RESUMEN
Bacterial pathogens utilize the factors of their hosts to infect them, but which factors they exploit remain poorly defined. Here, we show that a pathogenic Salmonella enterica serovar Typhimurium (STm) exploits host polyamines for the functional expression of virulence factors. An STm mutant strain lacking principal genes required for polyamine synthesis and transport exhibited impaired infectivity in mice. A polyamine uptake-impaired strain of STm was unable to inject effectors of the type 3 secretion system into host cells due to a failure of needle assembly. STm infection stimulated host polyamine production by increasing arginase expression. The decline in polyamine levels caused by difluoromethylornithine, which inhibits host polyamine production, attenuated STm colonization, whereas polyamine supplementation augmented STm pathogenesis. Our work reveals that host polyamines are a key factor promoting STm infection, and therefore a promising therapeutic target for bacterial infection.
Asunto(s)
Poliaminas , Salmonella typhimurium , Sistemas de Secreción Tipo III , Factores de Virulencia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/genética , Animales , Poliaminas/metabolismo , Ratones , Sistemas de Secreción Tipo III/metabolismo , Sistemas de Secreción Tipo III/genética , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Interacciones Huésped-Patógeno , Humanos , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/microbiología , FemeninoRESUMEN
The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Activación del Canal Iónico , Salmonella/metabolismo , Potenciales de la Membrana , Transporte de ProteínasRESUMEN
PURPOSE: To analyze the fertilization, developmental, and pregnancy potentials in oocytes with narrow perivitelline space. METHODS: Perivitelline space (PVS) of oocytes was evaluated at the time of ICSI, and those without sufficient PVS were judged as oocytes with narrow PVS (NPVS oocytes), and those with sufficient PVS formation were judged as oocytes with non-narrow PVS (non-NPVS oocytes). The analysis included 634 NPVS oocytes from 278 cycles and 12,121 non-NPVS oocytes from 1698 cycles. The fertilization and developmental potentials of NPVS and non-NPVS oocytes were compared by calculating odds ratios using a mixed-effects logistic regression model. We also compared the embryo transfer outcomes of those used for single vitrified-warmed blastocyst transfer after developing into the blastocyst stage. RESULTS: NPVS oocytes had higher odds ratios for degeneration (adjusted odds ratio [aOR], 1.555; 95% confidence interval [CI], 1.096-2.206; p = 0.0133) and 0PN (aOR, 1.387; 95% CI, 1.083-1.775; p = 0.0095), resulting in a lower 2PN rate (aOR, 0.761; 95% CI, 0.623-0.929; p = 0.0072). Even embryos with confirmed 2PN had lower odds ratios for cleavage (aOR, 0.501; 95% CI, 0.294-0.853; p = 0.0109) and blastocyst development (Gardner criteria; CC-AA) rates (aOR, 0.612; 95% CI, 0.476-0.788; p = 0.0001). Blastocysts developed from NPVS oocytes had significantly lower odds ratios for clinical pregnancy (aOR, 0.435; 95% CI, 0.222-0.854; p = 0.0156) than those developed from non-NPVS oocytes. CONCLUSIONS: Oocytes with NPVS have low fertilization and developmental potential, as well as low likelihood of pregnancy.
Asunto(s)
Transferencia de Embrión , Fertilización In Vitro , Metafase , Oocitos , Índice de Embarazo , Inyecciones de Esperma Intracitoplasmáticas , Humanos , Femenino , Embarazo , Oocitos/crecimiento & desarrollo , Adulto , Transferencia de Embrión/métodos , Inyecciones de Esperma Intracitoplasmáticas/métodos , Fertilización In Vitro/métodos , Blastocisto/citología , Fertilización , Desarrollo EmbrionarioRESUMEN
PURPOSE: To determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium could improve embryo-transfer outcomes in frozen-thawed blastocyst transfer. METHODS: Patients who underwent frozen-thawed blastocyst transfer (430 women, aged 30-39 years, 566 cycles) were analyzed. Frozen-thawed blastocysts were cultured in GM-CSF-containing medium or control medium for 3-5 h, followed by transfer to the uterus. The embryo-transfer outcomes in the two groups were measured and compared, and a propensity score matching (1:1) method was used to balance the differences in baseline characteristics. We analyzed 213 matched samples. RESULTS: In patients who underwent frozen-thawed blastocyst transfer with GM-CSF, the percentage of human chorionic gonadotropin-positive cases, biochemical pregnancies, clinical pregnancies, ongoing pregnancies, and live birth rates was 60.6%, 7.98%, 52.6%, 42.9%, and 40.9%, respectively, as compared with 45.1%, 3.29%, 41.8%, 31.1%, and 30.5%, respectively, for the control groups. The rates of human chorionic gonadotropin positivity (odds ratio [OR]: 1.87, 95% confidence interval: [CI]: 1.27-2.75), biochemical pregnancy (2.55, 1.04-6.29), clinical pregnancy (1.54, 1.05-2.27), ongoing pregnancy (1.64, 1.13-2.41), and live birth (1.67, 1.14-2.45) were significantly higher in the GM-CSF group than the control group. The incidence of pregnancy loss (22.3% vs. 27.0%) did not significantly differ between the groups. CONCLUSION: The use of a GM-CSF-containing medium for blastocyst-recovery culture improved the live birth rate as a result of increased implantation rate in the frozen-thawed blastocyst-transfer cycle. The use of GM-CSF-containing medium following blastocyst thawing could be an effective choice for improving the blastocyst-transfer outcomes.
Asunto(s)
Transferencia de Embrión , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Blastocisto , Gonadotropina Coriónica , Criopreservación , Femenino , Humanos , Embarazo , Índice de Embarazo , Estudios RetrospectivosRESUMEN
The bacterial flagellum is a supramolecular motility machine consisting of the basal body, the hook, and the filament. For construction of the flagellum beyond the cellular membranes, a type III protein export apparatus uses ATP and proton-motive force (PMF) across the cytoplasmic membrane as the energy sources to transport flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure. The protein export apparatus consists of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase complex. In addition, the basal body C ring acts as a sorting platform for the cytoplasmic ATPase complex that efficiently brings export substrates and type III export chaperone-substrate complexes from the cytoplasm to the export gate complex. In this book chapter, we will summarize our current understanding of molecular organization and assembly of the flagellar type III protein export apparatus.
Asunto(s)
Sistemas de Secreción Tipo III/biosíntesis , Sistemas de Secreción Tipo III/química , Proteínas Bacterianas , Flagelos , Transporte de Proteínas , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Sistemas de Secreción Tipo III/metabolismoRESUMEN
FlhA and FlhB are transmembrane proteins of the flagellar type III protein export apparatus, and their C-terminal cytoplasmic domains (FlhAC and FlhBC) coordinate flagellar protein export with assembly. FlhBC undergoes autocleavage between Asn-269 and Pro-270 in a well-conserved NPTH loop located between FlhBCN and FlhBCC polypeptides and interacts with the C-terminal domain of the FliK ruler when the length of the hook has reached about 55 nm in Salmonella As a result, the flagellar protein export apparatus switches its substrate specificity, thereby terminating hook assembly and initiating filament assembly. The mechanism of export switching remains unclear. Here, we report the role of FlhBC cleavage in the switching mechanism. Photo-cross-linking experiments revealed that the flhB(N269A) and flhB(P270A) mutations did not affect the binding affinity of FlhBC for FliK. Genetic analysis of the flhB(P270A) mutant revealed that the P270A mutation affects a FliK-dependent conformational change of FlhBC, thereby inhibiting the substrate specificity switching. The flhA(A489E) mutation in FlhAC suppressed the flhB(P270A) mutation, suggesting that an interaction between FlhBC and FlhAC is critical for the export switching. We propose that the interaction between FliKC and a cleaved form of FlhBC promotes a conformational change in FlhBC responsible for the termination of hook-type protein export and a structural remodeling of the FlhAC ring responsible for the initiation of filament-type protein export.IMPORTANCE The flagellar type III protein export apparatus coordinates protein export with assembly, which allows the flagellum to be efficiently built at the cell surface. Hook completion is an important morphological checkpoint for the sequential flagellar assembly process. The protein export apparatus switches its substrate specificity from the hook protein to the filament protein upon hook completion. FliK, FlhB, and FlhA are involved in the export-switching process, but the mechanism remains a mystery. By analyzing a slow-cleaving flhB(P270A) mutant, we provide evidence that an interaction between FliK and FlhB induces conformational rearrangements in FlhB, followed by a structural remodeling of the FlhA ring structure that terminates hook assembly and initiates filament formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/genética , Flagelos/genética , Proteínas de la Membrana/genética , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Especificidad por SustratoRESUMEN
The flagellar protein export apparatus switches its substrate specificity when hook length has reached approximately 55 nm in Salmonella. The C-terminal cytoplasmic domain of FlhB (FlhBC ) is involved in this switching process. FlhBC consists of FlhBCN and FlhBCC polypeptides. FlhBCC has a flexible C-terminal tail (FlhBCCT ). FlhBCC is involved in substrate recognition, and conformational rearrangements of FlhBCN -FlhBCC boundary are postulated to be required for the export switching. However, it remains unknown how it occurs. To clarify this question, we carried out mutational analysis of highly conserved residues in FlhBC . The flhB(E230A) mutation reduced the FlhB function. The flhB(E11S) mutation restored the protein transport activity of the flhB(E230A) mutant to the wild-type level, suggesting that the interaction of FlhBCN with the extreme N-terminal region of FlhB is required for flagellar protein export. The flhB(R320A) mutation affected hydrophobic interaction networks in FlhBCC , thereby increasing insolubility of FlhBC . The R320A mutation also affected the export switching, thereby producing longer hooks with the filament attached. C-terminal truncations of FlhBCCT induced a conformational change of FlhBCN -FlhBCC boundary, resulting in a loose hook length control. We propose that FlhBCCT may control conformational arrangements of FlhBCN -FlhBCC boundary through the hydrophobic interaction networks of FlhBCC .
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de la Membrana/genética , Salmonella typhi/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Transporte Biológico/genética , Análisis Mutacional de ADN/métodos , Flagelos/genética , Flagelos/fisiología , Proteínas de la Membrana/metabolismo , Mutación , Dominios Proteicos , Transporte de Proteínas/genética , Salmonella/genética , Salmonella/metabolismo , Salmonella typhi/metabolismo , Especificidad por SustratoRESUMEN
The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP-FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP-FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Análisis Mutacional de ADN , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Polimerizacion , Salmonella entericaRESUMEN
FliG is a rotor protein of the bacterial flagellar motor. FliG consists of FliGN , FliGM and FliGC domains. Intermolecular FliGM -FliGC interactions promote FliG ring formation on the cytoplasmic face of the MS ring. A conformational change in HelixMC connecting FliGM and FliGC is responsible for the switching between the counterclockwise (CCW) and clockwise (CW) rotational states of the FliG ring. However, it remains unknown how it occurs. Here, we carried out in vivo disulfide cross-linking experiments to see the effect of a CW-locked deletion (∆PAA) in FliG on the FliG ring structure in Salmonella enterica. Higher-order oligomers were observed in the membrane fraction of the fliG(∆PAA + G166C/G194C) strain upon oxidation with iodine in a way similar to FliG(G166C/G194C), indicating that the PAA deletion does not inhibit domain-swap polymerization of FliG. FliG(∆PAA + E174C) formed a cross-linked homodimer whereas FliG(E174C) did not, indicating that Glu174 in HelixMC of one FliG protomer is located much closer to that of its neighboring subunit in the CW motor than in the CCW motor. We will discuss possible helical rearrangements of HelixMC that induce a structural remodeling of the FliG ring upon flagellar motor switching.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Actividad Motora , Salmonella/metabolismo , Eliminación de Secuencia , Proteínas Bacterianas/genética , Reactivos de Enlaces Cruzados/química , Modelos Moleculares , Conformación Proteica , Salmonella/crecimiento & desarrollo , Relación Estructura-ActividadRESUMEN
FliI and FliJ form the FliI6FliJ ATPase complex of the bacterial flagellar export apparatus, a member of the type III secretion system. The FliI6FliJ complex is structurally similar to the α3ß3γ complex of F1-ATPase. The FliH homodimer binds to FliI to connect the ATPase complex to the flagellar base, but the details are unknown. Here we report the structure of the homodimer of a C-terminal fragment of FliH (FliHC2) in complex with FliI. FliHC2 shows an unusually asymmetric homodimeric structure that markedly resembles the peripheral stalk of the A/V-type ATPases. The FliHC2-FliI hexamer model reveals that the C-terminal domains of the FliI ATPase face the cell membrane in a way similar to the F/A/V-type ATPases. We discuss the mechanism of flagellar ATPase complex formation and a common origin shared by the type III secretion system and the F/A/V-type ATPases.
Asunto(s)
Proteínas Bacterianas/química , ATPasas de Translocación de Protón/química , Sistemas de Secreción Tipo III/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Flagelos/química , Flagelos/genética , Flagelos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Homología de Secuencia de Aminoácido , Electricidad Estática , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismoRESUMEN
The bacterial flagellar export switching machinery consists of a ruler protein, FliK, and an export switch protein, FlhB and switches substrate specificity of the flagellar type III export apparatus upon completion of hook assembly. An interaction between the C-terminal domain of FliK (FliKC ) and the C-terminal cytoplasmic domain of FlhB (FlhBC ) is postulated to be responsible for this switch. FliKC has a compactly folded domain termed FliKT3S4 (residues 268-352) and an intrinsically disordered region composed of the last 53 residues, FliKCT (residues 353-405). Residues 301-350 of FliKT3S4 and the last five residues of FliKCT are critical for the switching function of FliK. FliKCT is postulated to regulate the interaction of FliKT3S4 with FlhBC , but it remains unknown how. Here we report the role of FliKCT in the export switching mechanism. Systematic deletion analyses of FliKCT revealed that residues of 351-370 are responsible for efficient switching of substrate specificity of the export apparatus. Suppressor mutant analyses showed that FliKCT coordinates FliKT3S4 action with the switching. Site-directed photo-cross-linking experiments showed that Val-302 and Ile-304 in the hydrophobic core of FliKT3S4 bind to FlhBC . We propose that FliKCT may induce conformational rearrangements of FliKT3S4 to bind to FlhBC .
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Especificidad por Sustrato/genética , Secuencia de Aminoácidos , Transporte Biológico , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/metabolismo , Flagelos/microbiología , Proteínas de la Membrana/metabolismo , Dominios Proteicos , Estructura Terciaria de Proteína , Especificidad por Sustrato/fisiologíaRESUMEN
The bacterial flagellar motor rotates in both counterclockwise (CCW) and clockwise (CW) directions. FliG, FliM and FliN form the C ring on the cytoplasmic face of the MS ring made of a transmembrane protein, FliF. The C ring acts not only as a rotor but also as a switch of the direction of motor rotation. FliG consists of three domains: FliGN, FliGM and FliGC. FliGN directly binds to FliF. Intermolecular interactions between FliGM and FliGC drive FliG ring formation. FliGM is responsible for the interaction with FliM. FliGC is involved in the interaction with the stator protein MotA. Adaptive remodeling of the C ring occurs when the motor switches between the CCW and CW states. However, it remained unknown how. Here, we report the effects of a CW-locked deletion mutation (ΔPEV) in FliG of Thermotaoga maritia (Tm-FliG) on FliG-FliG and FliG-FliM interactions. The PEV deletion stabilized the intramolecular interaction between FliGM and FliGC, thereby suppressing the oligomerization of Tm-FliGMC in solution. This deletion also induced a conformational change of HelixMC connecting FliGM and FliGC to reduce the binding affinity of Tm-FliGMC for FliM. We will discuss adaptive remodeling of the C ring responsible for flagellar motor switching.
Asunto(s)
Proteínas Bacterianas/química , Flagelos/química , Proteínas Motoras Moleculares/química , Movimiento (Física) , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Proteínas Motoras Moleculares/ultraestructura , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The bacterial flagellar type III export chaperones not only act as bodyguards to protect their cognate substrates from aggregation and proteolysis in the cytoplasm but also ensure the order of export through their interactions with an export gate protein FlhA. FlgN chaperone binds to FlgK and FlgL with nanomolar affinity and transfers them to FlhA for their efficient and rapid transport for the formation of the hook-filament junction zone. However, it remains unknown how FlgN releases FlgK and FlgL at the FlhA export gate platform in a timely manner. Here, we have solved the crystal structure of Salmonella FlgN at 2.3 Å resolution and carried out structure-based functional analyses. FlgN consists of three α helices, α1, α2 and α3. Helix α1 adopts two distinct, extended and bent conformations through the conformational change of N-loop between α1 and α2. The N-loop deletion not only increases the probability of FlgN dimer formation but also abolish the interaction between FlgN and FlgK. Highly conserved Asn-92, Asn-95 and Ile-103 residues in helix α3 are involved in the strong interaction with FlgK. We propose that the N-loop coordinates helical rearrangements of FlgN with the association and dissociation of its cognate substrates during their export.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Flagelos/química , Flagelos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Transporte de Proteínas , Salmonella/metabolismo , Análisis de Secuencia de Proteína , Relación Estructura-ActividadRESUMEN
Size and folding of the cerebral cortex increased massively during mammalian evolution leading to the current diversity of brain morphologies. Various subtypes of neural stem and progenitor cells have been proposed to contribute differently in regulating thickness or folding of the cerebral cortex during development, but their specific roles have not been demonstrated. We report that the controlled expansion of unipotent basal progenitors in mouse embryos led to megalencephaly, with increased surface area of the cerebral cortex, but not to cortical folding. In contrast, expansion of multipotent basal progenitors in the naturally gyrencephalic ferret was sufficient to drive the formation of additional folds and fissures. In both models, changes occurred while preserving a structurally normal, six-layered cortex. Our results are the first experimental demonstration of specific and distinct roles for basal progenitor subtypes in regulating cerebral cortex size and folding during development underlying the superior intellectual capability acquired by higher mammals during evolution.
Asunto(s)
Encéfalo/fisiología , Diferenciación Celular , Corteza Cerebral/fisiología , Embrión de Mamíferos/fisiología , Proteínas de Filamentos Intermediarios/fisiología , Proteínas del Tejido Nervioso/fisiología , Células Madre/fisiología , Animales , Encéfalo/citología , Células Cultivadas , Corteza Cerebral/citología , Embrión de Mamíferos/citología , Hurones , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nestina , Células Madre/citologíaRESUMEN
UNLABELLED: Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed. IMPORTANCE: Human mycoplasma pneumonia is caused by a pathogenic bacterium, Mycoplasma pneumoniae This tiny, 2-µm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma "gliding motility" is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is directly involved in the gliding mechanism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycoplasma pneumoniae/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Movimiento , Mycoplasma pneumoniae/genética , Conformación ProteicaRESUMEN
Neurod1 is a transcription factor involved in several developmental programs of the gastrointestinal tract, pancreas, neurosensory, and central nervous system. In the brain, Neurod1 has been shown to be essential for neurogenesis as well as migration, maturation, and survival of newborn neurons during development and adulthood. Interestingly, Neurod1 expression is maintained in a subset of fully mature neurons where its function remains unclear. To study the role of Neurod1, systems are required that allow the temporal and spatial genetic manipulation of Neurod1-expressing cells. To this aim, we have generated four Neurod1-CreER(T2) mouse lines in which CreER(T2) expression, although at different levels, is restricted within areas of physiological Neurod1 expression and Neurod1 positive cells. In particular, the different levels of CreER(T2) expression in different mouse lines offers the opportunity to select the one that is more suited for a given experimental approach. Hence, our Neurod1-CreER(T2) lines provide valuable new tools for the manipulation of newborn neurons during development and adulthood as well as for studying the subpopulation of mature neurons that retain Neurod1 expression throughout life. In this context, we here report that Neurod1 is not only expressed in immature newborn neurons of the adult hippocampus, as already described, but also in fully mature granule cells of the dentate gyrus.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Integrasas/genética , Neurogénesis , Proteínas Recombinantes de Fusión/metabolismo , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Giro Dentado/citología , Giro Dentado/metabolismo , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Integrasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Assembly of the bacterial flagellar filament is strictly sequential; the junction proteins, FlgK and FlgL, are assembled at the distal end of the hook prior to the FliD cap, which supports assembly of as many as 30 000 FliC molecules into the filament. Export of these proteins requires assistance of flagellar chaperones: FlgN for FlgK and FlgL, FliT for FliD and FliS for FliC. The C-terminal cytoplasmic domain of FlhA (FlhAC ), a membrane component of the export apparatus, provides a binding-site for these chaperone-substrate complexes but it remains unknown how it co-ordinates flagellar protein export. Here, we report that the highly conserved hydrophobic dimple of FlhAC is involved in the export of FlgK, FlgL, FliD and FliC but not in proteins responsible for the structure and assembly of the hook, and that the binding affinity of FlhAC for the FlgN/FlgK complex is slightly higher than that for the FliT/FliD complex and about 14-fold higher than that for the FliS/FliC complex, leading to the proposal that the different binding affinities of FlhAC for these chaperone/substrate complexes may confer an advantage for the efficient formation of the junction and cap structures at the tip of the hook prior to filament formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/química , Salmonella typhimurium/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Secuencia Conservada , Chaperonas Moleculares/metabolismo , Unión Proteica , Multimerización de Proteína , Transporte de Proteínas , Salmonella typhimurium/genéticaRESUMEN
The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(ΔPAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(ΔPEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor.
Asunto(s)
Proteínas Bacterianas/química , Flagelos/fisiología , Salmonella enterica/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Flagelos/química , Modelos Genéticos , Datos de Secuencia Molecular , Fenotipo , Mutación Puntual , Estabilidad Proteica , Estructura Terciaria de Proteína , Rotación , Salmonella enterica/química , Salmonella enterica/fisiología , Thermotoga maritima/químicaRESUMEN
The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring and is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly. The highly conserved GYXLI motif (residues 368-372) of FlhAC induces dynamic domain motions of FlhAC required for efficient and robust flagellar protein export by the fT3SS, but it remains unknown whether this motif is also important for ordered protein export by the fT3SS. Here we analyzed two GYXLI mutants, flhA(GAAAA) and flhA(GGGGG), and provide evidence suggesting that the GYXLI motif in FlhAC requires the flagellar ATPase complex not only to efficiently remodel the FlhAC ring structure for the substrate specificity switching but also to correct substrate recognition errors that occur during flagellar assembly.
Asunto(s)
Proteínas Bacterianas , Proteínas de la Membrana , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Salmonella , ATPasas de Translocación de Protón/metabolismoRESUMEN
The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF-FliG fusion proteins and solved the near-atomic resolution structure of the S-ring-the upper part of the MS-ring-using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio, whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared with that of Salmonella, such as tilt angle differences of the RBM3 domain and the ß-collar region, which contribute to the vertical arrangement of the upper part of the ß-collar region despite the diversity in the RBM3 domain angles. Additionally, there is a decrease of the inter-subunit interaction between RBM3 domains, which influences the efficiency of the MS-ring formation in different bacterial species. Furthermore, although the inner-surface electrostatic properties of Vibrio and Salmonella S-rings are altered, the residues potentially interacting with other flagellar components, such as FliE and FlgB, are well structurally conserved in the Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species.IMPORTANCEUnderstanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the Vibrio S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied Salmonella S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the Vibrio and Salmonella S-rings highlighted the species-specific variations and the mechanism for the optimization of MS-ring formation in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to the advancement of bacterial flagellar biology.