Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group have shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-on-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening, that of metabolism and transport (32% of the mutants), and of enveloperelated genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression which in turn underlined the NF- κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS Opolysaccharide and T2SS mutants-induced responses were dependent on TLR2-TLR4- MyD88 activation suggested that LPS Opolysaccharide and PulA perturbed TLRdependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS Opolysaccharide and PulA T2SS could be new targets for designing new antimicrobials. Increasing TLR-governed defence responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of PI 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella containing vacuolae (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not colocalize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the downregulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation which may contribute to Klebsiella pathogenesis.

Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

We describe a protocol for the generation and validation of bacteria microarrays and their application to the study of specific features of the pathogen's surface and interactions with host receptors. Bacteria were directly printed on nitrocellulose-coated glass slides, using either manual or robotic arrayers, and printing quality, immobilization efficiency and stability of the arrays were rigorously controlled by incorporating a fluorescent dye into the bacteria. A panel of wild type and mutant strains of the human pathogen Klebsiella pneumoniae, responsible for nosocomial and community-acquired infections, was selected as model bacteria, and SYTO-13 was used as dye. Fluorescence signals of the printed bacteria were found to exhibit a linear concentration-dependence in the range of 1 x 10(8) to 1 x 10(9) bacteria per ml. Similar results were obtained with Pseudomonas aeruginosa and Acinetobacter baumannii, two other human pathogens. Successful validation of the quality and applicability of the established microarrays was accomplished by testing the capacity of the bacteria array to detect recognition by anti-Klebsiella antibodies and by the complement subcomponent C1q, which binds K. pneumoniae in an antibody-independent manner. The biotin/AlexaFluor-647-streptavidin system was used for monitoring binding, yielding strain-and dose-dependent signals, distinctive for each protein. Furthermore, the potential of the bacteria microarray for investigating specific features, e.g. glycosylation patterns, of the cell surface was confirmed by examining the binding behaviour of a panel of plant lectins with diverse carbohydrate-binding specificities. This and other possible applications of the newly developed arrays, as e.g. screening/evaluation of compounds to identify inhibitors of host-pathogen interactions, make bacteria microarrays a useful and sensitive tool for both basic and applied research in microbiology, biomedicine and biotechnology.

Performance and Fault Tolerance of Preconditioned Iterative Solvers on Low-Power ARM Architectures

As the complexity of computing systems grows, reliability and energy are two crucial challenges asking for holistic solutions. In this paper, we investigate the interplay among concurrency, power dissipation, energy consumption and voltage-frequency scaling for a key numerical kernel for the solution of sparse linear systems. Concretely, we leverage a task-parallel implementation of the Conjugate Gradient method, equipped with an state-of-the-art pre-conditioner embedded in the ILUPACK software, and target a low-power multi core processor from ARM.In addition, we perform a theoretical analysis on the impact of a technique like Near Threshold Voltage Computing (NTVC) from the points of view of increased hardware concurrency and error rate.

Deciphering tissue-induced Klebsiella pneumoniae lipid A structure

The host launches an antimicrobial defense program upon infection. A long-held belief is that pathogens prevent host recognition by remodeling their surface in response to different host microenvironments. Yet direct evidence that this happens in vivo is lacking. Here we report that the pathogen Klebsiella pneumoniae modifies one of its surface molecules, the lipopolysaccharide, in the lungs of mice to evade immune surveillance. These in vivo-induced changes are lost in bacteria grown after isolation from the tissues. These lipopolysaccharide modifications contribute to survival in vivo and mediate resistance to colistin, one of the last options to treat multidrug-resistant Klebsiella. This work opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Distribución de los recursos pesqueros de mayor abundancia a fines del invierno 1998. Crucero BIC Humboldt y BIC Jose Olaya Balandra 9808-09 de Paita a Tacna

Describe la distribución de abundancia de diversos recursos pelágicos y demersales: anchoveta (Engraulis ringens), sardina (Sardinops sagax), jurel (Trachurus picturatus murphyi), caballa (Scomber japonicus peruanus), samasa (Anchoa nasus), Vinciguerria lucetia, falso volador (Prionotus stephanophrys), bagre (Galeichthys peruvianus), pez cinta (Trichiurus lepturus) y múnida (Pleuroncodes monodon). Como plataformas de muestreo en el Cr. 9808-09, se utilizaron: para la zona Paita al Callao: el BIC Humboldt y la lancha IMARPE IV; y para la zona Los Palos (Tacna) al Callao, el BIC José Olaya Balandra y la lancha IMARPE V. Para la distribución de abundancia se utilizaron las ecosondas científicas SIMRAD EK 500 con frecuencias de 38 y 120 kHz de ambos buques, en un rango de detección de 3,5 a 400 m de profundidad, distribuidos en nueve capas de integración. Para el muestreo acústico se utilizó una grilla sistemática paralela con una separación de 12 mn entre cada transecto; se realizaron en total 352 laces de pesca. Las áreas de distribución de cada recurso se determinaron mediante un programa de software de interpolación de datos.

Aspectos biológicos pesqueros de los recursos pelágicos durante el crucero BIC Humboldt y BIC Jose Olaya Balandra 9808-09 entre los palos (Tacna) y Callao

Informe de los resultados de los análisis biológico-pesqueros realizados a las capturas efectuadas por el BIC José Olaya Balandra y la LP IMARPE V, durante el Crucero de Evaluación de Recursos Pelágicos 9808-09, del 20 de agosto al 18 de setiembre 1998, entre la frontera sur y el Callao. Se realizaron 160 lances de comprobación entre ambas embarcaciones. Se obtuvo una captura total de 7.682,6 kg, constituidas por anchoveta (Engraulis ringens) 8,8%; jurel (Trachurus picturatus) 0,1%; caballa (Scomber japonicus) 0,1%. Entre otras especies capturadas tenemos al falso volador (Prionotus stephanophrys) 35,5%; Vinciguerria lucetia 12,1%; samasa (Anchoa nasus) 7,1% y múnida (Pleuroncodes monodon) 10,9%.

Biomasas de las principales especies recursos pesqueros durante el verano 1999. Crucero BIC Jose Olaya Balandra 9902-03, de Tumbes a Tacna

Se describe la biomasa de los principales recursos encontrados durante este crucero de evaluación hidroacústica: anchoveta, sardina, jurel, caballa, samasa, vinciguerria, falso volador, bagre, pez cinta, entre otros.

Distribución de los recursos pesqueros de mayor abundancia en verano 1999. Crucero BIC Jose Olaya Balandra 9902-03, de Tumbes a Tacna

Describe la distribución de los recursos pesqueros: anchoveta (Engraulis ringens), sardina (Sardinops sagax), jurel (Trachurus picturatus murphyi), caballa (Scomber japonicus), samasa (Anchoa nasus), vinciguerria (Vinciguerria lucetia), falso volador (Prionotus stephanophrys), bagre (Galeichthys peruvianus), pota (Dosidicus gigas), múnida (Pleuroncodes monodon) y pez cinta (Trichiurus lepturus), que por su abundancia fueron considerados en esta evaluación. Para la prospección marina de Punta Zorritos (Tumbes) a Morro Sama (Tacna) se utilizó al BIC José Olaya Balandra y para los muestreos costeros a la LP IMARPE V, entre los días 10 de febrero y 1° de abril de 1999. Para la determinación de la distribución se utilizó la ecosonda científica SIMRAD EK 500 de 38 y 120 kHz, en un rango de detección de 2,5 a 250 m de profundidad. Para el muestreo acústico se utilizó una grilla sistemática paralela con una separación de 15 mn entre cada transecto; se realizaron en total 336 lances de pesca para muestreos acústicos y biológicos. Las áreas de distribución de cada especie se determinaron mediante un programa de software de interpolación de datos.

Eficiencia y comportamiento de las redes de arrastre pelágicas en el estudio de recursos pelágicos. Crucero BIC Jose Olaya Balandra y BIC Humboldt 9906

Los resultados del Crucero 9906 de Evaluación Hidroacústica de Recursos Pelágicos a bordo del BIC Humboldt y del BIC José Olaya Balandra en el área cubierta entre Paita (05°00 'S) y Bahía Independencia (14°15°S), del 13 al 28 de junio de 1999, indican que las redes de arrastre pelágicas 124/1800 y 494/800 tuvieron buena eficiencia y comportamiento en 100 lances de comprobación. La captura total de los dos buques fue de 11.243.3 kg, correspondiendo a la anchoveta 8.564,1 kg; sardina 35,49 kg; jurel 22,2 kg; caballa 131 kg; samasa 25,47 kg; vinciguerria 1.242,31 kg y otros recursos 1.213,64 kg. El índice de captura (CPUE) del BIC José Olaya Balandra fue de 748,98 kg/h y del BIC Humboldt 72,31 kg/h. El mayor porcentaje de mezcla de los 100 lances con recursos pelágicos correspondió a la anchoveta con otros recursos a 10 mn de distancia de la costa.

Distribución y concentración de la Merluza (Merluccius gayi peruanus) en enero 1999. Crucero BIC Jose Olaya Balandra 9901

Durante el verano de 1999 la distribución de la merluza (Merluccius gayi peruanus) se presentó muy restringida en relación con el patrón convencionalmente previsible.

Estudio del macrozoobento durante el crucero BIC Jose Olaya Balandra 9901

Se estudiaron muestras obtenidas en cinco estaciones, ubicados en el sublitoral peruano de fonfo blando entre los 8°10' y 4°00' al norte de Salaverry; frete a Pimentel, Paita y Máncora-.

Red de arrastre pelágica 124/1800 y resultados del filtrado de los paneles de malla 150 y 75 mm en la prspección de la vinciguerria Crucero BIC Jose Olaya Balandra 9910, del Callao a Chancay

Se presentaron los resultados de la modificación y adaptaciones de la red pelágica 124/1800 para la captura del recurso Vinciguerria.

Distribución vertical de la vinciguerria, Estrategias de pesca y comportamiento de la red de arrastre pelágica tipo 124/1800. Crucero Bic Jose Olaya Balandra 9910

Durante este estudio se registraron valores de ecointegración y CPUE altos durante la noche, cuando los peces estaban más concentrados cerca de la superficie en una capa de 50 metros de profundidad.