RESUMEN
Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine-rich repeats. LrrkA knockout (KO) cells kill ingested Klebsiella pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: Intraphagosomal proteolysis is inefficient, and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA, or far1. Together, these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA, and Kil2. This study is the first identification of a signalling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells.
Asunto(s)
Dictyostelium/enzimología , Ácido Fólico/metabolismo , Fagosomas/microbiología , Fosfotransferasas/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Dictyostelium/genética , Dictyostelium/microbiología , Regulación Bacteriana de la Expresión Génica , Espacio Intracelular/microbiología , Klebsiella pneumoniae/metabolismo , Leucina/química , Fagocitosis , Fosfotransferasas/genética , Dominios Proteicos , Proteínas Protozoarias/genéticaRESUMEN
Bacterial sensing, ingestion, and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. The cellular mechanisms involved in intracellular killing, their relative importance, and their specificity towards different bacteria are however poorly defined. In this study, we used Dictyostelium discoideum, a phagocytic cell model amenable to genetic analysis, to identify new gene products involved in intracellular killing. A random genetic screen led us to identify the role of Vps13F in intracellular killing of Klebsiella pneumoniae. Vps13F knock-out (KO) cells exhibited a delayed intracellular killing of K. pneumoniae, although the general organization of the phagocytic and endocytic pathway appeared largely unaffected. Transcriptomic analysis revealed that vps13F KO cells may be functionally similar to previously characterized fspA KO cells, shown to be defective in folate sensing. Indeed, vps13F KO cells showed a decreased chemokinetic response to various stimulants, suggesting a direct or indirect role of Vps13F in intracellular signaling. Overstimulation with excess folate restored efficient killing in vps13F KO cells. Finally, genetic inactivation of Far1, the folate receptor, resulted in inefficient intracellular killing of K. pneumoniae. Together, these observations show that stimulation of Dictyostelium by bacterial folate is necessary for rapid intracellular killing of K. pneumoniae.
Asunto(s)
Dictyostelium/microbiología , Dictyostelium/fisiología , Ácido Fólico/metabolismo , Klebsiella pneumoniae/fisiología , Fagocitosis/genética , Proteínas Protozoarias/genética , Receptor 1 de Folato/genética , Técnicas de Inactivación de Genes , Fagocitosis/fisiología , Transducción de Señal/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Mycobacterium tuberculosis bacilli display two signature features: acid-fast staining and the capacity to induce long-term latent infections in humans. However, the mechanisms governing these two important processes remain largely unknown. Ser/Thr phosphorylation has recently emerged as an important regulatory mechanism allowing mycobacteria to adapt their cell wall structure/composition in response to their environment. Herein, we evaluated whether phosphorylation of KasB, a crucial mycolic acid biosynthetic enzyme, could modulate acid-fast staining and virulence. Tandem mass spectrometry and site-directed mutagenesis revealed that phosphorylation of KasB occurred at Thr334 and Thr336 both in vitro and in mycobacteria. Isogenic strains of M. tuberculosis with either a deletion of the kasB gene or a kasB_T334D/T336D allele, mimicking constitutive phosphorylation of KasB, were constructed by specialized linkage transduction. Biochemical and structural analyses comparing these mutants to the parental strain revealed that both mutant strains had mycolic acids that were shortened by 4-6 carbon atoms and lacked trans-cyclopropanation. Together, these results suggested that in M. tuberculosis, phosphorylation profoundly decreases the condensing activity of KasB. Structural/modeling analyses reveal that Thr334 and Thr336 are located in the vicinity of the catalytic triad, which indicates that phosphorylation of these amino acids would result in loss of enzyme activity. Importantly, the kasB_T334D/T336D phosphomimetic and deletion alleles, in contrast to the kasB_T334A/T336A phosphoablative allele, completely lost acid-fast staining. Moreover, assessing the virulence of these strains indicated that the KasB phosphomimetic mutant was attenuated in both immunodeficient and immunocompetent mice following aerosol infection. This attenuation was characterized by the absence of lung pathology. Overall, these results highlight for the first time the role of Ser/Thr kinase-dependent KasB phosphorylation in regulating the later stages of mycolic acid elongation, with important consequences in terms of acid-fast staining and pathogenicity.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Ácidos Micólicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Animales , Proteínas Bacterianas/genética , Dominio Catalítico/genética , Pared Celular/metabolismo , Metabolismo de los Lípidos/genética , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Técnicas Microbiológicas/métodos , Modelos Moleculares , Mycobacterium tuberculosis/genética , Ácidos Micólicos/química , Fosforilación , Coloración y Etiquetado/métodos , Tuberculosis/diagnóstico , Tuberculosis/metabolismo , Tuberculosis/microbiología , VirulenciaRESUMEN
GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette-Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.
Asunto(s)
Antituberculosos , Proteínas Bacterianas/metabolismo , Glucosiltransferasas/metabolismo , Mycobacterium tuberculosis/enzimología , Proteínas Bacterianas/genética , Catálisis , Prueba de Complementación Genética , Glucosiltransferasas/genética , Mycobacterium bovis/enzimología , Mycobacterium bovis/genética , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Streptomyces coelicolor/enzimología , Streptomyces coelicolor/genéticaRESUMEN
Recent efforts have underlined the role of Serine/Threonine Protein Kinases (STPKs) in growth, pathogenesis and cell wall metabolism in mycobacteria. Herein, we demonstrated that the Mycobacterium tuberculosis EthR, a transcriptional repressor that regulates the activation process of the antitubercular drug ethionamide (ETH) is a specific substrate of the mycobacterial kinase PknF. ETH is a prodrug that must undergo bioactivation by the monooxygenease EthA to exert its antimycobacterial activity and previous studies reported that EthR represses transcription of ethA by binding to the ethA-ethR intergenic region. Mass spectrometry analyses and site-directed mutagenesis identified a set of four phosphoacceptors, namely Thr2, Thr3, Ser4 and Ser7. This was further supported by the complete loss of PknF-dependent phosphorylation of a phosphoablative EthR mutant protein. Importantly, a phosphomimetic version of EthR, in which all phosphosites were replaced by Asp residues, exhibited markedly decreased DNA-binding activity compared with the wild-type protein. Together, these findings are the first demonstration of EthR phosphorylation and indicate that phosphorylation negatively affects its DNA-binding activity, which may impact ETH resistance levels in M. tb.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Antituberculosos/metabolismo , Proteínas Bacterianas/genética , Etionamida/metabolismo , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/genética , Fosforilación , Proteínas Represoras/química , Proteínas Represoras/genética , Serina/metabolismo , Treonina/metabolismo , Tuberculosis/microbiologíaRESUMEN
Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Salmonella Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high resolution. While macrophages polarize toward M1-like phenotype to control early infection, during later stages, Salmonella persists inside non-inflammatory clustered macrophages. Transcriptomic profiling of macrophages showed a highly dynamic signature during infection characterized by a switch from pro-inflammatory to anti-inflammatory/pro-regenerative status and revealed a shift in adhesion program. In agreement with this specific adhesion signature, macrophage trajectory tracking identifies motionless macrophages as a permissive niche for persistent Salmonella. Our results demonstrate that zebrafish model provides a unique platform to explore, in a whole organism, the versatile nature of macrophage functional programs during bacterial acute and persistent infections.
Asunto(s)
Interacciones Huésped-Patógeno , Pez Cebra , Animales , Macrófagos/microbiología , Salmonella typhimurium , FenotipoRESUMEN
The Staphylococcus aureus serine/threonine protein kinase Stk1 (also known as PknB) affects different key pathways such as cell wall metabolism, antibiotic susceptibility, and regulation of virulence. Here we report that the catabolite control protein A (CcpA), a highly conserved regulator of carbon catabolite repression and virulence in a number of gram-positive pathogens, was efficiently phosphorylated in vitro and in vivo by Stk1 in S. aureus, whereas the CcpA homologues of Bacillus subtilis and Bacillus anthracis were not affected by the Stk1 orthologue PrkC. Mass spectrometry and mutational analyses identified Thr-18 and Thr-33 as the phosphoacceptors; both are located in the DNA binding domain of this protein. Electrophoretic mobility shift assays demonstrated that the CcpA DNA binding activity was completely abrogated for the phosphorylated CcpA. The physiological relevance of CcpA phosphorylation was assessed by generating CcpA phosphoablative (T18A/T33A) or phosphomimetic (T18D/T33D) mutants. In contrast to the wild-type and phosphoablative ccpA alleles, introduction of the phosphomimetic ccpA allele in a ΔccpA mutant failed to restore the parental biofilm formation profile and the transcription of citZ and hla to levels seen with the wild type. The strong up regulation of ccpA transcripts and CcpA level in the ccpA mutant trans-complemented with the phosphomimetic CcpA variant suggest furthermore that CcpA acts as a negative regulator of its own expression. Together, these findings demonstrate that Stk1-driven phosphorylation of CcpA inhibits its DNA binding activity toward its regulon in S. aureus, representing a novel regulatory mechanism of CcpA activity in addition to the well known regulation via HprKP/Hpr in this clinically important pathogen.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/fisiología , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Represoras/biosíntesis , Staphylococcus aureus/fisiología , Transcripción Genética/fisiología , Factores de Virulencia/biosíntesis , Sustitución de Aminoácidos , Bacillus subtilis/fisiología , Proteínas Bacterianas/genética , Escherichia coli/fisiología , Mutación Missense , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/genética , Factores de Virulencia/genéticaRESUMEN
Pathogenic mycobacteria survive within macrophages by residing in phagosomes, which they prevent from maturing and fusing with lysosomes. Although several bacterial components were seen to modulate phagosome processing, the molecular regulatory mechanisms taking part in this process remain elusive. We investigated whether the phagosome maturation block (PMB) could be modulated by signaling through Ser/Thr phosphorylation. Here, we demonstrated that mycolic acid cyclopropane synthase PcaA, but not MmaA2, was phosphorylated by mycobacterial Ser/Thr kinases at Thr-168 and Thr-183 both in vitro and in mycobacteria. Phosphorylation of PcaA was associated with a significant decrease in the methyltransferase activity, in agreement with the strategic structural localization of these two phosphoacceptors. Using a BCG ΔpcaA mutant, we showed that PcaA was required for intracellular survival and prevention of phagosome maturation in human monocyte-derived macrophages. The physiological relevance of PcaA phosphorylation was further assessed by generating PcaA phosphoablative (T168A/T183A) or phosphomimetic (T168D/T183D) mutants. In contrast to the wild-type and phosphoablative pcaA alleles, introduction of the phosphomimetic pcaA allele in the ΔpcaA mutant failed to restore the parental mycolic acid profile and cording morphotype. Importantly, the PcaA phosphomimetic strain, as the ΔpcaA mutant, exhibited reduced survival in human macrophages and was unable to prevent phagosome maturation. Our results add new insight into the importance of mycolic acid cyclopropane rings in the PMB and provide the first evidence of a Ser/Thr kinase-dependent mechanism for modulating mycolic acid composition and PMB.
Asunto(s)
Proteínas Bacterianas/metabolismo , Metiltransferasas/metabolismo , Mycobacterium tuberculosis/enzimología , Ácidos Micólicos/metabolismo , Fagosomas , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia Conservada , Ciclopropanos/metabolismo , Interacciones Huésped-Patógeno , Humanos , Macrófagos/microbiología , Metiltransferasas/química , Metiltransferasas/genética , Viabilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mycobacterium bovis/enzimología , Mycobacterium bovis/metabolismo , Mycobacterium bovis/fisiología , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/químicaRESUMEN
S-Adenosylhomocysteine hydrolase (SahH) is known as an ubiquitous player in methylation-based process that maintains the intracellular S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) equilibrium. Given its crucial role in central metabolism in both eukaryotes and prokaryotes, it is assumed that SahH must be regulated, albeit little is known regarding molecular mechanisms governing its activity. We report here that SahH from Mycobacterium tuberculosis can be phosphorylated by mycobacterial Ser/Thr protein kinases and that phosphorylation negatively affects its enzymatic activity. Mass spectrometric analyses and site-directed mutagenesis identified Thr2 and Thr221 as the two phosphoacceptors. SahH_T2D, SahH_T221D and SahH_T2D/T221D, designed to mimic constitutive phosphorylation, exhibited markedly decreased activity compared to the wild-type enzyme. Both residues are fully conserved in other mycobacterial SahH orthologues, suggesting that SahH phosphorylation on Thr2 and Thr221 may represent a novel and presumably more general mechanism of regulation of the SAH/SAM balance in mycobacteria.
Asunto(s)
Adenosilhomocisteinasa/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , Procesamiento Proteico-Postraduccional , Serina/metabolismo , Treonina/metabolismo , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Serina/química , Serina/genética , Treonina/química , Treonina/genéticaRESUMEN
Inflammation is a hallmark of the physiological response to aggressions. It is orchestrated by a plethora of molecules that detect the danger, signal intracellularly, and activate immune mechanisms to fight the threat. Understanding these processes at a level that allows to modulate their fate in a pathological context strongly relies on in vivo studies, as these can capture the complexity of the whole process and integrate the intricate interplay between the cellular and molecular actors of inflammation. Over the years, zebrafish has proven to be a well-recognized model to study immune responses linked to human physiopathology. We here provide a systematic review of the molecular effectors of inflammation known in this vertebrate and recapitulate their modes of action, as inferred from sterile or infection-based inflammatory models. We present a comprehensive analysis of their sequence, expression, and tissue distribution and summarize the tools that have been developed to study their function. We further highlight how these tools helped gain insights into the mechanisms of immune cell activation, induction, or resolution of inflammation, by uncovering downstream receptors and signaling pathways. These progresses pave the way for more refined models of inflammation, mimicking human diseases and enabling drug development using zebrafish models.
RESUMEN
One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/metabolismo , Serina/metabolismo , Treonina/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Femenino , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Mycobacterium tuberculosis/citología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Operón/genética , Fosforilación , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Corynebacteria grow by wall extension at the cell poles, with DivIVA being an essential protein orchestrating cell elongation and morphogenesis. DivIVA is considered a scaffolding protein able to recruit other proteins and enzymes involved in polar peptidoglycan biosynthesis. Partial depletion of DivIVA induced overexpression of cg3264, a previously uncharacterized gene that encodes a novel coiled coil-rich protein specific for corynebacteria and a few other actinomycetes. By partial depletion and overexpression of Cg3264, we demonstrated that this protein is an essential cytoskeletal element needed for maintenance of the rod-shaped morphology of Corynebacterium glutamicum, and it was therefore renamed RsmP (rod-shaped morphology protein). RsmP forms long polymers in vitro in the absence of any cofactors, thus resembling eukaryotic intermediate filaments. We also investigated whether RsmP could be regulated post-translationally by phosphorylation, like eukaryotic intermediate filaments. RsmP was phosphorylated in vitro by the PknA protein kinase and to a lesser extent by PknL. A mass spectrometric analysis indicated that phosphorylation exclusively occurred on a serine (Ser-6) and two threonine (Thr-168 and Thr-211) residues. We confirmed that mutagenesis to alanine (phosphoablative protein) totally abolished PknA-dependent phosphorylation of RsmP. Interestingly, when the three residues were converted to aspartic acid, the phosphomimetic protein accumulated at the cell poles instead of making filaments along the cell, as observed for the native or phosphoablative RsmP proteins, indicating that phosphorylation of RsmP is necessary for directing cell growth at the cell poles.
Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/metabolismo , Corynebacterium glutamicum/ultraestructura , Proteínas del Citoesqueleto/metabolismo , Proteínas Bacterianas/genética , Corynebacterium glutamicum/genética , Proteínas del Citoesqueleto/genética , Electroforesis en Gel Bidimensional , Evolución Molecular , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano/genética , Microscopía Electrónica de Transmisión , Mutagénesis Sitio-Dirigida , Fosforilación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition.
Asunto(s)
Acido Graso Sintasa Tipo II/metabolismo , Hidroliasas/metabolismo , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Regulación hacia Abajo , Acido Graso Sintasa Tipo II/genética , Hidroliasas/genética , Datos de Secuencia Molecular , Fosforilación , Serina/metabolismo , Treonina/metabolismoRESUMEN
LrrkA is a Dictyostelium discoideum kinase with leucine-rich repeats. LrrkA stimulates Kil2 and intra-phagosomal killing of ingested bacteria in response to folate. In this study, we show that genetic inactivation of lrrkA also causes a previously unnoticed phenotype: lrrkA KO cells exhibit enhanced phagocytosis and cell motility compared to parental cells. This phenotype is cell autonomous, is reversible upon re-expression of LrrkA, and is not due to an abnormal response to inhibitory quorum-sensing factors secreted by D. discoideum in its medium. In addition, folate increases motility in parental D. discoideum cells, but not in lrrkA KO cells, suggesting that LrrkA plays a pivotal role in the cellular response to folate. On the contrary, lrrkA KO cells regulate gene transcription in response to folate in a manner indistinguishable from parental cells. Overall, based on analysis of mutant phenotypes, we identify gene products that participate in the control of intracellular killing, cell motility, and gene transcription in response to folate. These observations reveal a mechanism by which D. discoideum encountering bacterially-secreted folate can migrate, engulf, and kill bacteria more efficiently.
RESUMEN
Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.
Asunto(s)
Fragmentos de Péptidos/química , Fosfoproteínas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Fragmentos de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tripsina/metabolismoRESUMEN
Dictyostelium discoideum has been used largely as a model organism to study the organization and function of the endocytic pathway. Here we describe dense structures present in D. discoideum endocytic compartments, which we named pycnosomes. Pycnosomes are constitutively secreted in the extracellular medium, from which they can be recovered by differential centrifugation. We identified the most abundant protein present in secreted pycnosomes, that we designated SctA. SctA defines a new family of proteins with four members in D. discoideum, and homologous proteins in other protists and eumetazoa. We developed a monoclonal antibody specific for SctA and used it to further characterize secreted and intracellular pycnosomes. Within cells, immunofluorescence as well as electron microscopy identified pycnosomes as SctA-enriched dense structures in the lumen of endocytic compartments. Pycnosomes are occasionally seen in continuity with intra-endosomal membranes, particularly in U18666A-treated cells where intraluminal budding is highly enhanced. While the exact nature, origin and cellular function of pycnosomes remain to be established, this study provides a first description of these structures as well as a characterization of reagents that can be used for further studies.
Asunto(s)
Dictyostelium/metabolismo , Endosomas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Transporte Biológico , Dictyostelium/inmunología , Dictyostelium/ultraestructura , Endocitosis , Endosomas/inmunología , Endosomas/ultraestructura , Membranas Intracelulares/metabolismo , Ratones , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismoRESUMEN
The protein Rv0020c from Mycobacterium tuberculosis, also called FhaA, is one of the major substrates of the essential Ser/Thr protein kinase (STPK) PknB. The protein is composed of three domains and is phosphorylated on a unique site in its N terminus. We solved the solution structure of both N- and C-terminal domains and demonstrated that the approximately 300 amino acids of the intermediate domain are not folded. We present evidence that the FHA, a phosphospecific binding domain, of Rv0020c does not interact with the phosphorylated catalytic domains of PknB, but with the phosphorylated juxtamembrane domain that links the catalytic domain to the mycobacterial membrane. We also demonstrated that the degree and the pattern of phosphorylation of this juxtamembrane domain modulates the affinity of the substrate (Rv0020c) toward its kinase (PknB).