Periodic Report Summary - STOPLATENT-TB (Latent tuberculosis: New tools for the detection and clearance of dormant mycobacterium tuberculosis)
Project context and objectives:
It was estimated that one third of the human population harbour latent tuberculosis. This is why latent tuberculosis caused by Mycobacterium tuberculosis is considered to be one of the more successful infections. The clinical definition of latency is clear, however the bacterial biology underlying this clinical situation remains poorly understood.
Dormant tubercle bacilli are difficult to detect (they are considered non-cultivable) and are also refractory to standard treatments which are usually effective against actively dividing bacilli. These conditions prevent the clearance of the dormant bacilli from infected tissues.
It is classically considered that dormant bacilli remain undetectable inside granulomas, the central defences of the tissue-host against tuberculosis. However, data was accumulated (some from laboratories of participants) indicating that non-granulomatous including extra-pulmonary tissues could also carry dormant bacilli.
Project results:
The project was carried out dynamically and members of the consortium interacted fluently and helped each other for the general success of the objectives. Promising results were obtained and some of them were published and others were confirmed prior to their dissemination.
The project aimed to test the knowledge on relationships of lipid metabolism with the establishment of latent infection. Several innovative models were developed on that basis for the study of non-dividing bacilli in the analysis of the latent infection. The wide range of models applied involved from in vitro models, which consider the presence of lipids as carbon source the key conditions for growing, to ex vivo infection of adipocytes, where the availability of lipids is clear, and finally, to in vivo models. All these conditions were developed within the STOPLATENT-TB project:
1. in vitro: the bacteria were grown in liquid media in the presence of different lipid-cocktails including cholesterol as a sole carbon source (Beneficiary 6 [J. Gonzalez-y-Merchand-IPN-ENCB] and Beneficiary 7 [P. del Portillo-CorpoGen]). Stress in vitro conditions, which were supposed to be related to the non-dividing persistent stage of the bacilli, such as hypoxia or acidic pH, were implemented under lipid enriched media. Transcriptomic and proteomic were analysed in the bacterial cultures;
2. ex vivo: the kinetics growth in adipocytes, according to the method developed by Beneficiary 8 (O. Neyrolles-CNRS) was evaluated and compared with the growth in other cells, such as macrophages or pre-adipocytes. The comparison of the cell and bacteria responses when macrophages were infected with actively dividing and non-dividing bacilli was also evaluated;
3. in vivo: several new models of tuberculosis infection in animals, putatively suitable for the study of latent infection, were developed within the project. These were novel prototypes that, besides being useful for the STOPLATENT-TB consortium, they were also applied to the collaborations with other research groups.
Objective a) In order to gain insight into the metabolic activity of the bacilli under in vitro models developed, a novel approach was undertaken, namely RNAseq, to determine the transcriptomic profile of M. tuberculosis under stress in vitro. Beneficiary 7 (P. del Portillo-CorpoGen) implemented the protocol in order to have a general picture of the expression profile of the bacilli growing in FA-EM. Detection of M. tuberculosis was viable but non-cultivable in human samples demonstrated that most of these bacilli had metabolic activity under such conditions (Beneficiary 1 [M.J. Garcia-UAM]) and that activity includes products required for cell division.
Objective b). Beneficiary 6 (J. Gonzalez-y-Merchand-IPN-ENCB) analysed the morphological changes of the M. tuberculosis bacilli cultured in different conditions (log and stationary phases, as well as NRP1 and NRP2 phases) by scanning electron microscopy. Besides, they compared cultures in standard Dubos media with cultures in Fatty acids-enriched media. The metabolic change of bacilli under environmental conditions enriched in lipids influences the phenotypic appearance, as demonstrated by electron microscopy.
Objective c) Efforts were made to detect fluorescent bacilli during latent infection in vivo. This was a difficult task that required main changes in the adopted approach. Those changes included not only modified protocol for the detection of fluorescence (Beneficiary 5 [A. Coates-SG-UL]) but also the use of bioluminescence as another label product (Beneficiary 4 [P.J. Cardona-IGTP]). These procedures proved to be technically very demanding. On the contrary, the detection of putatively dormant bacilli during infection in human clinical samples was possible by qRT-PCR, a procedure that is independent of the immunological status of the patient.
Objective d) Beneficiary 2 (L.Fattorini-ISS) tested drugs combinations able to kill aerobic (A), replicating, populations, as well as hypoxic (H), non-replicating bacilli. Overall, a differential activity of drugs against A and H bacilli was observed. The observations of this Beneficiary showed that drug susceptibility of M. tuberculosis varied depending on the growth stage of the organism, and novel drugs showed more activity against dormant M. tuberculosis, a useful data to take into account in the design of other drug's combinations for latent TB.
Potential impact:
The project was anticipated to be widely applicable in several aspects of the disease under study, i.e. tuberculosis:
a) the project allowed the development of new experimental animal models in order to better and faster detect which therapeutic regimens are more efficient;
b) development of new diagnostic tools: a new target specific to detect viable non cultivable bacilli during latency was identified;
c) development of new drugs and drug therapies: novel drug combinations focused on the sterilisation of active and latent infection were identified and new therapies were tested for the treatment of latent infections;
d) development of new control strategies: mechanisms for the establishment of latency could potentially give information for the design of better vaccines
This knowledge was anticipated to promote more efficient control policies against tuberculosis.
It was estimated that one third of the human population harbour latent tuberculosis. This is why latent tuberculosis caused by Mycobacterium tuberculosis is considered to be one of the more successful infections. The clinical definition of latency is clear, however the bacterial biology underlying this clinical situation remains poorly understood.
Dormant tubercle bacilli are difficult to detect (they are considered non-cultivable) and are also refractory to standard treatments which are usually effective against actively dividing bacilli. These conditions prevent the clearance of the dormant bacilli from infected tissues.
It is classically considered that dormant bacilli remain undetectable inside granulomas, the central defences of the tissue-host against tuberculosis. However, data was accumulated (some from laboratories of participants) indicating that non-granulomatous including extra-pulmonary tissues could also carry dormant bacilli.
Project results:
The project was carried out dynamically and members of the consortium interacted fluently and helped each other for the general success of the objectives. Promising results were obtained and some of them were published and others were confirmed prior to their dissemination.
The project aimed to test the knowledge on relationships of lipid metabolism with the establishment of latent infection. Several innovative models were developed on that basis for the study of non-dividing bacilli in the analysis of the latent infection. The wide range of models applied involved from in vitro models, which consider the presence of lipids as carbon source the key conditions for growing, to ex vivo infection of adipocytes, where the availability of lipids is clear, and finally, to in vivo models. All these conditions were developed within the STOPLATENT-TB project:
1. in vitro: the bacteria were grown in liquid media in the presence of different lipid-cocktails including cholesterol as a sole carbon source (Beneficiary 6 [J. Gonzalez-y-Merchand-IPN-ENCB] and Beneficiary 7 [P. del Portillo-CorpoGen]). Stress in vitro conditions, which were supposed to be related to the non-dividing persistent stage of the bacilli, such as hypoxia or acidic pH, were implemented under lipid enriched media. Transcriptomic and proteomic were analysed in the bacterial cultures;
2. ex vivo: the kinetics growth in adipocytes, according to the method developed by Beneficiary 8 (O. Neyrolles-CNRS) was evaluated and compared with the growth in other cells, such as macrophages or pre-adipocytes. The comparison of the cell and bacteria responses when macrophages were infected with actively dividing and non-dividing bacilli was also evaluated;
3. in vivo: several new models of tuberculosis infection in animals, putatively suitable for the study of latent infection, were developed within the project. These were novel prototypes that, besides being useful for the STOPLATENT-TB consortium, they were also applied to the collaborations with other research groups.
Objective a) In order to gain insight into the metabolic activity of the bacilli under in vitro models developed, a novel approach was undertaken, namely RNAseq, to determine the transcriptomic profile of M. tuberculosis under stress in vitro. Beneficiary 7 (P. del Portillo-CorpoGen) implemented the protocol in order to have a general picture of the expression profile of the bacilli growing in FA-EM. Detection of M. tuberculosis was viable but non-cultivable in human samples demonstrated that most of these bacilli had metabolic activity under such conditions (Beneficiary 1 [M.J. Garcia-UAM]) and that activity includes products required for cell division.
Objective b). Beneficiary 6 (J. Gonzalez-y-Merchand-IPN-ENCB) analysed the morphological changes of the M. tuberculosis bacilli cultured in different conditions (log and stationary phases, as well as NRP1 and NRP2 phases) by scanning electron microscopy. Besides, they compared cultures in standard Dubos media with cultures in Fatty acids-enriched media. The metabolic change of bacilli under environmental conditions enriched in lipids influences the phenotypic appearance, as demonstrated by electron microscopy.
Objective c) Efforts were made to detect fluorescent bacilli during latent infection in vivo. This was a difficult task that required main changes in the adopted approach. Those changes included not only modified protocol for the detection of fluorescence (Beneficiary 5 [A. Coates-SG-UL]) but also the use of bioluminescence as another label product (Beneficiary 4 [P.J. Cardona-IGTP]). These procedures proved to be technically very demanding. On the contrary, the detection of putatively dormant bacilli during infection in human clinical samples was possible by qRT-PCR, a procedure that is independent of the immunological status of the patient.
Objective d) Beneficiary 2 (L.Fattorini-ISS) tested drugs combinations able to kill aerobic (A), replicating, populations, as well as hypoxic (H), non-replicating bacilli. Overall, a differential activity of drugs against A and H bacilli was observed. The observations of this Beneficiary showed that drug susceptibility of M. tuberculosis varied depending on the growth stage of the organism, and novel drugs showed more activity against dormant M. tuberculosis, a useful data to take into account in the design of other drug's combinations for latent TB.
Potential impact:
The project was anticipated to be widely applicable in several aspects of the disease under study, i.e. tuberculosis:
a) the project allowed the development of new experimental animal models in order to better and faster detect which therapeutic regimens are more efficient;
b) development of new diagnostic tools: a new target specific to detect viable non cultivable bacilli during latency was identified;
c) development of new drugs and drug therapies: novel drug combinations focused on the sterilisation of active and latent infection were identified and new therapies were tested for the treatment of latent infections;
d) development of new control strategies: mechanisms for the establishment of latency could potentially give information for the design of better vaccines
This knowledge was anticipated to promote more efficient control policies against tuberculosis.