the first insights into the molecular mechanism of resuscitation from dormancy involving a product of RpfB/RipA‐mediated peptidoglycan cleavage.

Resuscitation-promoting Factors (Rpf): In Search of Inhibitors

Resuscitation promoting factors (Rpf) are a family of proteins secreted by actively growing actinobacteria, including Mycobacterium tuberculosis. Experimental evidence suggests that Rpfs play a distinct role in bacterial resuscitation and re-growth as well as reactivation of chronic tuberculosis in mice. The striking similarity of the Rpfs structure to cell wall hydrolysing enzymes has provided a basis for the development of novel low molecular weight inhibitors of Rpfs activity. In particular, recently characterised nitrophenylthiocyanate compounds could be considered as a promising scaffold for generation of therapeutic agents targeting reactivation of latent tuberculosis. This review describes recent progress in understanding of molecular mechanisms of Rpf biological activity.

Dormant forms of mycobacteria

Dormant states of bacteria with drastically decreased metabolic activity, enhanced resistance to harmful factors, and absence of cell division is a form for surviving unfavorable environmental conditions. This state does not necessarily imply formation of highly differentiated spores and cysts; it has been demonstrated for non-spore-forming bacteria, including pathogenic ones. The latency of a number of infectious diseases is generally believed to be related to the capacity of bacteria (including Mycobacterium tuberculosis, an infective agent of tuberculosis) to produce dormant forms. Indeed, some results of histological investigation and modeling of latent infections in animals, as well as results obtained with in vitro models, support the hypothesis of production of dormant forms by tuberculosis bacteria. In the present review, existing experimental models of dormant form production in mycobacteria are considered, as well as modern data concerning the mechanisms of their formation and their relation to the “nonculturable” state. The mechanisms of reversion to culturability and the role of extracellular factors in reactivation of dormant forms are discussed in detail.

The Role of Intercellular Contacts in the Initiation of Growth and in the Development of a Transiently Nonculturable State by Cultures of Rhodococcus rhodochrous Grown in Poor Media

It was found that the growth of Rhodococcus rhodochrous cells in a modified Saton’s medium strongly depends on the rate of culture agitation in the flask: agitation at 250 rpm in flasks with baffles stops cell multiplication, whereas slight agitation leads to pronounced culture growth. The growth retardation phenomenon was reversible and did not manifest itself in exponential-phase cultures or when the cells were grown in a rich medium; furthermore, it was not connected with the degree of culture aeration. When agitated at a moderate rate, the bacterial cells formed aggregates in the lag phase, which broke up into single cells in the exponential phase. The inhibitory effect of vigorous agitation was removed by the addition, to the medium, of the supernatant (SN) of a log-phase culture grown in the same medium with moderate agitation. Vigorous agitation is thought to interfere with cell contact, whose establishment is necessary for the development of an R. rhodochrous culture in a poor medium, which occurs in the form of (micro) cryptic growth. When grown in a modified Saton’s medium, R. rhodochrous cells were capable of transition, in the prolonged stationary phase, to a resting and transiently nonculturable state. Such cells could be resuscitated by incubation in a liquid medium with the addition of the supernatant or the Rpf secreted protein. The formation of transiently nonculturable cells was only possible under the conditions of a considerable agitation rate (250–300 rpm), which prevented secondary (cryptic) growth of the culture. This circumstance indicates the importance of intercellular contacts not only for the initiation of growth but also for the transition of the bacteria to a dormant state.

Influence of oxidative and nitrosative stress on accumulation of diphosphate intermediates of the non-mevalonate pathway of isoprenoid biosynthesis in corynebacteria and mycobacteria

Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) — an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) — most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82–92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes, well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.

Generation of dormant forms by Mycobacterium smegmatis in the poststationary phase during gradual acidification of the medium

Persistent forms of the wild-type strain of Mycobacterium smegmatis and its mutants with inactivated devR and hlp genes were investigated. devR encodes the regulatory protein responsible for the formation of nonreplicating mycobacterial forms under hypoxia, and hlp codes for a histone-like protein. It has been found that a gradual decrease of pH in M. smegmatis wild-type poststationary cultures resulted in the formation of a special type of persisters. They significantly differed from vegetative cells in their properties and were represented by shortened ovoid forms with thickened cell walls. According to atomic force microscopy data, the size of the ovoid forms and vegetative cells was 1.2 × 0.9 μm and 3.7 × 0.8 μm, respectively. The metabolism level was markedly decreased in ovoid cells: the incorporation of [5,6-3H]uracil and thymidine was decreased 200- and 50-fold, respectively. The intracellular ATP content was lowered threefold. The ovoid forms that emerged in poststationary cultures in Sauton’s medium when the medium pH value was gradually decreased to 6.0 retained for a long time (9 months) the capacity to resume growth on rich solid and liquid medium. Compared to vegetative cells, the ovoid forms exhibited an elevated resistance to heating (60–80°C) and antibiotics (hygromycin, kanamycin, and tetracycline). The ovoid forms of the M. smegmatis wild-type strain were classified as dormant forms based on their survival capacity, resistance to deleterious factors, and structural peculiarities. The ovoid forms generated in poststationary cultures upon decreasing the pH value to 5.0 or below lost the colony-forming capacity. It was established that the capacity to form ovoid cells upon gradual decrease in the pH value to 6.0 was reduced in ΔdevR and hlp-0 mutants compared to the wild-type strain (generation of 5–6 and 40% dormant forms, respectively) The amount of M. smegmatis dormant cells formed correlated with the acidification degree of the medium. The model developed can be used in tests of new antibacterial preparations that effectively inhibit resuscitating mycobacterial dormant forms that persist in the host organism.