COVID-19: paradigmas sanitarios revisados / COVID-19: revised healthcare paradigms

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The microbiome as a human organ.

The human organism is a complex structure composed of cells belonging to all three domains of life on Earth, Eukarya, Bacteria and Archaea, as well as their viruses. Bacterial cells of more than a thousand taxonomic units are condensed in a particular functional collective domain, the intestinal microbiome. The microbiome constitutes the last human organ under active research. Like other organs, and despite its intrinsic complexity, the microbiome is readily inherited, in a process probably involving 'small world' power law dynamics of construction in newborns. Like any other organ, the microbiome has physiology and pathology, and the individual (and collective?) health might be damaged when its collective population structure is altered. The diagnostic of microbiomic diseases involves metagenomic studies. The therapeutics of microbiome-induced pathology include microbiota transplantation, a technique increasingly available. Perhaps a new medical specialty, microbiomology, is being born.

Fungi sensing environmental stress.

Cells need to adapt to the external environment in order to survive. Signal transduction pathways are crucial mechanisms that allow cells to sense and respond to extracellular stimuli. Among the signal transduction pathways, we point out the cascades mediated by mitogen-activated protein kinases (MAPKs). The MAPKs are conserved from yeast to human and play relevant roles in the physiology of the cell. In pathogenic fungi these MAPK pathways control virulence factors. This review describes the MAPK cascades described in Candida albicans, the most frequently isolated fungus, from fungal systemic infections among individuals in developed countries.

The MAP kinase signal transduction network in Candida albicans.

MAP (mitogen-activated protein) kinase-mediated pathways are key elements in sensing and transmitting the response of cells to environmental conditions by the sequential action of phosphorylation events. In the fungal pathogen Candida albicans, different routes have been identified by genetic analysis, and especially by the phenotypic characterization of mutants altered in the Mkc1, Cek1/2 and Hog1 MAP kinases. The cell integrity (or MKC1-mediated) pathway is primarily involved in the biogenesis of the cell wall. The HOG pathway participates in the response to osmotic stress while the Cek1 pathway mediates mating and filamentation. Their actual functions are, however, much broader and Mkc1 senses several types of stress, while Hog1 is also responsive to other stress conditions and participates in two morphogenetic programmes: filamentation and chlamydospore formation. Furthermore, it has been recently shown that Cek1 participates in a putative pathway involved in the construction of the cell wall and which seems to be operative under basal conditions. As these stimuli are frequently encountered in the human host, they provide a reasonable explanation for the significant reduction in pathogenicity that several signal transduction mutants show in certain animal models of virulence. MAPK pathways therefore represent an attractive multienzymic system for which novel antifungal therapy could be designed.

The Cek1 and Hog1 mitogen-activated protein kinases play complementary roles in cell wall biogenesis and chlamydospore formation in the fungal pathogen Candida albicans.

The Hog1 mitogen-activated protein (MAP) kinase mediates an adaptive response to both osmotic and oxidative stress in the fungal pathogen Candida albicans. This protein also participates in two distinct morphogenetic processes, namely the yeast-to-hypha transition (as a repressor) and chlamydospore formation (as an inducer). We show here that repression of filamentous growth occurs both under serum limitation and under other partially inducing conditions, such as low temperature, low pH, or nitrogen starvation. To understand the relationship of the HOG pathway to other MAP kinase cascades that also play a role in morphological transitions, we have constructed and characterized a set of double mutants in which we deleted both the HOG1 gene and other signaling elements (the CST20, CLA4, and HST7 kinases, the CPH1 and EFG1 transcription factors, and the CPP1 protein phosphatase). We also show that Hog1 prevents the yeast-to-hypha switch independent of all the elements analyzed and that the inability of the hog1 mutants to form chlamydospores is suppressed when additional elements of the CEK1 pathway (CST20 or HST7) are altered. Finally, we report that Hog1 represses the activation of the Cek1 MAP kinase under basal conditions and that Cek1 activation correlates with resistance to certain cell wall inhibitors (such as Congo red), demonstrating a role for this pathway in cell wall biogenesis.

Identification of a nuclear export signal in the KSHV latent protein LANA2 mediating its export from the nucleus.

LANA2 is a latent protein detected in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected B cells that inhibits p53-dependent transcriptional transactivation and apoptosis and PKR-dependent apoptosis, suggesting an important role in the transforming activity of the virus. It has been reported that LANA2 localizes into the nucleus of both KSHV-infected B cells and transiently transfected HeLa cells. In this study, we show that LANA2 is a nucleocytoplasmic shuttling protein that requires a Rev-type nuclear export signal located in the C-terminus to direct the protein to the cytoplasm, through an association with the export receptor CRM1. In addition, a functional protein kinase B (PKB)/Akt phosphorylation motif partially overlapping with the nuclear export signal was identified. Nuclear exclusion of LANA2 was negatively regulated by the phosphorylation of threonine 564 by Akt. The ability of LANA2 to shuttle between nucleus and cytoplasm has implications for the function of this viral protein.

The importance of the phagocytes' innate response in resolution of the infection induced by a low virulent Candida albicans mutant.

We have reported that a Candida albicans mkc1Delta/mkc1Delta mutant, deleted in the Mkc1p mitogen-activated protein kinase, an essential element of the cell integrity signalling pathway, has reduced virulence in a murine model of systemic infection. We analyse here the immunological basis for this feature in view of its failure to vaccinate. Firstly, the influence of the Th response was analysed by infecting different knockout mice, revealing the importance of interferon-gamma in the resolution of mkc1 systemic infection. Secondly, the role of innate immunity was studied. The infection of neutropenic mice revealed that the candidacidal activity of neutrophils is crucial during the first 3 days of infection for the mutant strain. Macrophages played a critical role in the clearance of infection. Although a similar anti-Candida activity was found for both fungal strains with naïve macrophages, activated macrophages discriminated between both strains. In vitro experiments revealed that the mutant strain displayed a greater susceptibility to nitric oxide (NO), a reduced inhibitory effect on macrophage NO production and an increased capacity of macrophage stimulation by cell wall extracts. The importance of NO in systemic infection with the mutant strain was confirmed by the strong increase in the susceptibility of aminoguanidine (an iNOs inhibitor)-treated mice.

The latency protein LANA2 from Kaposi's sarcoma-associated herpesvirus inhibits apoptosis induced by dsRNA-activated protein kinase but not RNase L activation.

Kaposi's sarcoma-associated herpesvirus (KSHV) uses several strategies to counteract the interferon (IFN) system. In this study, the relationship of the protein LANA2 from KSHV to the IFN-activated protein kinase (PKR) and 2-5A system was analysed. It was found that LANA2 could not abrogate apoptosis or RNA degradation mediated by the 2-5A system. However, expression of LANA2 inhibited apoptosis triggered by PKR. LANA2 also counteracted the PKR-mediated inhibition of protein synthesis and partially blocked PKR-induced phosphorylation of eIF-2alpha. Analysis of PKR-induced activation of caspases 3 and 9 revealed that LANA2 abrogated activation of caspase 3 but not of caspase 9. These findings show that LANA2 is able to interfere with downstream events triggered by PKR. Hence, LANA2 should be considered as a KSHV defence protein against IFN.

Strategies for the identification of virulence determinants in human pathogenic fungi.

The incidence of fungal infections is increasing in different countries. The current available therapy of these infections does not satisfy all requirements in terms of specificity and therapeutic index, a fact that has stimulated the scientific community to identify fungal virulence determinants. Several pathogenic fungi are opportunistic and, therefore, identification of virulence genes is difficult, given their close relationship with host cells. In recent years, the development of genetic tools in several pathogenic fungi has enabled the development of genetic strategies for their identification. These include several strategies based on the phenotypic analysis of strains or environmental conditions in which the expression of the putative gene(s) is either altered or deleted; and this is accomplished through the development of in vitro or in vivo systems. In the near future, this research will produce a better picture of fungal pathogenesis and therefore define novel promising targets in antifungal therapy.

Large-scale identification of putative exported proteins in Candida albicans by genetic selection.

In all living organisms, secreted proteins play essential roles in different processes. Of special interest is the construction of the fungal cell wall, since this structure is absent from mammalian cells. The identification of the proteins involved in its biogenesis is therefore a primary goal in antifungal research. To perform a systematic identification of such proteins in Candida albicans, we carried out a genetic screening in which in-frame fusions with an intracellular allele of invertase gene SUC2 of Saccharomyces cerevisiae can be used to select and identify putatively exported proteins in the heterologous host S. cerevisiae. Eighty-three clones were selected, including 11 previously identified genes from C. albicans as well as 41 C. albicans genes that encode proteins homologous to already described proteins from related organisms. They include enzymes involved in cell wall synthesis and protein secretion. We also found membrane receptors and transporters presumably related to the interaction of C. albicans with the environment as well as extracellular enzymes and proteins involved in different morphological transitions. In addition, 11 C. albicans open reading frames (ORFs) identified in this screening encode proteins homologous to unknown or putative proteins, while 5 ORFs encode novel secreted proteins without known homologues in other organisms. This screening procedure therefore not only identifies a set of targets of interest in antifungal research but also provides new clues for understanding the topological locations of many proteins involved in processes relevant to the pathogenicity of this microorganism.

Analysis of the serologic response to systemic Candida albicans infection in a murine model.

Two different strains of mice with different susceptibilities to systemic candidiasis (BALB/c and CBA/H) were infected with Candida albicans SC5314. Immune sera were obtained on different days post-infection and assayed against two-dimensional polyacrylamide gel electrophoresis separation of cytoplasmic extracts obtained from protoplasts. More than 31 immunoreactive proteins were detected. Some of them were identified and found to correspond to (i) glycolytic enzymes, such as fructose biphosphate aldolase, triose phosphate isomerase (TPIS), glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase (PGK), enolase (ENO1) and pyruvate kinase, (ii) other metabolic enzymes, such as methionine synthase (METE), inosine-5'-monophosphate dehydrogenase (IMH3), alcohol dehydrogenase and aconitate hydratase and (iii) heat shock proteins: HS71 (or Ssa1p) and HS75 (or Ssb1p), both from the HSP70 family. This work reports for the first time antigenic properties for IMH3 and TPIS. Different profiles of antibody expression, depending on the mouse strain and the course of infection, were observed. ENO1 was the most immunogenic protein in infected BALB/c mice (the most resistant strain). On the other hand, sera from CBA/H mice (a more susceptible strain) showed a strong increase in reactivity along the infection against METE, HS75 and PGK. Many of these immunoreactive proteins have also been detected using sera from human patients with systemic candidiasis, thus indicating the usefulness of the murine model for studying the antibody response in systemic candidiasis. In this work we demonstrate that the combination of two-dimensional electrophoresis with immunoblotting using murine immune sera can be an important tool for the identification of C. albicans antigens and for monitoring the evolution of the disease.

Pim1, a MAP kinase involved in cell wall integrity in Pichia pastoris.

Mitogen-activated protein kinases (MAPKs) are key enzymes in the signal transduction pathways of eukaryotes. We report the isolation of a Pichia pastoris gene, PIM1, which encodes the first MAPK to be identified in this yeast. Pim1 shows the greatest similarity to fungal MAPKs involved in the maintenance of cell integrity. Disruption of the PIM1 gene results in an osmoremediable thermosensitive phenotype reminiscent of that observed in mutants affected in the MAPK Slt2/ Mpk1 of Saccharomyces cerevisiae, which is involved in ensuring cell wall integrity. Furthermore, pim1 mutants are hypersensitive to caffeine and cell wall-destabilising compounds. Pim1 is phosphorylated at two sites, and thereby activated, in response to heat stress, caffeine and agents that alter the fungal cell wall, which is consistent with a role in adaptation to these conditions. These results support the idea that the MAPK-based mechanisms which regulate cell wall integrity are conserved in yeast species. Pim1 is also doubly phosphorylated in S. cerevisiae in response to stimuli that activate the cell integrity pathway in this yeast. In addition, Pim1 is able to activate the transcription of a reporter gene in one-hybrid experiments, as does its S. cerevisiae counterpart, Slt2. Interestingly, however, Pim1 does not rescue the mutant phenotype of an slt2delta strain. This indicates some functional divergence in MAPK modulation and signal transmission by cell integrity pathways and provides a tool that may contribute to a better understanding of MAPK signalling.

A single-copy suppressor of the Saccharomyces cerevisae late-mitotic mutants cdc15 and dbf2 is encoded by the Candida albicans CDC14 gene.

The Saccharomyces cerevisiae CDC15, DBF2, TEM1 and CDC14 genes encode regulatory proteins that play a crucial role in the latest stages of the M phase of the cell cycle. By complementation of a S. cerevisiae cdc15-lyt1 mutant with a Candida albicans centromeric-based genomic library, we have isolated a homologue of the protein phosphatase-encoding gene CDC14. The sequence analysis of the C. albicans CDC14 gene reveals a putative open reading frame of 1626 base pairs interrupted by an intron located close to the 5' region. Analysis of C. albicans cDNA proved that the intron is processed in vivo. The CaCDC14 gene shares 49% of amino acid sequence identity with the S. cerevisiae CDC14 gene, 46% with Schizosaccharomyces pombe homologue, 35% with Caenorhabditis elegans and 37% and 38% with human CDC14A and CDC14B genes, respectively. As expected, the C. albicans CDC14 gene complemented a S. cerevisiae cdc14-1 mutant. We found that this gene was able to efficiently suppress not only a S.cerevisiae cdc15-lyt1 mutant but also a dbf2-2 mutant in a low number of copies and allowed growth, although very slightly, of a tem1 deletant. Overexpression of the human CDC14A and CDC14B genes complemented, although very poorly, S. cerevisiae cdc15-lyt1 and dbf2-2 mutants, suggesting a conserved function of these genes throughout phylogeny. The sequence of CaCDC14 was deposited in the EMBL database under Accession No. AJ243449.

Two different NO-dependent mechanisms account for the low virulence of a non-mycelial morphological mutant of Candida albicans.

We have previously described the low virulence of a Candida albicans morphological mutant: 92'. We have now used this strain to examine the role of phagocytes in its pathogenesis. Our results show that C. albicans 92' cannot evade innate host macrophage defence mechanisms as efficiently as the parental strain. In addition to the high susceptibility to phagocytosis by peritoneal macrophages, the NO produced by macrophages is a very important element in the low virulence of this agerminative mutant, a thesis supported by in vivo and in vitro experiments. Whereas the parental strain was able to inhibit macrophage NO production, the mutant was quite inefficient at reducing NO production by macrophages. In addition, the mutant showed high sensitivity to a NO generator. Treatment of mice with aminoguanidine (a preferred inducible NO synthase inhibitor) caused 90% mortality in 92' systemic infection, thus supporting a role for NO in the low virulence of this strain. Our data show that both the low inhibitory effect of 92' on macrophage NO production and the higher sensitivity to NO underlie the low virulence of this strain.

Choline-binding domain as a novel affinity tag for purification of fusion proteins produced in Pichia pastoris.

The choline-binding domain (ChoBD) of the carboxy-terminal region of the Streptococcus pneumoniae amidase LYTA (C-LYTA) presents a strong affinity for tertiary amines. We report a method for single-step purification of proteins expressed in the methylotrophic yeast Pichia pastoris based on the fusion of C-LYTA to the protein of interest. We show that C-LYTA can be efficiently expressed and secreted in this host. Tagged proteins fused to this binding domain can be purified on inexpensive DEAE matrices. It therefore provides a useful system for the purification of recombinant proteins with high specificity suitable for industrial purposes.

Virulence genes in the pathogenic yeast Candida albicans.

In recent years, the incidence of fungal infections has been rising all over the world. Although the amount of research in the field of pathogenic fungi has also increased, there is still a need for the identification of reliable determinants of virulence. In this review, we focus on identified Candida albicans genes whose deletant strains have been tested in experimental virulence assays. We discuss the putative relationship of these genes to virulence and also outline the use of new different systems to examine the precise effect in virulence of different genes.

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae.

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of beta1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect beta1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.

Signal transduction pathways and cell-wall construction in Candida albicans.

Signal transduction pathways are the molecular mechanisms responsible for detecting and transmitting changes in the surrounding environment to the nucleus where appropriate responses are generated. The cell wall is the most external structure of the fungal cell and, in pathogenic fungi, is responsible for specifically interacting with the mammalian host cell in a highly dynamic interplay. Recent work has shown the role that some signal transduction pathways, involving members of the MAP kinase family, have in this process in the nonpathogenic model organism Saccharomyces cerevisiae. However, as yet little is known about these phenomena in pathogenic fungi. The aim of this review is to characterize the existing signal transduction pathways in Candida albicans and their relationship with the cell-wall construction.

A proteomic approach for the study of Saccharomyces cerevisiae cell wall biogenesis.

In fungi, cell shape is determined by the presence of a rigid cell wall which separates the cell from the extracellular medium. This highly dynamic structure is essential for the maintenance of cell integrity and is involved in several phenomena such as flocculation, adherence and pathogenicity. The composition of the fungal cell wall is well known, but issues such as the assembly and remodeling of its components remain poorly understood. In an attempt to study the de novo construction of the yeast cell wall, we have undertaken a large-scale proteomic approach to analyze the proteins secreted by regenerating protoplasts. Upon incubation of protoplasts in regenerating conditions, numerous proteins are secreted into the culture medium. These presumably include proteins destined for the cell wall, comprising both structural proteins as well as enzymes involved in cell wall biogenesis. This work reports the establishment of a reference map of proteins secreted by regenerating protoplasts by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and their identification by mass spectrometry. Thirty-two different proteins have been identified, including known cell wall proteins, glycolytic enzymes, heat shock proteins, and proteins involved in several other processes. Using this approach, novel proteins possibly involved in cell wall construction have also been identified. This reference map will allow comparative analyses to be carried out on a selected collection of mutants affected in the cell wall.