How does tb reproduce

Combined with the high variability associated to this technique, prone to high errors when working in Biosafety Level 3 lab with very high volume of samples and the fact that colony counts do not correlate to the dilution factor; this method needs to be carefully considered. Even more problematic is the fact that in cell biology studies that investigate the intracellular localization of Mtb, there is in general little information about the viability status of the bacteria.

The main obstacle in elucidating the precise fate of Mtb has been the difficulty in unequivocally establishing if a single Mtb localised in a defined compartment e. Even under conditions where the majority of bacteria are killed, the ability of a minor pool to replicate, or to remain alive in a non-replicating state can have major implications for the future course of the infection.

Measuring Mtb viability in host cells after infection. A , Currently, the conventional method to evaluate viability of Mtb relies on bacterial CFU enumeration on agar plates. This is a time-consuming approach due to the slow growth rate of Mtb that takes about 3—6 weeks to observe visible colonies on agar plates. One of its main disadvantages of this method is that clumps of bacteria cells can be miscounted as single colonies. Then, BlaC will hydrolyze the lactam ring to activate the fluorophore, and DprE1 will covalently bind the anchor unit for fluorescence immobilization.

E , A FITC-trehalose probe that exploits the processing by Mtb Ag85 enzymes is specifically incorporated into Mtb growing in vitro and within macrophages. In the last years, to overcome the problems associated to the CFU assay and trying to obtain information about the physiological state of Mtb within cells, image-based approaches, mostly based on fluorescence, have been developed.

A similar dual reporter strategy, that does not measure directly bacterial viability, has been used by combining promoters that respond to specific environmental cues in host cells and in vivo. In this system, one promoter drives constitutive expression of a fluorophore whereas a second promoter that is regulated by environmental cues controls the expression of a different fluorophore. Using this approach, it has been possible to define how Mtb respond to stress and nutrients in host cells and in vivo Tan et al.

Because it utilises highly stable GFP as a readout, with this system the spatiotemporal resolution is limited. Another small molecular probe that uses a very elegant dual-targeting strategy has been developed to specifically label mycobacteria Cheng et al. This probe discriminates between live and dead M.

The probe works when bacteria are pre-treated and used to infect mouse macrophages and could potentially work to monitor viability of Mtb within host cells Fig. In long term experiments needed for monitoring Mtb replication, the system could be potentially diluted over time. Overall, it is not trivial to discriminate between live and dead bacilli when looking at infected cells and it depends largely on how Mtb viability is defined. Most of the systems have some difficulties with the interpretation of the results and ideally, a combination of more than one method is required.

Other probes exploited specific sugars that are only present in mycobacteria. These enzymes are sufficiently promiscuous to process a variety of trehalose analogs that are fluorescently labelled and trehalose-probe analogs are also efficiently anchored to Mtb.

Trehalose probes are selectively taken up by live cells and label live Mtb in infected mammalian cells.

These probes were able to differentially label relevant populations of intracellular Mtb that reflects intracellular localisation Backus et al. These probes have not been extensively used to label replicating Mtb in macrophages.

Some additional trehalose probes have been developed to detect Mtb in vitro and sputum and could represent another useful probe to monitor intracellular replication of Mtb Kamariza et al. In the last few years, many groups have developed fluorescence reporters in Mtb to monitor bacterial replication in vitro.

Macrophages are a niche for Mtb and resting macrophages in vitro provide very good conditions for bacterial replication. Understanding the location where Mtb replicates but also the conditions that restrict growth is critical to further delineate the cellular immune response to Mtb.

This aspect however has been less investigated and establishing if a single Mtb localised in a defined compartment is able to replicate has been difficult. To measure bacterial replication quantitatively and at the single-cell level in host cells has been challenging due to the slow replication of mycobacteria and the limited availability of live cell imaging systems in Biosafety Level 3.

One way to follow intracellular replication of Mtb is by monitoring active growth of fluorescent Mtb through time at single cell level in fixed cells Fig.

With these methodologies is however not possible to determine if bacteria are alive or dead as discussed above. Moreover, without spatiotemporal resolution, it is difficult to define whether Mtb was actually growing within the same cell due to cell death and efferocytosis. Measuring Mtb intracellular replication. A , One approach to follow Mtb replication involves the quantification of fluorescent Mtb strains through time at single cell level in fixed cells.

Although this method can be adapted to high-content imaging, it does not take into account cellular heterogeneity and does not allow to fully distinguish live from dead bacteria. B , Shows the Luciferin-luciferase reporter.

This system is useful for studying cell populations but does not allow for single-cell analysis. C , A more time-consuming approach that it does allow spatiotemporal resolution is to evaluate intracellular bacteria replication by live cell imaging. D , Shows genetically encoded fluorescent reporter in combination with quantitative time-lapse microscopy. It uses a destabilized variant of green fluorescent protein GFPdes with a plasmid expressing a stable red fluorescent protein DsRed2 from a constitutive promoter, as internal control.

In this example, stationary-phase populations exhibit a drop in GFPdes expression followed by the appearance of a subset of non-replicating bacteria displaying high levels of fluorescence. Luciferin-luciferase systems are widely utilised in mycobacterial research Andreu et al. These reporters mainly exploits the firefly, G. In addition, bacterial luciferases have also been employed and the functional expression of the whole Lux operon in Mtb and M.

Luciferase-based bacterial replication are useful for monitoring Mtb replication in bulk but do not allow for single cell studies in cell-based models of infection Fig. On the other hand, live cell studies allow for spatiotemporal resolution at the single cell level Fig. In some of these studies, that are rather limited, specific cellular environments that are favourable for Mtb replication were defined Lerner et al. In these studies, it was observed in real time that once Mtb induced host cell death, it was able to rapidly grow inside the dead infected macrophage regardless of the differentiation or activation status.

Replication inside dead cells was significantly faster than in the extracellular environment and once host cell death occurred, other macrophages internalized the dead infected cells, and this promptly led to their own death, amplifying the cell death response.

Once there is substantial bacterial replication, correlative approaches were of great advantage to precisely define subcellular compartments permissive for bacterial replication such as necrotic cells Lerner et al. Alternatively, fluorescent reporters and probes can be used in order to monitor Mtb replication in host cells. One strategy used to monitor replication and phenotypic variation at the single-cell rates of rRNA transcription in Mtb is using a destabilised variant of GFP and a plasmid expressing the stable red fluorescent protein DsRed2 Manina, Dhar and McKinney Using this reporter strain, and quantitative time-lapse microscopy combined with a genetically encoded fluorescent reporter rRNA-GFP des it is possible to investigate the single-cell dynamics of Mtb replication and rRNA expression in vitro and in vivo.

These studies revealed a wide range of phenotypic heterogeneity even in bacteria grown under nutrient-rich conditions in vitro and increased heterogeneity in bacteria exposed to diverse stresses. For instance, stationary-phase populations showed a drop in rRNA-GFP des expression followed by the appearance of a subset of non-replicating bacteria displaying high levels of fluorescence Fig.

These observations confirmed that stationary-phase bacteria maintain a high level of de novo protein production, in agreement with studies in other bacteria Rosenberg et al. This reporter has been used mostly in vitro but one study also investigated mycobacteria in infected macrophages in drug screening studies looking for compounds that affect intracellular bacteria growth Sorrentino et al.

One of the caveats of this system is the low fluorescent signal from GFP des and strategies using GFP des in tandem could potentially increase signal to noise levels. Another replication reporter has been developed and used to study the effect of vaccination on the Mtb replication status, at the single bacillus level.

In most of the cases time courses are performed with multiple fixed-samples at pre-defined timepoints. However, it is clear that some of the interactions are transient and dynamic. In recent years, several imaging approaches have been established to study the spatiotemporal detection of Mtb Schnettger et al.

This represents an opportunity to study in more detail the precise roles of the different cellular pathways and their interplay with intracellular organelles for deciding the fate of Mtb in human macrophages.

The precise cellular mechanisms that control Mtb replication in human macrophages, and in particular the mechanisms by which Mtb usurps the anti-bacterial host cell defence system, have not been completely elucidated.

It is likely that the differentially localised populations of intracellular Mtb dynamically interact with cellular organelles during infection Fig. Spatiotemporal interactions between distinct Mtb populations and cellular organelles will dictate whether Mtb replicates, its growth restricted or eventually killed Fig. Given this background, there is a need to functionally define the precise locations where Mtb remains in host cells and their contribution to TB pathogenesis.

In parallel, it will be important to identify the bacterial factors that allow Mtb to survive and eventually replicate in host cells and tissues. Spatiotemporal regulation of Mtb replication or control. In host cells, Mtb initially resides inside phagosomes where bacteria subvert phagosomal function. Some Mtb will effectively be targeted to late phagosomes where bacteria will be exposed to an acidic and proteolytic environment. At some point during the infection cycle, Mtb will damage phagosomes and access the cytosol.

During these events, bacteria-containing compartments and cytosolic bacteria is actively interacting with a plethora of host cell organelles. The interactions between the different Mtb populations and host cell organelles will determine if Mtb replicates, or its growth is restricted or eventually eliminated by the host cell. Outstanding questions are shown emphasizing central themes in the cell biology of Mtb-host cell interactions requiring further research.

The scarcity of knowledge in certain areas of host cell-Mtb interactions is likely a consequence of a lack of technologies that allow spatiotemporal resolution at the single cell level. For example, although the long-standing dogma in the field states that the lysosomal environment is detrimental for Mtb replication, several studies with mycobacteria challenge the notion that lysosomes are the sites where bacterial killing occurs Armstrong and Hart ; Jordao et al.

There is no clear correlation between Mtb delivery to phagolysosomes and control of bacterial replication. A previous study showed that during Mtb replication in human macrophages, the number of bacteria in membrane compartments positive for CD63 which is a frequently used marker of fusion of phagosomes with late endosomes and lysosomes - or LAMPpositive phagosomes increased, suggesting that Mtb was able to replicate within compartments that have acquired the markers expected for lysosomes Welin et al.

Additional reports also showed that a proportion of Mtb was positive for Rab7 and other late endocytic markers Seto et al. However, heterogeneity and lack of single cell studies made it difficult to define where Mtb replicates. For instance, it is not possible to exclude that Mtb replicated in a non-fusogenic phagosome and consequently had been taken up into an autophagosome that has matured. Interestingly, the transmembrane serine protease MarP, which was identified in a screen for acid tolerance determinants, is also required for virulence Vandal et al.

This is consistent with the idea that Mtb experiences acid stress in vivo and that the pathogen possess factors to counteract an acidic environment Vandal et al. From a therapeutic perspective, the intracellular lifestyle of Mtb represents a crucial stage in the disease and successful drug discovery programmes have to include in vitro studies using infected human host cells Young et al.

Conflict of interest. None declared. National Center for Biotechnology Information , U. Published online Mar Claudio Bussi and Maximiliano G Gutierrez.

Author information Article notes Copyright and License information Disclaimer. Received Dec 16; Accepted Mar This article has been cited by other articles in PMC.

Keywords: Mycobacterium tuberculosis , macrophage, phagosome, autophagy, Tuberculosis. Open in a separate window.

Figure 1. Figure 2. In a damaged compartment: causes and consequences In addition to the bacterial Sec and Tat secretion pathways, Mtb possesses the ESX secretion system, a Type VII secretion system T7SS which is found to be non-essential for growth in vitro , but is required for bacterial virulence in vivo Hsu et al.

In the cytosol The emerging concept is that after Mtb-induced membrane damage occurs, there is communication or access to the cytosol and eventually the host cell will try to repair the damage via different mechanisms Fig.

In time The recently formed Mtb phagosome e. Figure 3. Interactions with the endocytic pathways Pathogenic mycobacteria have evolved mechanisms to interfere with both glyco lipid and protein-mediated mechanisms that regulate the trafficking of bacteria to lysosomal organelles for destruction Pieters Interactions with the autophagic pathway Whereas there is a large body of literature linking xenophagy as an anti-mycobacterial pathway Deretic ; Gutierrez et al.

Interactions with other organelles It has been postulated that interactions between Mtb and LD are important for the infection. Challenges to study human macrophage-Mtb interactions in vitro Human macrophages Although Mtb is a human pathogen, the majority of the in vitro host-pathogen studies in TB have been carried out in mouse cells.

Figure 4. Localisation of Mtb within host cells Determining the localisation of Mtb within human macrophages and other host cells is critical to understand how cellular pathways contribute to bacterial control or replication.

Figure 5. Intracellular Mtb viability At present, researchers mostly rely on rates of replication or killing of the mycobacteria at the population level e. Figure 6. Intracellular Mtb replication In the last few years, many groups have developed fluorescence reporters in Mtb to monitor bacterial replication in vitro.

Figure 7. Spatiotemporal studies In most of the cases time courses are performed with multiple fixed-samples at pre-defined timepoints. Concluding remarks: Mtb survival and replication in space and time The precise cellular mechanisms that control Mtb replication in human macrophages, and in particular the mechanisms by which Mtb usurps the anti-bacterial host cell defence system, have not been completely elucidated. Figure 8.

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By identifying this protein we are now able to expose the hiding bacteria, which will allow the macrophages to destroy them. TB is called the ultimate killer. It is the leading cause of death among infectious diseases in the world today and is responsible for one in four adult preventable deaths, according to the World Health Organization WHO.

Ten million new cases of TB arise every year, killing close to two million people worldwide annually. Every 20 seconds TB kills someone and approximately 4, people die every day. The current treatment regime involves taking multiple medications over an extended period of time. Materials provided by University of British Columbia. Once you have inhaled the bacterium, the bacterium lodges in the lung tissue. Healthy individuals may contract latent TB, but the disease may not become active until months or years later, at a time when the immune system becomes weak for some reason.

However, people with weakened immune systems are at a greater risk for developing active TB right away. When they breathe in the bacterium, it settles in their lungs and starts growing because their immune systems cannot fight the infection. In these instances, TB disease may develop within days or weeks after the infection.

When a person gets active TB disease, it means TB bacteria are multiplying and attacking the lung s or other parts of the body, such as the lymph nodes, bones, kidney, brain, spine and even the skin. From the lungs, TB bacteria move through the blood or lymphatic system to different parts of the body.

The chances of getting infected by the TB germ are highest for people that are in close contact with others who are infected. This includes:. People at the highest risk for developing active TB disease are those with a weak immune system, including:.

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