RESUMO
Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Antibacterianos/farmacologia , Microscopia Crioeletrônica , DNA Polimerase Dirigida por DNA , Humanos , Mycobacterium tuberculosis/genética , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
Whole-cell screening of Mycobacterium tuberculosis (Mtb) remains a mainstay of drug discovery, but subsequent target elucidation often proves difficult. Conditional mutants that underexpress essential genes have been used to identify compounds with known mechanism of action by target-based whole-cell screening (TB-WCS). Here, the feasibility of TB-WCS in Mtb was assessed by generating mutants that conditionally express pantothenate synthetase (panC), diaminopimelate decarboxylase (lysA), and isocitrate lyase (icl1). The essentiality of panC and lysA, and conditional essentiality of icl1 for growth on fatty acids, was confirmed. Depletion of PanC and Icl1 rendered mutants hypersensitive to target-specific inhibitors. Stable reporter strains were generated for use in high-throughput screening, and their utility was demonstrated by identifying compounds that display greater potency against a PanC-depleted strain. These findings illustrate the power of TB-WCS as a tool for tuberculosis drug discovery.
Assuntos
Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboxiliases/antagonistas & inibidores , Carboxiliases/genética , Carboxiliases/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Isocitrato Liase/antagonistas & inibidores , Isocitrato Liase/genética , Isocitrato Liase/metabolismo , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.
Assuntos
Antitoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Ribonucleases/metabolismo , Antitoxinas/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Mycobacterium tuberculosis/genética , Ribonucleases/genéticaRESUMO
In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA') and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA' and DnaE2, as well as with the beta-clamp. Moreover, disruption of the ImuB-beta clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA', imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the alpha-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA'-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Mutagênese Insercional/genética , Mycobacterium tuberculosis/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Biocatálise , Domínio Catalítico , Dano ao DNA , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the beta-clamp, consistent with its canonical C-terminal beta-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N(2)-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , 4-Nitroquinolina-1-Óxido/farmacologia , Acroleína/farmacologia , Animais , Proteínas de Bactérias/genética , Benzopirenos/farmacologia , Células Cultivadas , Feminino , Humanos , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Nitrofurazona/farmacologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Quinolonas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-HíbridoRESUMO
Intracellular replication of Salmonella enterica requires the formation of a unique organelle termed Salmonella-containing vacuole (SCV). The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2-T3SS) has a crucial role in the formation and maintenance of the SCV. The SPI2-T3SS translocates a large number of effector proteins that interfere with host cell functions such as microtubule-dependent transport. We investigated the function of the effector SseF and observed that this protein is required to maintain the SCV in a juxtanuclear position in infected epithelial cells. The formation of juxtanuclear clusters of replicating Salmonella required the recruitment of dynein to the SCV but SseF-deficient strains were highly reduced in dynein recruitment to the SCV. We performed a functional dissection of SseF and defined domains that were important for translocation and the specific effector functions of this protein. Of particular importance was a hydrophobic domain in the C-terminal half that contains three putative transmembrane (TM) helices. Deletion of one of these TM helices ablated the effector functions of SseF. We observed that this domain was essential for the proper intracellular positioning of the SCV to a juxtanuclear, Golgi-associated localization. These data show that SseF, in concert with the effector proteins SifA and SseG mediate the precise positioning of the SCV by differentially modulating the recruitment of microtubule motor proteins to the SCV.
Assuntos
Proteínas de Bactérias/fisiologia , Salmonella/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Primers do DNA , Dineínas/metabolismo , Imunofluorescência , Células HeLa , Humanos , Salmonella/crescimento & desenvolvimento , Salmonella/metabolismoRESUMO
Intracellular survival and replication within eukaryotic host cells is of central importance for the pathogenesis of infections caused by Salmonella enterica. Intracellular Salmonella translocates a set of effector proteins by means of a type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI2) that manipulates normal host-cell functions. Intracellular survival and replication is linked to the function of the SPI2-T3SS, but recent observations show that many additional cellular functions are targeted by this virulence system. In this review, we focus on the recent observations on the interference of intracellular Salmonella with functions of the innate and adaptive immune system and the modification of endocytic and exocytic cellular transport. The common molecular basis of the different SPI2-dependent phenotypes could be the interference with cellular transport along microtubules.
Assuntos
Células Dendríticas/imunologia , Salmonella enterica/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Células Dendríticas/microbiologia , Ilhas Genômicas , Humanos , Imunidade Ativa , Imunidade Inata , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologiaRESUMO
During intracellular life, Salmonella enterica proliferate within a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the microtubule cytoskeleton and cellular transport. To characterize the interaction of intracellular Salmonella with host cell transport processes, we utilized various model systems to follow microtubule-dependent transport. The vesicular stomatitis virus glycoprotein (VSVG) is a commonly used marker to follow protein transport from the Golgi to the plasma membrane. Using a VSVG-GFP fusion protein, we observed that virulent intracellular Salmonella alter exocytotic transport and recruit exocytotic transport vesicles to the SCV. This virulence function was dependent on the function of the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) and more specifically on a subset of SPI2 effector proteins. Furthermore, the Golgi to plasma membrane traffic of the shingolipid C(5)-ceramide was redirected to the SCV by virulent Salmonella. We propose that Salmonella modulates the biogenesis of the SCV by deviating this compartment from the default endocytic pathway to an organelle that interacts with the exocytic pathway. This observation might reveal a novel element of the intracellular survival and replication strategy of Salmonella.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ilhas Genômicas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Salmonella enterica/genética , Salmonella enterica/metabolismo , Animais , Transporte Biológico Ativo , Compartimento Celular , Linhagem Celular , Exocitose , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Microtúbulos/metabolismo , Microtúbulos/microbiologia , Modelos Biológicos , Transporte Proteico , Salmonella enterica/patogenicidade , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Vesículas Secretórias/metabolismo , Vesículas Secretórias/microbiologia , Transdução de Sinais , Vacúolos/metabolismo , Vacúolos/microbiologia , Proteínas do Envelope Viral/metabolismoRESUMO
Bacteroides fragilis Bf1 possesses two enzymes having glutamate dehydrogenase (GDH) activity. One is dual cofactor NAD(P)H-dependent, while the other has NADH-specific activity. The gene encoding the NADH-GDH (gdhB) was cloned by complementation of the glutamate auxotrophic mutant Escherichia coli MX3004 and the recombinant protein was characterized with respect to the GDH activities present in the parental organism grown under different nitrogen conditions. The NAD(P)H-dependent GDH of B. fragilis was confirmed to be most active under high ammonia conditions, but the NADH-specific GDH levels were increased by high peptide concentrations in the growth medium and not regulated by the levels of ammonia. Northern blotting analysis showed that gdhB regulation was at the transcription level, with a single transcript of approximately 1.6 kb being produced. GDH activity was demonstrated by zymography of the parental and recombinant enzymes. The recombinant GDH was NADH-specific and co-migrated with the equivalent enzyme band from B. fragilis cell extracts. The gdhB structural gene comprises 1335 bp and encodes a protein of 445 aa (49 kDa). Comparisons of the derived protein sequence with that of GDH from other bacteria indicated that significant sequence homology and conservation of functional domains exists with enzymes of Family I-type hexameric GDH proteins.