The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection.

Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection.

Development of an Adaptive Acquisition and Transmission System for Digital Processing of ECG Signals under Variable n-QAM Schemes / Desarrollo de un Sistema Adaptativo de Adquisición y Transmisión para el Procesamiento Digital de Señales de ECG Bajo Esquemas Variables n-QAM

Abstract In the post-pandemic era, it is critical to monitor and transmit biomedical signals, specifically ECG. This study aims to develop a platform that enables signal acquisition, adaptation, and transmission using different n-QAM modulation schemes. The system comprises an acquisition stage implemented in the 2.5 GHz band employing the Olimex module and electrodes equipped with an Ag/AgCl type sensor. To effectively manage appropriate bandwidths during implementation of the various n-QAM modulation schemes, an adaptive algorithm was developed and applied to the system. The power amplifier was operated in the linear region to enhance the crest factor and achieve an ACPR close to 30 dBc, demonstrating an appropriate demodulation of the electrocardiogram (ECG) signal, it is feasible to shift to modulation schemes above 64-QAM in order to detect high frequencies and perform a subsequent Fourier analysis. As a telemedicine proposal, the developed system offers flexibility in signal acquisition, data storage, and digitalization, in addition to a multivariable n-QAM scheme; the hardware implementation ensures n-QAM scheme compatibility. For the purpose of contributing to telemedicine via RF transmission, the system was executed on an AD9361 transceiver, which removes the requirement for a traditional signal vector generator and enables optimal control of the tones to be transmitted.

Resumen El monitoreo y transmisión de señales biomédicas, particularmente ECG, es fundamental en la era pospandemia, este trabajo de investigación se centra en el desarrollo de una plataforma para la adquisición, adaptación y transmisión de señales bajo diversos esquemas de modulación n-QAM. El sistema incluye una etapa de adquisición mediante el módulo Olimex y electrodos con un sensor tipo Ag/AgCl. Se desarrolló un algoritmo adaptativo a los diversos esquemas de modulación n-QAM para la gestión de anchos de banda apropiados durante una implementación en la banda de 2,5 GHz, al amplificador de potencia se operó en la región lineal para mejorar el factor de cresta y obtener un ACPR cercano a 30 dBc, se realizó una demodulación adecuada de la señal ECG y es posible migrar a esquemas de modulación superiores a 64-QAM si se requiere detectar altas frecuencias y un posterior análisis de Fourier. El sistema desarrollado como propuesta de Telemedicina brinda versatilidad para la adquisición de señales, digitalización, almacenamiento de datos y un esquema multivariable n-QAM, la implementación en hardware proporcionó una adecuada adaptabilidad para esquemas n-QAM. El sistema se implementó sobre un transceptor AD9361 que elimina el tradicional generador vectorial de señal y permite un control óptimo de los tonos a enviar, para aporte en el área de Telemedicina a través de transmisión RF.

Nucleotide channel of RNA-dependent RNA polymerase used for intermolecular uridylylation of protein primer.

Poliovirus VPg is a 22 amino acid residue peptide that serves as the protein primer for replication of the viral RNA genome. VPg is known to bind directly to the viral RNA-dependent RNA polymerase, 3D, for covalent uridylylation, yielding mono and di-uridylylated products, VPg-pU and VPg-pUpU, which are subsequently elongated. To model the docking of the VPg substrate to a putative VPg-binding site on the 3D polymerase molecule, we performed a variety of structure-based computations followed by experimental verification. First, potential VPg folded structures were identified, yielding a suite of predicted beta-hairpin structures. These putative VPg structures were then docked to the region of the polymerase implicated by genetic experiments to bind VPg, using grid-based and fragment-based methods. Residues in VPg predicted to affect binding were identified through molecular dynamics simulations, and their effects on the 3D-VPg interaction were tested computationally and biochemically. Experiments with mutant VPg and mutant polymerase molecules confirmed the predicted binding site for VPg on the back side of the polymerase molecule during the uridylylation reaction, opposite to that predicted to bind elongating RNA primers.

Modulation of the host transcriptome by Coxiella burnetii nuclear effector Cbu1314.

Coxiella burnetii is a Gram-negative, obligate intracellular pathogen that directs the formation of a parasitophorous vacuole derived from the host lysosomal network. Biogenesis and maintenance of this replicative compartment is dependent on bacterial protein synthesis and results in differential expression of specific host genes. However, the mechanisms by which the pathogen induces changes in the host transcriptome is poorly understood. In the current study we identified a Dot/Icm secreted effector, Cbu1314, which encodes two nuclear localization signals that are required for nuclear localization and association with host chromatin. Chromatin immunoprecipitation (ChIP) and deep sequencing revealed that Cbu1314 associated with host genes involved in transcription, cell signaling, and the immune response. RNA sequencing of cells overexpressing Cbu1314 demonstrated that Cbu1314 modulates the host transcriptome and these transcriptional changes required a functional nuclear localization signal. Of the differentially expressed genes, sixteen were also identified as Cbu1314 targets using ChIP sequencing. Collectively these results suggest that Cbu1314 associates with host chromatin and plays a role in modulating the host transcriptome.

Interstitial contacts in an RNA-dependent RNA polymerase lattice.

Catalytic activities can be facilitated by ordered enzymatic arrays that co-localize and orient enzymes and their substrates. The purified RNA-dependent RNA polymerase from poliovirus self-assembles to form two-dimensional lattices, possibly facilitating the assembly of viral RNA replication complexes on the cytoplasmic face of intracellular membranes. Creation of a two-dimensional lattice requires at least two different molecular contacts between polymerase molecules. One set of polymerase contacts, between the "thumb" domain of one polymerase and the back of the "palm" domain of another, has been previously defined. To identify the second interface needed for lattice formation and to test its function in viral RNA synthesis, we used a hybrid approach of electron microscopic and biochemical evaluation of both wild-type and mutant viral polymerases to evaluate computationally generated models of this second interface. A unique solution satisfied all constraints and predicted a two-dimensional structure formed from antiparallel arrays of polymerase fibers that use contacts from the flexible amino-terminal region of the protein. Enzymes that contained mutations in this newly defined interface did not form lattices and altered the structure of wild-type lattices. When reconstructed into virus, mutations that disrupt lattice assembly exhibited growth defects, synthetic lethality or both, supporting the function of the oligomeric lattice in infected cells. Understanding the structure of polymerase lattices within the multimeric RNA-dependent RNA polymerase complex should facilitate antiviral drug design and provide a precedent for other positive-strand RNA viruses.