RESUMO
Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H-NS-like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H-NS, Lsr2 binds DNA in sequence-dependent and non-specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA-binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP-sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Bactérias/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Proteínas de Ligação a DNA/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Regulação Bacteriana da Expressão Gênica/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/fisiologia , Treonina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Chemically-induced dimerization (CID) systems are essential tools to interrogate and control biological systems. AcVHH is a single domain antibody homo-dimerizing upon caffeine binding. AcVHH has a strong potential for clinical applications through caffeine-mediated in vivo control of therapeutic gene networks. Here we provide the structural basis for caffeine-induced homo-dimerization of acVHH.
Assuntos
Anticorpos/química , Cafeína/química , Dimerização , Humanos , Domínios de Imunoglobulina , Modelos Químicos , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Engineered bacteria promise to revolutionize diagnostics and therapeutics, yet many applications are precluded by the limited number of detectable signals. Here we present a general framework to engineer synthetic receptors enabling bacterial cells to respond to novel ligands. These receptors are activated via ligand-induced dimerization of a single-domain antibody fused to monomeric DNA-binding domains (split-DBDs). Using E. coli as a model system, we engineer both transmembrane and cytosolic receptors using a VHH for ligand detection and demonstrate the scalability of our platform by using the DBDs of two different transcriptional regulators. We provide a method to optimize receptor behavior by finely tuning protein expression levels and optimizing interdomain linker regions. Finally, we show that these receptors can be connected to downstream synthetic gene circuits for further signal processing. The general nature of the split-DBD principle and the versatility of antibody-based detection should support the deployment of these receptors into various hosts to detect ligands for which no receptor is found in nature.
Assuntos
Escherichia coli/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Bactérias/genética , Cafeína/farmacologia , Parede Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Expressão Gênica/efeitos dos fármacos , Engenharia Genética , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Serina Endopeptidases/genética , Anticorpos de Domínio Único/genética , Transativadores/química , Transativadores/genéticaRESUMO
Mycobacterium tuberculosis (Mtb) encodes several bacterial effectors impacting the colonization of phagocytes. LppM (Rv2171) is both implicated in phagocytosis and able to efficiently block phagosomal acidification in the macrophage, two key processes contributing to Mtb persistence. We show that LppM is anchored to the mycobacterial cell wall by a C-terminal membrane domain. However, the protein also exists as a truncated protein secreted into the culture medium. The LppM solution structure we solve here displays no similarity with other Mtb lipoproteins also involved in phagosomal maturation (i.e., LprG). In addition, we demonstrate that the protein may be able to bind rare molecular species of phosphatidylinositol mannosides, bacterial compounds known to affect the host immune response. Thus, our data demonstrate a dual localization of LppM and provide a unique perspective on the regulation of protein secretion and localization in Mtb.
