The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth

The receptor-like protein kinase PknB from Mycobacterium tuberculosis is encoded by the distal gene in a highly conserved operon, present in all actinobacteria, that may control cell shape and cell division. Genes coding for a PknB-like protein kinase are also found in many more distantly related gram-positive bacteria. Here, we report that the pknB gene can be disrupted by allelic replacement in M. tuberculosis and the saprophyte Mycobacterium smegmatis only in the presence of a second functional copy of the gene. We also demonstrate that eukaryotic Ser/Thr protein kinase inhibitors, which inactivate PknB in vitro with a 50% inhibitory concentration in the submicromolar range, are able to kill M. tuberculosis H37Rv, M. smegmatis mc(2)155, and Mycobacterium aurum A+ with MICs in the micromolar range. Furthermore, significantly higher concentrations of these compounds are required to inhibit growth of M. smegmatis strains overexpressing PknB, suggesting that this protein kinase is the molecular target. These findings demonstrate that the Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth and support the development of protein kinase inhibitors as new potential antituberculosis drugs.

Single-cell studies of Mycobacterium smegmatis cell cycle using time-lapse fluorescence microscopy

Joëlle Xiao Yuan Ven

Cell division is one of the most primordial cellular processes and is essential for life propagation. It is composed of crucial events, namely genome duplication, chromosomes segregation and membrane separation. Failure of these processes will have dramatic impacts on the development of any organisms, including cell death, polyploidy, or cell cycle arrest. In the case of bacterial pathogens, blocking cell division efficiently using targeted drugs is of interest to cure bacterial infections. However, despite its ancestral origin, the mechanisms of cell division have diverged amongst organisms. Given its crucial role, as well as the potential therapeutic applications, cell division is an intensive field of research and many aspects are yet to be fully understood. Amongst bacteria, the phyla Proteobacteria and Firmicutes have been intensively studied using their respective model organisms Escherichia coli and Bacillus subtilis. However, there is a paucity of research on cell division in Actinobacteria, the phylum that contains the worldâs leading pathogen, Mycobacterium tuberculosis. In particular, the molecular players involved in the process of chromosome segregation and the timing of cell division have not been fully investigated. In the well-studied bacteria E. coli and B. subtilis the Min system determines the division site localization, while the nucleoid occlusion system (Noc or SlmA, respectively) determines the timing of division. In mycobacteria, however, no homologs for the Min or Noc systems have been identified thus far. However, the partitioning (par) genes are currently thought to be responsible for symmetric chromosome segregation. In addition, FtsK has been identified as a homolog of the B. subtilis protein SpoIIIE, which is responsible for rescuing DNA in the forming septum and pulling it in the forespore during starvation-induced sporulation. Overall, the role and dynamics of DNA, where FtsK might play a role, during mycobacterial cell division, still remain to be uncovered. Current methods to study DNA localization using snapshot microscopy typically utilise DNA dyes, such as DAPI, Hoechst, or SYTO. However, these compounds impair cell growth, preventing the dynamic study of mycobacterial division. To circumvent this shortcoming, I constructed a Mycobacterium smegmatis reporter strain expressing a Dendra2 fusion protein in-frame to the histone-like protein Hlp, and compared it to conventional DNA dyes. Additionally, I characterized FtsK and, for the first time, demonstrated its essentiality in M. smegmatis. As FtsK is essential I adapted a dual repression system to knock down FtsK, which proved to be a synthetic rescue with the par genes deletion. Using a custom-made microfluidic device and long-term time-lapse microscopy, the distribution and dynamics of both Hlp and FtsK fluorescent reporter proteins were followed at the single-cell level. This approach allowed the spatial and temporal characterization of these proteins with respect to other septal genes in wild-type cells as well as in Îpar mutants. Finally, I propose a new model where the par genes are important for chromosome orientation and FtsK is important for chromosome segregation. Altogether, this thesis represents a step forward towards a better understanding of the mechanisms underlying cell division in mycobacteria and in the long-term, a potential source of novel treatments to control the division of M. tuberculosis, for instance by targeting FtsK.

EPFL2019

Macro-array and bioinformatic analyses reveal mycobacterial 'core' genes, variation in the ESAT-6 gene family and new phylogenetic markers for the Mycobacterium tuberculosis complex

Stewart Cole

To better understand the biology and the virulence determinants of the two major mycobacterial human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, their genome sequences have been determined recently. In silico comparisons revealed that among the 1439 genes common to both M. tuberculosis and M. leprae, 219 genes code for proteins that show no similarity with proteins from other organisms. Therefore, the latter 'core' genes could be specific for mycobacteria or even for the intracellular mycobacterial pathogens. To obtain more information as to whether these genes really were mycobacteria-specific, they were included in a focused macro-array, which also contained genes from previously defined regions of difference (RD) known to be absent from Mycobacterium bovis BCG relative to M. tuberculosis. Hybridization of DNA from 40 strains of the M. tuberculosis complex and in silico comparison of these genes with the near-complete genome sequences from Mycobacterium avium, Mycobacterium marinum and Mycobacterium smegmatis were undertaken to answer this question. The results showed that among the 219 conserved genes, very few were not present in all the strains tested. Some of these missing genes code for proteins of the ESAT-6 family, a group of highly immunogenic small proteins whose presence and number is variable among the genomically highly conserved members of the M. tuberculosis complex. Indeed, the results suggest that, with few exceptions, the 'core' genes conserved among M. tuberculosis H37Rv and M. leprae are also highly conserved among other mycobacterial strains, which makes them interesting potential targets for developing new specific anti-mycobacterial drugs. In contrast, the genes from RD regions showed great variability among certain members of the M. tuberculosis complex, and some new specific deletions in Mycobacterium canettii, Mycobacterium microti and seal isolates were identified and further characterized during this study. Together with the distribution of a particular 6 or 7 bp micro-deletion in the gene encoding the polyketide synthase pks15/1, these results confirm and further extend the revised phylogenetic model for the M. tuberculosis complex recently presented.

2004

The Phosphate Specific Transport (Pst) System in Fast- and Slow-Growing Mycobacteria

Cyntia De Piano

Although mycobacteria are known to accumulate polyphosphate (PolyP), a linear polymer of orthophosphate, when subjected to stresses, the exact roles of this molecule are not yet clearly understood. We found that deletion of the pstA gene encoding for a transmembrane domain of the high-affinity phosphate-specific ABC transporter system (Pst system) resulted in accumulation of high amounts of PolyP during growth in phosphate (Pi)-replete conditions in Mycobacterium tuberculosis and Mycobacterium smegmatis. These PolyP stores were gradually used during Pi starvation by the ApstA mutants in order to sustain growth and this allowed the mutant strains to reach a higher cell density than the wild-type (WT) parental strain. Neither the mRNA nor the protein levels of polyphosphate kinase 1 (PPK1) could explain the PolyP accumulation observed in the mutant, suggesting that another regulatory mechanism is involved. The ApstA mutant of M. smegmatis was hypersensitive to several in vitro stresses, including detergent, oxidative stress, and cell wall targeting antibiotics. Deletion of the main enzyme involved in PolyP synthesis, PPK1, in the ApstA mutant background of M. smegmatis completely reversed the PolyP accumulation and partially reversed the stress sensitivity as well as the derepression of PHO regulon genes observed in the ApstA mutant. The mechanism of PolyP accumulation in M. smegmatis was investigated using a genetic approach. We found that this mechanism differs from the (p)ppGpp-mediated PolyP accumulation observed in Escherichia coli, since deletion of the RelA enzyme in a ApstA mutant background did not prevent PolyP accumulation. The mechanism of PolyP degradation during Pi starvation in the M. smegmatis ApstA mutant was also analyzed. Deletion of both polyphosphatase enzymes (PPX1 and PPX2) as well as the polyphosphate kinase 2 (PPK2) enzyme did not have any impact on PolyP degradation. PPK1 is a bifunctional enzyme that can either synthesize or degrade PolyP. Our current hypothesis is that PPK1 might perform the reverse reaction (PolyP degradation) during Pi starvation. The aim of the second part of my project was to make a comparative study of the expression and regulation of the phosphate specific transporter (pst) operon in fast-growing [M. smegmatis] and slow-growing [M. tuberculosis] mycobacteria. To achieve this goal, pst reporter strains of M. smegmatis and M. tuberculosis were constructed by precise insertion of a modified dest_gfp gene (provided by Giulia Manina) encoding a destabilized green fluorescent protein (dest_GFP) into the corresponding pst locus as transcriptional ) fusions. The transcriptional reporter construct [p) was introduced in both the WT and the ApstA mutant backgrounds. The kinetics of induction of the pst-gfp reporters in WT and ApstA mutant strains in response to Pi starvation was analyzed. Population-averaged measurements were made using qRT-PCR and Western blotting. Single-cell measurements using flow cytometry and timelapse fluorescence microscopy were also performed. For the microscopy experiments, the bacteria were cultured in custom-made microfluidic devices that permit rapid switching of the culture medium, e.g., switching between Pi-replete and Pi-depleted media. Because these studies require precise environmental control and resolution of individual and temporal phenotypes, my principal experimental tool was the integrated microfluidics and timelapse fluorescence microscopy systems that have recently been developed in the laboratory. Although there have been many studies published on the population-averaged behavior of bacteria responding to external stresses such as Pi limitation, we are not aware of any studies addressing the individuality of such responses using a real-time single-cell approach. We observed that the kinetics of induction of the Pst operon upon Pi starvation scaled with the growth rate of the bacteria and peak induction occurred within about two generations for both smegmatis and tuberculosis. Interestingly, we found that the basal level of Pst expression and the steady state level reached upon induction by Pi starvation were similar in tuberculosis and M. smegmatis, although the kinetics of induction were much faster in M. smegmatis. These studies contributed to improve the understanding of the regulation and the kinetics of response of the PHO regulon in fast- and slow-growing mycobacteria. The finding that PolyP plays a role in this process could also apply to other organisms where the PHO regulon is regulated in a similar way.

EPFL2013