Cell aggregation: a mechanism of pathogenic Leptospira to survive in fresh water

Transmission of leptospirosis is facilitated by the survival of pathogenic leptospires in moist environments outside their mammalian host. In the present study, the survival mechanisms of Leptospira interrogans serovar Canicola in aqueous conditions and lack of nutrients were investigated. In distilled water, leptospires were able to remain motile for 110 days (pH 7.2). However, when incubated in a semi-solid medium composed of distilled water and 0.5% purified agarose (pH 7.2), they survived 347 days. In this viscous environment, aggregates of live spirochetes were observed. Neither antibiotics (e.g. tetracycline and ampicillin) nor nutrients inhibited leptospiral aggregation. Immunoblot analysis suggested that cells incubated in water down-regulate the expression of LipL31, an inner-membrane protein, but retain expression of other membrane proteins. These studies provide insights into the mechanisms by which pathogenic Leptospira survives for prolonged periods of time in natural aqueous environments, a key stage in the leptospiral lifecycle. [Int Microbiol 2004; 7(1):35–39

Fruit calcium: transport and physiology

Published: 29 April 2016Calcium has well-documented roles in plant signaling, water relations and cell wall interactions. Significant research into how calcium impacts these individual processes in various tissues has been carried out; however, the influence of calcium on fruit ripening has not been thoroughly explored. Here, we review the current state of knowledge on how calcium may impact the development, physical traits and disease susceptibility of fruit through facilitating developmental and stress response signaling, stabilizing membranes, influencing water relations and modifying cell wall properties through cross-linking of de-esterified pectins. We explore the involvement of calcium in hormone signaling integral to the physiological mechanisms behind common disorders that have been associated with fruit calcium deficiency (e.g., blossom end rot in tomatoes or bitter pit in apples). This review works toward an improved understanding of how the many roles of calcium interact to influence fruit ripening, and proposes future research directions to fill knowledge gaps. Specifically, we focus mostly on grapes and present a model that integrates existing knowledge around these various functions of calcium in fruit, which provides a basis for understanding the physiological impacts of sub-optimal calcium nutrition in grapes. Calcium accumulation and distribution in fruit is shown to be highly dependent on water delivery and cell wall interactions in the apoplasm. Localized calcium deficiencies observed in particular species or varieties can result from differences in xylem morphology, fruit water relations and pectin composition, and can cause leaky membranes, irregular cell wall softening, impaired hormonal signaling and aberrant fruit development. We propose that the role of apoplasmic calcium-pectin crosslinking, particularly in the xylem, is an understudied area that may have a key influence on fruit water relations. Furthermore, we believe that improved knowledge of the calcium-regulated signaling pathways that control ripening would assist in addressing calcium deficiency disorders and improving fruit pathogen resistance.Bradleigh Hocking, Stephen D. Tyerman, Rachel A. Burton and Matthew Gilliha

The molecular and structural characterization of the PTS1 glycosomal protein import pathway in Leishmania donovani /

In Leishmania glycosomes compartmentalize a variety of essential biochemical and metabolic pathways required for parasite viability. Biogenesis and maintenance of glycosomes involves a family of proteins called peroxins, however the molecular mechanisms governing the early events of this pathway have not been fully established.A structural-functional analysis of the receptor LdPEX5 revealed the formation of a tetrameric structure stabilized by coiled-coil motifs. A biophysical approach showed that the tetrameric structure of LdPEX5 dissociates to dimers upon binding to the PTS1 ligand. However, the tetrameric LdPEX5 is more thermodynamically favorable to bind. Lastly, LdPEX14 modulates the LdPEX5-PTS1 interaction as the presence of LdPEX14 decreases the Kd of LdPEX5-PTS1 by ∼10 folds.PTS1-loaded LdPEX5 docks onto the glycosomal membrane via the membrane-associated LdPEX14, an interaction that was characterized by molecular mapping and biophysical approaches. In higher eukaryotes this PEX5-PEX14 interaction involves conserved WXXXF/Y pentapeptide motifs found on PEX5 and a signature sequence found on PEX14. These three repeats in LdPEX5 do not appear to be crucial for interaction with LdPEX14 thus suggesting a unique molecular mechanism mediating the docking event. On the other hand, the conserved signature motif is crucial for interaction with LdPEX5.The topology and nature of the interaction of LdPEX14 with the glycosomal membrane is not clear. In vivo expression of FLAG-LdPEX14-HA together with proteinase digestion confirmed that both N- and C-termini are cytosolic facing. Mapping analysis revealed that the first 63 N-terminal residues of LdPEX14 are critical for anchoring LdPEX14 to the glycosomal membrane. Interestingly, expression of the first 74 amino acids of LdPEX14 is toxic to the parasites.Finally, the structure of LdPEX14 on the glycosomal membrane was addressed by molecular mapping and biophysical techniques. Partial trypsin digestion of recombinant LdPEX14 and molecular mapping suggested that the first 250 residues of LdPEX14 were involved in the formation of the complex. Biophysical approaches and cross-linking assays suggest that this complex may consist of ∼12-100 LdPEX14 subunits. Interestingly, the structure of LdPEX14 appears to be modulated by LdPEX5.Considering all the results, these findings have established important molecular information concerning early events in the targeting and import of PTS1 proteins into the glycosome

Modulation of the Leishmania donovani Peroxin 5 Quaternary Structure by Peroxisomal Targeting Signal 1 Ligands

The import of proteins containing the peroxisomal targeting signal 1 (PTS1) into the Leishmania glycosome is dependent on the docking of the PTS1-loaded LdPEX5 cytosolic receptor with LdPEX14 on the glycosome surface. Here we show that, in the absence of PTS1, LdPEX5 is a tetramer that is stabilized by two distinct interaction domains; the first is a coiled-coil motif encompassing residues 277 to 310, whereas the second domain is localized to residues 1 to 202. By using microcalorimetry, surface plasmon resonance, and size exclusion chromatography techniques, we show that PTS1 peptide binding to LdPEX5 tetramers promotes their dissociation into dimeric structures, which are stabilized by a coiled-coil interaction. Moreover, we demonstrated that the resulting LdPEX5-PTS1 complex is remarkably stable and exhibits extremely slow dissociation kinetics. However, binding of LdPEX14 to LdPEX5 modulates the LdPEX5-PTS1 affinity as it decreases the thermodynamic dissociation constant for this latter complex by 10-fold. These changes in the oligomeric state of LdPEX5 and in its affinity for PTS1 ligand upon LdPEX14 binding may explain how, under physiological conditions, LdPEX5 can function to deliver and unload its cargo to the protein translocation machinery on the glycosomal membrane

Peroxin 5–peroxin 14 association in the protozoan Leishmania donovani involves a novel protein–protein interaction motif

Import of proteins with a PTS1 (peroxisomal targeting signal 1) into the Leishmania glycosomal organelle involves docking of a PTS1-laden LdPEX5 [Leishmania donovani PEX5 (peroxin 5)] receptor to LdPEX14 on the surface of the glycosomal membrane. In higher eukaryotes, the PEX5–PEX14 interaction is mediated by a conserved diaromatic WXXXY/F motif. Site-directed and deletion mutageneses of the three WXXXY/F repeats in LdPEX5 did not abolish the LdPEX5–LdPEX14 association. Analysis of the equilibrium dissociation constant (K(d)) revealed that ldpex5-W53A (Trp(53)→Ala), ldpex5-W293A, ldpex5-W176,293A and ldpex5-W53,176,293A mutant receptors were capable of binding LdPEX14 with affinities comparable with wild-type LdPEX5. That the diaromatic motifs were not required for the LdPEX5–LdPEX14 interaction was further verified by deletion analysis that showed that ldpex5 deletion mutants or ldpex5 fragments lacking the WXXXY/F motifs retained LdPEX14 binding activity. Mapping studies of LdPEX5 indicated that the necessary elements required for LdPEX14 association were localized to a region between residues 290 and 323. Finally, mutational analysis of LdPEX14 confirmed that residues 23–63, which encompass the conserved signature sequence AX(2)FLX(7)SPX(6)FLKGKGL/V present in all PEX14 proteins, are essential for LdPEX5 binding

Antropologías hechas en Ecuador. El quehacer antropológico (Volumen IV)

Al igual que en otros países, en Ecuador la antropología no es solo una disciplina, son varias genealogías que obedecen a temas diversos con enfoques interdisciplinarios y que cambian de acuerdo al contexto social, económico y político; pero a diferencia de la región, registra pocas escuelas de antropología y centros de formación de profesionales en el área. Esta recopilación de textos muestra la diversidad y las múltiples facetas de las antropologías ecuatorianas. La antropología ecuatoriana no se agota en estas historiografías y resalta aquellas genealogías del pensamiento ecuatoriano, nutrido por reflexiones desde las escuelas clásicas de la antropología, que dialogan fuertemente con el contexto nacional y que, particularmente, tienen la capacidad de recrearse a la luz de las necesidades reales de la gente con quienes se co-construye el conocimiento