Phage Displayed Short Peptides against Cells of Candida albicans Demonstrate Presence of Species, Morphology and Region Specific Carbohydrate Epitopes

Candida albicans is a commensal opportunistic pathogen, which can cause superficial infections as well as systemic infections in immuocompromised hosts. Among nosocomial fungal infections, infections by C. albicans are associated with highest mortality rates even though incidence of infections by other related species is on the rise world over. Since C. albicans and other Candida species differ in their susceptibility to antifungal drug treatment, it is crucial to accurately identify the species for effective drug treatment. Most diagnostic tests that differentiate between C. albicans and other Candida species are time consuming, as they necessarily involve laboratory culturing. Others, which employ highly sensitive PCR based technologies often, yield false positives which is equally dangerous since that leads to unnecessary antifungal treatment. This is the first report of phage display technology based identification of short peptide sequences that can distinguish C. albicans from other closely related species. The peptides also show high degree of specificity towards its different morphological forms. Using fluorescence microscopy, we show that the peptides bind on the surface of these cells and obtained clones that could even specifically bind to only specific regions of cells indicating restricted distribution of the epitopes. What was peculiar and interesting was that the epitopes were carbohydrate in nature. This gives insight into the complexity of the carbohydrate composition of fungal cell walls. In an ELISA format these peptides allow specific detection of relatively small numbers of C. albicans cells. Hence, if used in combination, such a test could help accurate diagnosis and allow physicians to initiate appropriate drug therapy on time.

Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFN gamma and TNF alpha levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.

Biotechnological potential of plant-associated endophytic fungi:hope versus hype

The potential of endophytes, particularly endophytic fungi, capable of demonstrating desirable functional traits worth exploitation using red biotechnology is well established. However, these discoveries have not yet translated into industrial bioprocesses for commercial production of biopharmaceuticals using fungal endophytes. Here, we define the current challenges in transforming curiosity driven discoveries into industrial scale endophyte biotechnology. The possible practical, feasible, and sustainable strategies that can lead to harnessing fungal endophyte-mediated pharmaceutical products are discussed.

Rethinking production of Taxol (R) (paclitaxel) using endophyte biotechnology

Taxol (R) (generic name paclitaxel) represents one of the most clinically valuable natural products known to mankind in the recent past. More than two decades have elapsed since the notable discovery of the first Taxol (R) producing endophytic fungus, which was followed by a plethora of reports on other endophytes possessing similar biosynthetic potential. However, industrial-scale Taxol (R) production using fungal endophytes, although seemingly promising, has not seen the light of the day. In this opinion article, we embark on the current state of knowledge on Taxol (R) biosynthesis focusing on the chemical ecology of its producers, and ask whether it is actually possible to produce Taxol (R) using endophyte biotechnology. The key problems that have prevented the exploitation of potent endophytic fungi by industrial bioprocesses for sustained production of Taxol (R) are discussed.

Biogenic nanosilver incorporated reverse osmosis membrane for antibacterial and antifungal activities against selected pathogenic strains: An enhanced eco-friendly water disinfection approach

Reverse osmosis (RO) membranes have been used extensively in water desalination plants, waste water treatment in industries, agricultural farms and drinking water production applications. The objective of this work is to impart antibacterial and antifungal activities to commercially available RO membrane used in water purification systems by incorporating biogenic silver nanoparticles (AgNPs) synthesized using Rosa indica wichuriana hybrid leaf extract. The morphology and surface topography of uncoated and AgNPs-coated RO membrane were studied using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Elemental composition of the AgNPs-coated RO membrane was analyzed by energy-dispersive X-ray spectroscopy (EDAX). The functional groups were identified by Fourier Transform Infrared spectroscopy (FT-IR). Hydrophilicity of the uncoated and AgNPs-coated RO membrane was analyzed using water contact angle measurements. The thermal properties were studied by thermogravimetric analysis (TGA). The AgNPs incorporated RO membrane exhibited good antibacterial and antifungal activities against pathogenic bacterial strains such as E. coli, S. aureus, M. luteus, K. pneumoniae, and P. aeruginosa and fungal strains such as Candida tropicalis, C. krusei, C. glabrata, and C. albicans.

Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris

Background: Candida auris is a multidrug resistant, emerging agent of fungemia in humans. Its actual global distribution remains obscure as the current commercial methods of clinical diagnosis misidentify it as C. haemulonii. Here we report the first draft genome of C. auris to explore the genomic basis of virulence and unique differences that could be employed for differential diagnosis. Results: More than 99.5 % of the C. auris genomic reads did not align to the current whole (or draft) genome sequences of Candida albicans, Candida lusitaniae, Candida glabrata and Saccharomyces cerevisiae; thereby indicating its divergence from the active Candida clade. The genome spans around 12.49 Mb with 8527 predicted genes. Functional annotation revealed that among the sequenced Candida species, it is closest to the hemiascomycete species Clavispora lusitaniae. Comparison with the well-studied species Candida albicans showed that it shares significant virulence attributes with other pathogenic Candida species such as oligopeptide transporters, mannosyl transfersases, secreted proteases and genes involved in biofilm formation. We also identified a plethora of transporters belonging to the ABC and major facilitator superfamily along with known MDR transcription factors which explained its high tolerance to antifungal drugs. Conclusions: Our study emphasizes an urgent need for accurate fungal screening methods such as PCR and electrophoretic karyotyping to ensure proper management of fungemia. Our work highlights the potential genetic mechanisms involved in virulence and pathogenicity of an important emerging human pathogen namely C. auris. Owing to its diversity at the genomic scale; we expect the genome sequence to be a useful resource to map species specific differences that will help develop accurate diagnostic markers and better drug targets.

Sonic hedgehog-responsive lipoxygenases and cyclooxygenase-2 modulate Dectin-1-induced inflammatory cytokines

Immune responses during fungal infections are predominately mediated by 5/15-lipoxygenases (LO)-or cyclooxygenase (COX)-2-catalysed bioactive eicosanoid metabolites like leukotrienes, lipoxins and prostaglandins. Although few host mediators of fungi-triggered eicosanoid production have been established, the molecular mechanism of expression and regulation of 5-LO, 15-LO and COX-2 are not well-defined. Here, we demonstrate that, macrophages infected with representative fungi Candida albicans, Aspergillus flavus or Aspergillus fumigatus or those treated with Curdlan, a selective agonist of pattern recognition receptor for fungi Dectin-1, displays increased expression of 5-LO, 15-LO and COX-2. Interestingly, Dectin-1-responsive Syk pathway activates mTOR-sonic hedgehog (SHH) signaling cascade to stimulate the expression of these lipid metabolizing enzymes. Loss-of-function analysis of the identified intermediaries indicates that while Syk-mTOR-SHH pathway-induced 5-LO and 15-LO suppressed the Dectin-l-responsive pro-inflammatory signature cytokines like TNE-alpha, IL-1 beta and IL-12, Syk-mTOR-SHH-induced COX-2 positively regulated these cytokines. Dectin-1-stimulated IL-6, however, is dependent on 5-LO, 15-LO and COX-2 activity. Together, the current study establishes Dectin-1-arbitrated host mediators that direct the differential regulation of immune responses during fungal infections and thus are potential candidates of therapeutic intervention. (C) 2015 Elsevier Ltd. All rights reserved.

An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp-CrP14, obtained from stem tissues, and Talaromyces radicus-CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 mu g/ml and 20 mu g/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus-CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus-CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 mu g/l) in modified M2 medium and of vinblastine (70 mu g/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.

Targeting human telomeric G-quadruplex DNA with curcumin and its synthesized analogues under molecular crowding conditions

The formation of telomeric G-quadruplexes has been shown to inhibit telomerase activity. Indeed, a number of small molecules capable of p-stacking with G-tetrads have shown the ability to inhibit telomerase activity through the stabilization of G-quadruplexes. Curcumin displays a wide spectrum of medicinal properties ranging from anti-bacterial, anti-viral, anti-protozoal, anti-fungal and anti-inflammatory to anti-cancer activity. We have investigated the interactions of curcumin and its structural analogues with the human telomeric sequence AG(3)(T(2)AG(3))(3) under molecular crowding conditions. Experimental studies indicated the existence of a AG(3)(T(2)AG(3))(3)/curcumin complex with binding affinity of 0.72 x 10(6) M-1 under molecular crowding conditions. The results from UV-visible absorption spectroscopy, a fluorescent TO displacement assay, circular dichroism and molecular docking studies, imply that curcumin and their analogues interact with G-quadruplex DNA via groove binding. While other analogs of curcumin studied here bind to G-quadruplexes in a qualitatively similar manner their affinities are relatively lower in comparison to curcumin. The Knoevenagel condensate, a methoxy-benzylidene derivative of curcumin, also exhibited significant binding to G-quadruplex DNA, although with two times decreased affinity. Our study establishes the potential of curcumin as a promising natural product for G-quadruplex specific ligands.

Targeting human telomeric G-quadruplex DNA with curcumin and its synthesized analogues under molecular crowding conditions

The formation of telomeric G-quadruplexes has been shown to inhibit telomerase activity. Indeed, a number of small molecules capable of p-stacking with G-tetrads have shown the ability to inhibit telomerase activity through the stabilization of G-quadruplexes. Curcumin displays a wide spectrum of medicinal properties ranging from anti-bacterial, anti-viral, anti-protozoal, anti-fungal and anti-inflammatory to anti-cancer activity. We have investigated the interactions of curcumin and its structural analogues with the human telomeric sequence AG(3)(T(2)AG(3))(3) under molecular crowding conditions. Experimental studies indicated the existence of a AG(3)(T(2)AG(3))(3)/curcumin complex with binding affinity of 0.72 x 10(6) M-1 under molecular crowding conditions. The results from UV-visible absorption spectroscopy, a fluorescent TO displacement assay, circular dichroism and molecular docking studies, imply that curcumin and their analogues interact with G-quadruplex DNA via groove binding. While other analogs of curcumin studied here bind to G-quadruplexes in a qualitatively similar manner their affinities are relatively lower in comparison to curcumin. The Knoevenagel condensate, a methoxy-benzylidene derivative of curcumin, also exhibited significant binding to G-quadruplex DNA, although with two times decreased affinity. Our study establishes the potential of curcumin as a promising natural product for G-quadruplex specific ligands.

Evolución de la eficiencia reproductiva en la finca piloto San José del municipio de Santo Tomás, Chontales. Area modelo del proyecto de mejoramiento de la productividad ganadera para los productores de pequeña y mediana escala

El presente estudio se realizo con el objetivo de determinar la evolución de la eficiencia reproductiva en la finca piloto San José del municipio de Santo tomas, del departamento de Chontales. Evolución de la Eficiencia Reproductiva en la Finca Piloto San José, en el Municipio de Santo Tomas Chontales. Area modelo del proyecto de Mejoramiento de la Productividad Ganadera para los Productores de Pequeña y Mediana Escala. La finca se sitúa entre las coordenadas 13º28’51’’ latitud norte y 70º77’02’’ longitud este, con altura de 420 msnm, con una precipitación promedio anual de 1600 a 2000 mm, con temperatura media anual de 25º a 27ºC. El presente estudio se evaluaron los diferentes índices reproductivos de la finca piloto San José, haciendo uso de los registros que se levantaron durante la etapa de ejecución del proyecto, realizando monitoreos periódicos como: pesajes de ganado y diagnósticos reproductivos, también se realizaba pesaje de leche y prueba de mastitis, estas actividades se realizan una vez al mes, pero con diferencias de 15 días por actividades. La producción total de leche obtenida en la finca fue de 49.500kg de leche durante un año, cuando el IPP fue de 12 meses. Cuando el IPP llego a los 24 meses la producción de leche fue de 27,000Kg. Se obtuvo que entre menor fueron los IPP y los ingresos de las finca fueron mayores. Cuando se alargaron los dias de ordeño también se alargaron los dias de secado. En el año 2005 el promedio del IPC era de 8.5 meses y en el año 2008 se redujo a 4.7 meses. En el año 2005 el IPP era de 18 meses y para el año 2008 se redujo a 14 meses. Para el invierno del 2005 se tenía promedios de 9 partos en invierno con relación al de verano que fue de 3 partos, luego en el verano del 2008 los partos se redujeron a 4 partos, pero en invierno aumentaron a 15 partos por época. El IPC para el 2005 correspondía a un 22 %, para el año 2008 se logro reducir a un 7.5 %. El IPP en el año 2005 fue del 45.7 % y para el 2008 se redujo a un22.4 %, prácticamente se redujo a un 50 %. En la finca piloto en el 2005 se contaba con 12 animales en ordeño y al año 2008 se incremento su número de animales productivos a 19 animales. La producción promedio por vaca siempre se mantuvo estable entre los 4 y 5 litros de leche por vaca, aumentado solamente la producción total de leche por día.

Diagnóstico zoosanitario del hato lechero en el Centro Integral de Investigación, Innovación, Producción, Extensión y Enseñanza Agropecuaria las Lomas durante el período de Marzo-Junio 2014

El presente estudio se llevó acabo con el objetivo de realizar un diagnóstico zoosanitario del hato lechero en el centro integral de investigación, innovación, producción, extensión, y enseñanza agropecuaria Las Lomas durante el periodo marzo-junio 2014. Los datos generados fueron sometidos a estadística descriptiva utilizando el programa EXCEL, descritos cualitativamente, y a través de porcentajes. El tamaño de la población para este trabajo fue de 318 bovinos de los cuales se seleccionaron en diferentes categorías. En el caso de la triada clínica se obtuvo resultados normales en las distintas categorías del hato con promedios para frecuencia respiratoria por ejemplo en vacas lactando de 29.94, frecuencia respiratoria en vacas horas una media de 64 y para temperatura en terneros de destete una media de 38.53. En los animales que se les tomo muestras para enviarlas al laboratorio se encontró que el 50% de estos resultaron positivos a infección por parásitos de los géneros coccidias spp, 26%, nematodos de la superfamilia strongyloidea, 2% y la superfamilia trichostrongyloidea, 14% y hemoparasitos como babesia ssp con un 8 %, consideramos que no son resultados alarmantes pero con la presencia de ellos se corre el riesgo de que se desarrolle la enfermedad en el resto del hato. En cuanto a los resultados de la prueba de mastitis resultaron positivos 11 vacas que corresponde al 22.44% de las 49 vacas muestreadas, cabemos señalar que no son resultados altos pero que en conjunto con las malas condiciones en las salas de ordeño y las malas prácticas durante el ordeño conllevan a producir una leche de mala calidad, consideramos que el estado general de los animales es bueno ya que a la vez presentaron una condición corporal promedio de 3.5 en las diferentes categorías, y con los datos obtenidos nos dio pauta para la elaboración de un plan de sanidad animal que contribuye a dar un mejor manejo en el hato.

Ocurrencia de Staphilococcus patógenos en leche mamítica en el Departamento de Managua y prueba de sensibilidad a antibióticos

El presente trabajo se realizó en los labortarios de la Escuela Nacional de Agricultura y Ganadería, durante los meses comprendidos entre Junio y Noviembre de 1966. Se examinaron nueve fincas en el Departamento de Managua con un total de 926 vacas en producción lactea. Para la determinacion de mastitis, se uso la prueba de Kloz Gerber y para el aislamiento de staphilococcus patogeno los medios de Hanitol Salt, Agar Sangre, Caldo nutritivo y para la prueba de coagulase el plasma sanguineo. Para la prueba de sensibilidad se usaron los antibioticos siguientes: Oxitetraciclina, CLortetraciclina, Penicilina, Dihidroestreptomicina, Puradantina, Cloranfenicol. De 926 vacas examinadas, 49 (5.29%) presentaron mastitis producidas por staphilococcus patógeno. De las vacas que fueron disgnosticadas con mastitis, el 42.25% fue originada por staphilococcus patogeno. La raza que presento mayor susceptibilidad al staphilococcus patogeno fue la Pardo Suizo y la de menos susceptibilidad, la vaca criolla. La penicila, fue el antibiotico que mostro mayor promedio de zona de inhibicion al staphilococcus patogeno. Los staphilicoccus patogenos aislados de leches de vacas mamiticas, no presentaron en este trabajo, resistencia absoluta a los antibioticos usados.

Edema mamario ante y post parto en vacas lecheras en la Finca San José de las Ramplas, Mateare, Managua, mayo – agosto 2013

Con el objeto de evaluar la prevalencia del edema mamario bovino, se desarrolló el presente estudio de caso en la Finca San José de las Ramplas, municipio de Mateare, Managua. Se analizaron 4 casos positivos a edema de la glándula mamaria ante y post parto, de un total de 34 vacas lactantes en el periodo de mayo-agosto del 2013. El edema mamario consiste en la acumulación excesiva de fluidos en el espacio intercelular que puede generar efectos negativos en el animal, tales como: estrés, dolor, aumento en la susceptibilidad por heridas a causa de la tensión de la piel, mayor probabilidad de sufrir mastitis, daño al pezón, ruptura del ligamento suspensorio de la ubre, disminución de la producción de leche e inconvenientes en el ordeño. El diagnóstico de la alteración se realizó a partir de la inspección de los casos positivos detectados mediante examen clínico y el análisis comparativo con las tablas 1 y 2 de calificación de presencia de edema mamario; se realizaron pruebas complementarias para diagnóstico de mastitis, la anamnesis y conjuntamente el llenado de la hoja clínica; recolectando los datos para analizar las causas y factores predisponentes que conllevaron a esta patología en las vacas; obteniendo una prevalencia de 11.76 % con respecto a la población de la categoría lactante. Esta patología se presenta comúnmente en vaquillas y vacas en el preparto, post parto y primigestas, causada por el cambio brusco en la alimentación, escaso consumo de agua, y primer parto a mayor edad. Para prevenir esta patología, se recomienda conocer el manejo del hato bovino, indagar sobre la correcta alimentación e implementar los programas nutricionales de acuerdo con los recursos que existen en la finca y que suplan las necesidades de cada categoría, implementar un plan sanitario que incluya la suplementación con vitaminas y minerales de acuerdo a su estado productivo y reproductivo para el correcto funcionamiento del bovino según sus necesidades y capacitar al personal de la finca en cuanto a las normas de bioseguridad y manejo adecuado de cada categoría del hato.