RESUMEN
Whole blood based assays, particularly interferon gamma (IFN-γ) release assays (IGRAs), are used for the diagnosis of both bovine and human tuberculosis (TB). The aim of the current study was to evaluate a panel of cytokines and chemokines for potential use as diagnostic readouts indicative of Mycobacterium bovis (M. bovis) infection in cattle. A gene expression assay was used to determine the kinetics of the response to M. bovis purified protein derivative and a fusion protein consisting of ESAT-6, CFP10, and Rv3615c upon aerosol infection with â¼104 cfu of M. bovis. The panel of biomarkers included: IFN-γ, CXCL9, CXCL10, CCL2, CCL3, TNF-α, IL-1α, IL-1ß, IL-1Ra, IL-22, IL-21 and IL-13. Protein levels of IFN-γ, CXCL9, and CXCL10 were determined by ELISA. Findings suggest that CXCL9, CXCL10, IL-21, IL-13, and several acute phase cytokines may be worth pursuing as diagnostic biomarkers of M. bovis infection in cattle.
Asunto(s)
Enfermedades de los Bovinos/diagnóstico , Citocinas/genética , Inmunidad Celular , Tuberculosis Bovina/diagnóstico , Tuberculosis Bovina/inmunología , Animales , Biomarcadores/sangre , Bovinos , Enfermedades de los Bovinos/sangre , Enfermedades de los Bovinos/inmunología , Quimiocina CXCL10/sangre , Quimiocina CXCL9/sangre , Citocinas/inmunología , Expresión Génica , Interferón gamma , Ensayos de Liberación de Interferón gamma , Masculino , Mycobacterium bovis , Tuberculosis Bovina/sangreRESUMEN
Walker, AJ, McFadden, BA, Sanders, DJ, Rabideau, MM, Hofacker, ML, and Arent, SM. Biomarker response to a competitive season in Division I female soccer players. J Strength Cond Res 33(10): 2622-2628, 2019-The purpose of this study was to evaluate effects of training load (TL) on performance and biomarkers of health, performance, and recovery in Division I female soccer players throughout a competitive season. Subjects (N = 25, Mage = 20 ± 1.1 years) were monitored before the start of preseason and every 4-weeks thereafter (T1-T5). A battery of performance tests was administered before the start of preseason (P1) and end-of-season (P2), including body composition (percent body fat [%BF], fat free mass [FFM], and fat mass), vertical jump (VJ), and VO2max. Blood draws were conducted at every time point (T1-T5) to assess free and total cortisol (CORTF and CORTT), prolactin (PRL), T3, IL-6, creatine kinase (CK), sex-hormone binding globulin, omega-3 (n-3FA), vitamin-D (Vit-D), iron (Fe), hematocrit (HcT), ferritin (Fer), percent saturation (%Sat), and total iron-binding capacity (TIBC). Daily exercise energy expenditure (EEE) and TL were determined. There were significant declines in VO2max, VJ, weight, and %BF from P1-P2 (p < 0.05) with no significant differences in FFM. Training load and EEE significantly decreased from T1-T3 (p < 0.05). Significant increases were seen in CORTT, CORTF, PRL, T3, IL-6, CK, and TIBC throughout the season (p < 0.05). Significant decreases were seen in n-3FA, Fe, Fer, %Sat, and Hct throughout the season (p < 0.05). Female athletes experience significant physiological changes following high TL and EEE associated with preseason and appear to be further exacerbated by the cumulative effects of the season. Unique insights provided by biomarkers enable athletes and coaches to be cognizant of the physiological changes that are occurring throughout the season.
Asunto(s)
Rendimiento Atlético/fisiología , Ejercicio Físico/fisiología , Esfuerzo Físico/fisiología , Fútbol/fisiología , Adolescente , Biomarcadores/sangre , Composición Corporal , Peso Corporal , Creatina Quinasa/sangre , Prueba de Esfuerzo , Ácidos Grasos Omega-3/sangre , Femenino , Ferritinas/sangre , Hematócrito , Humanos , Hidrocortisona/sangre , Interleucina-6/sangre , Hierro/sangre , Oxígeno/sangre , Consumo de Oxígeno , Acondicionamiento Físico Humano/fisiología , Prolactina/sangre , Globulina de Unión a Hormona Sexual/metabolismo , Triyodotironina/sangre , Vitamina D/sangre , Adulto JovenRESUMEN
Francisella tularensis is the causative agent of tularemia and a potential biowarfare agent. The virulence of F. tularensis is decreased by deletion of guaB, the gene encoding IMP dehydrogenase (IMPDH), suggesting that this enzyme is a target for antibacterial design. Here we report that F. tularensis growth is blocked by inhibitors of bacterial IMPDHs. Seventeen compounds from two different frameworks, designated the D and Q series, display antibacterial activities with MICs of <1 µM. These compounds are also active against intracellular infections. Surprisingly, antibacterial activity does not correlate with IMPDH inhibition. In addition, the presence of guanine does not affect the antibacterial activity of most compounds, nor does the deletion of guaB These observations suggest that antibacterial activity derives from inhibition of another target(s). Moreover, D compounds display antibacterial activity only against F. tularensis, suggesting the presence of a unique target or uptake mechanism. A ΔguaB mutant resistant to compound D73 contained a missense mutation (Gly45Cys) in nuoB, which encodes a subunit of bacterial complex I. Overexpression of the nuoB mutant conferred resistance to D73 in both wild-type and ΔguaB strains. This strain was not resistant to Q compounds, suggesting that a different off-target mechanism operates for these compounds. Several Q compounds are also effective against Mycobacterium tuberculosis, in which a second target has also been implicated, in addition to IMPDH. The fortuitous presence of multiple targets with overlapping structure-activity relationships presents an intriguing opportunity for the development of robust antibiotics that may avoid the emergence of resistance.
Asunto(s)
Antibacterianos/farmacología , Benzoxazoles/farmacología , Francisella tularensis/efectos de los fármacos , IMP Deshidrogenasa/antagonistas & inhibidores , Ftalazinas/farmacología , Animales , Línea Celular , Complejo I de Transporte de Electrón/genética , Humanos , IMP Deshidrogenasa/genética , Ratones , Pruebas de Sensibilidad Microbiana , Relación Estructura-Actividad , Tularemia/tratamiento farmacológico , Tularemia/microbiologíaRESUMEN
FipB, an essential virulence factor in the highly virulent Schu S4 strain of F. tularensis subsp. tularensis, shares sequence similarity with Disulfide Bond formation (Dsb) proteins, which can have oxidoreductase, isomerase, or chaperone activity. To further explore FipB's role in virulence potential substrates were identified by co-purification and 2D gel electrophoresis, followed by protein sequencing using mass spectrometry. A total of 119 potential substrates were identified. Proteins with predicted enzymatic activity were prevalent, and there were 19 proteins that had been previously identified as impacting virulence. Among the potential substrates were IglC, IglB, and PdpB, three components of the Francisella Type Six Secretion System (T6SS), which is also essential for virulence. T6SS are widespread in Gram-negative pathogens, but have not been reported to be dependent on Dsb-like proteins for assembly or function. The presented results suggest that FipB affects IglB and IglC substrates differently. In a fipB mutant there were differences in free sulfhydryl accessibility of IglC, but not IglB, when compared to wild-type bacteria. However, for both proteins FipB appears to act as a chaperone that facilitates proper folding and conformation. Understanding the role FipB plays the assembly and structure in this T6SS may reveal critical aspects of assembly that are common and novel among this widely distributed class of secretion systems.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Francisella tularensis/patogenicidad , Sistemas de Secreción Tipo VI/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Línea Celular , Francisella tularensis/química , Francisella tularensis/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Análisis de Secuencia de Proteína , Sistemas de Secreción Tipo VI/química , Sistemas de Secreción Tipo VI/genética , Virulencia/genética , Factores de Virulencia/químicaRESUMEN
FipB, an essential virulence factor of Francisella tularensis, is a lipoprotein with two conserved domains that have similarity to disulfide bond formation A (DsbA) proteins and the amino-terminal dimerization domain of macrophage infectivity potentiator (Mip) proteins, which are proteins with peptidyl-prolyl cis/trans isomerase activity. This combination of conserved domains is unusual, so we further characterized the enzymatic activity and the importance of the Mip domain and lipid modification in virulence. Unlike typical DsbA proteins, which are oxidases, FipB exhibited both oxidase and isomerase activities. FipA, which also shares similarity with Mip proteins, potentiated the isomerase activity of FipB in an in vitro assay and within the bacteria, as measured by increased copper sensitivity. To determine the importance of the Mip domain and lipid modification of FipB, mutants producing FipB proteins that lacked either the Mip domain or the critical cysteine necessary for lipid modification were constructed. Both strains replicated within host cells and retained virulence in mice, though there was some attenuation. FipB formed surface-exposed dimers that were sensitive to dithiothreitol (DTT), dependent on the Mip domain and on at least one cysteine in the active site of the DsbA-like domain. However, these dimers were not essential for virulence, because the Mip deletion mutant, which failed to form dimers, was still able to replicate intracellularly and retained virulence in mice. Thus, the Mip domains of FipB and FipA impart additional isomerase functionality to FipB, but only the DsbA-like domain and oxidase activity are essential for its critical virulence functions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Francisella tularensis/metabolismo , Tularemia/microbiología , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cobre , Francisella tularensis/efectos de los fármacos , Francisella tularensis/patogenicidad , Regulación Enzimológica de la Expresión Génica , Isomerasas/genética , Isomerasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Estructura Terciaria de Proteína , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
The lipoprotein encoded by the Francisella tularensis subsp. tularensis locus FTT1103 is essential for virulence; an FTT1103 deletion mutant is defective in uptake and intracellular survival, and mice survive high dose challenges of greater than 10(8) bacteria. This protein has two conserved domains; one is found in a class of virulence proteins called macrophage infectivity potentiator (Mip) proteins, and the other in oxidoreductase Disulfide Bond formation protein A (DsbA)-related proteins. We have designated the protein encoded by FTT1103 as FipB for Francisellainfectivity potentiator protein B. The locus FTT1102 (fipA), which is upstream of fipB, also has similarity to same conserved Mip domain. Deletion and site-specific mutants of fipA and fipB were constructed in the Schu S4 strain, and characterized with respect to intracellular replication and in vivo virulence. A nonpolar fipA mutant demonstrated reduced survival in host cells, but was only slightly attenuated in vivo. Although FipB protein was present in a fipA mutant, the abundance of the three isoforms of FipB was altered, suggesting that FipA has a role in post-translational modification of FipB. Similar to many DsbA homologues, FipB contains a cysteine-any amino acid-any amino acid-cysteine (CXXC) motif. This motif was found to be important for FipB's role in virulence; a deletion mutant complemented with a gene encoding a FipB protein in which the first cysteine was changed to an alanine residue (AXXC) failed to restore intracellular survival or in vivo virulence. Complementation with a gene that encoded a CXXA containing FipB protein was significantly defective in intracellular growth; however, only slightly attenuated in vivo.