Ricardo Escalante

The global regulator Crc modulates metabolism, susceptibility to antibiotics and virulence in Pseudomonas aeruginosa

The capacity of a bacterial pathogen to produce a disease in a treated host depends on the formers virulence and resistance to antibiotics. Several scattered pieces of evidence suggest that these two characteristics can be influenced by bacterial metabolism. This potential relationship is particularly important upon infection of a host, a situation that demands bacteria adapt their physiology to their new environment, making use of newly available nutrients. To explore the potential cross-talk between bacterial metabolism, antibiotic resistance and virulence, a Pseudomonas aeruginosa model was used. This species is an important opportunistic pathogen intrinsically resistant to many antibiotics. The role of Crc, a global regulator that controls the metabolism of carbon sources and catabolite repression in Pseudomonas, was analysed to determine its contribution to the intrinsic antibiotic resistance and virulence of P. aeruginosa. Using proteomic analyses, high-throughput metabolic tests and functional assays, the present work shows the virulence and antibiotic resistance of this pathogen to be linked to its physiology, and to be under the control (directly or indirectly) of Crc. A P. aeruginosa strain lacking the Crc regulator showed defects in type III secretion, motility, expression of quorum sensing-regulated virulence factors, and was less virulent in a Dictyostelium discoideum model. In addition, this mutant strain was more susceptible to beta-lactams, aminoglycosides, fosfomycin and rifampin. Crc might therefore be a good target in the search for new antibiotics.

Structural and functional studies of a family of Dictyostelium discoideum developmentally regulated, prestalk genes coding for small proteins

BackgroundThe social amoeba Dictyostelium discoideum executes a multicellular development program upon starvation. This morphogenetic process requires the differential regulation of a large number of genes and is coordinated by extracellular signals. The MADS-box transcription factor SrfA is required for several stages of development, including slug migration and spore terminal differentiation.ResultsSubtractive hybridization allowed the isolation of a gene, sigN (SrfA-induced gene N), that was dependent on the transcription factor SrfA for expression at the slug stage of development. Homology searches detected the existence of a large family of sigN-related genes in the Dictyostelium discoideum genome. The 13 most similar genes are grouped in two regions of chromosome 2 and have been named Group1 and Group2 sigN genes. The putative encoded proteins are 87–89 amino acids long. All these genes have a similar structure, composed of a first exon containing a 13 nucleotides long open reading frame and a second exon comprising the remaining of the putative coding region. The expression of these genes is induced at10 hours of development. Analyses of their promoter regions indicate that these genes are expressed in the prestalk region of developing structures. The addition of antibodies raised against SigN Group 2 proteins induced disintegration of multi-cellular structures at the mound stage of development.ConclusionA large family of genes coding for small proteins has been identified in D. discoideum. Two groups of very similar genes from this family have been shown to be specifically expressed in prestalk cells during development. Functional studies using antibodies raised against Group 2 SigN proteins indicate that these genes could play a role during multicellular development.

Autophagy in Dictyostelium Genes and pathways, cell death and infection

The use of simple organisms to understand the molecular and cellular function of complex processes is instrumental for the rapid development of biomedical research. A remarkable example has been the discovery in S. cerevisiae of a group of proteins involved in the pathways of autophagy. Orthologues of these proteins have been identified in humans and experimental model organisms. Interestingly, some mammalian autophagy proteins do not seem to have homologues in yeast but are present in Dictyostelium, a social amoeba with two distinctive life styles, a unicellular stage in nutrient-rich conditions that differentiates upon starvation into a multicellular stage that depends on autophagy. This review focuses on the identification and annotation of the putative Dictyostelium autophagy genes and on the role of autophagy in development, cell death and infection by bacterial pathogens.

Autophagy dysfunction and ubiquitin-positive protein aggregates in Dictyostelium cells lacking Vmp1

Ubiquitin-positive protein aggregates are a hallmark of many degenerative diseases. Their presence can be induced by dysfunction in protein degradation pathways such as proteasome and autophagy. We now report several lines of evidence suggesting a defect in autophagy in Dictyostelium cells lacking Vmp1 (Vacuole membrane protein 1), an endoplasmic reticulum (ER)-resident protein involved in pathological processes such as cancer and pancreatitis. vmp1- null cells are unable to survive starvation or undergo autophagic-cell-death under the appropriate inductive signals. Moreover, confocal studies using the autophagy marker Atg8 and previous transmission electron microscopy analysis showed defects in autophagosome formation. Although Vmp1 is localized in the ER we found co-localization with Atg8 suggesting a contribution of both Vmp1 and ER in autophagosome biogenesis or maturation. Interestingly, vmp1- mutant cells showed accumulation of huge ubiquitin-positive protein aggregates containing the autophagy marker GFP-Atg8 and the putative Dictyostelium p62 homologue as described in many degenerative human diseases. The analysis of other Dictyostelium autophagic mutants (atg1-, atg5-, atg6-, atg7- and atg8-) showed a correlation in the severity of their corresponding phenotypes and the presence of ubiquitin-positive protein aggregates suggesting that the deleterious effects associated with development of these aggregates might contribute to the complex phenotypes observed in autophagy deficient mutants. Our results suggest that Vmp1 is required for the clearance of these ubiquitinated protein aggregates through autophagy and highlight a potential role for Vmp1 in protein-aggregation diseases.

MidA is a putative methyltransferase that is required for mitochondrial complex I function

Dictyostelium and human MidA are homologous proteins that belong to a family of proteins of unknown function called DUF185. Using yeast two-hybrid screening and pull-down experiments, we showed that both proteins interact with the mitochondrial complex I subunit NDUFS2. Consistent with this, Dictyostelium cells lacking MidA showed a specific defect in complex I activity, and knockdown of human MidA in HEK293T cells resulted in reduced levels of assembled complex I. These results indicate a role for MidA in complex I assembly or stability. A structural bioinformatics analysis suggested the presence of a methyltransferase domain; this was further supported by site-directed mutagenesis of specific residues from the putative catalytic site. Interestingly, this complex I deficiency in a Dictyostelium midA− mutant causes a complex phenotypic outcome, which includes phototaxis and thermotaxis defects. We found that these aspects of the phenotype are mediated by a chronic activation of AMPK, revealing a possible role of AMPK signaling in complex I cytopathology.

Dictyostelium transcriptional responses to Pseudomonas aeruginosa: common and specific effects from PAO1 and PA14 strains

BackgroundPseudomonas aeruginosa is one of the most relevant human opportunistic bacterial pathogens. Two strains (PAO1 and PA14) have been mainly used as models for studying virulence of P. aeruginosa. The strain PA14 is more virulent than PAO1 in a wide range of hosts including insects, nematodes and plants. Whereas some of the differences might be attributable to concerted action of determinants encoded in pathogenicity islands present in the genome of PA14, a global analysis of the differential host responses to these P. aeruginosa strains has not been addressed. Little is known about the host response to infection with P. aeruginosa and whether or not the global host transcription is being affected as a defense mechanism or altered in the benefit of the pathogen. Since the social amoeba Dictyostelium discoideum is a suitable host to study virulence of P. aeruginosa and other pathogens, we used available genomic tools in this model system to study the transcriptional host response to P. aeruginosa infection.ResultsWe have compared the virulence of the P. aeruginosa PAO1 and PA14 using D. discoideum and studied the transcriptional response of the amoeba upon infection. Our results showed that PA14 is more virulent in Dictyostelium than PA01using different plating assays. For studying the differential response of the host to infection by these model strains, D. discoideum cells were exposed to either P. aeruginosa PAO1 or P. aeruginosa PA14 (mixed with an excess of the non-pathogenic bacterium Klebsiella aerogenes as food supply) and after 4 hours, cellular RNA extracted. A three-way comparison was made using whole-genome D. discoideum microarrays between RNA samples from cells treated with the two different strains and control cells exposed only to K. aerogenes. The transcriptomic analyses have shown the existence of common and specific responses to infection. The expression of 364 genes changed in a similar way upon infection with one or another strain, whereas 169 genes were differentially regulated depending on whether the infecting strain was either P. aeruginosa PAO1 or PA14. Effects on metabolism, signalling, stress response and cell cycle can be inferred from the genes affected.ConclusionOur results show that pathogenic Pseudomonas strains invoke both a common transcriptional response from Dictyostelium and a strain specific one, indicating that the infective process of bacterial pathogens can be strain-specific and is more complex than previously thought.

Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1(-) Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1(-) cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.

The MADS-box transcription factor SrfA is required for actin cytoskeleton organization and spore coat stability during Dictyostelium sporulation

The MADS-box transcription factor SrfA is involved in spore differentiation in Dictyostelium [Development 125 (1998) 3801]. Mutant spores show an altered morphology and loss of viability. A detailed structural analysis of mutant spores has been performed to gain insight into the specific aspects of spore differentiation in which SrfA is involved. Two main structural defects have been observed. One is the formation of high order actin structures, the so-called actin rods. SrfA mutant spores showed the initial stages of rod formation but no mature rods were found in older spores either in the nucleus or the cytoplasm. Moreover, phosphorylation of actin, that is believed to stabilize the actin rods, is strongly reduced in the mutant. The other defect observed was the formation of the spore coat. Young srfA- spores show basically normal trilaminar coat structures suggesting that release of prespore vesicles and basic assembly of the coat takes place in the absence of SrfA. However, the outer layer gets wavier as the spore ages and suffers a progressive degradation suggesting a late defect in the stability of the spore coat. Taken together, these results suggest that SrfA is involved in late events of spore maturation necessary for spore stability.

In situ hybridization analyses of Na, K-ATPase alpha-subunit expression during early larval development of Artemia franciscana.

The spatial pattern of expression of the mRNA encoded by the Na,K-ATPase alpha-subunit cDNA clone pArATNa136 was determined by in situ hybridization of first, second, and third instar Artemia franciscana larvae. This mRNA was expressed at high levels in the salt gland, the antennal gland, and the end of the midgut, which are the three main osmoregulatory organs in Artemia at these stages of development. The pattern of expression was similar at the three stages of development analyzed, although the level of expression increased during development, especially in the salt and antennal glands. The expression of the mRNA coding for another Na, K-ATPase alpha-subunit isoform, the proposed alpha 2-isoform, was also determined and was shown to be limited to the salt gland. These results suggest that the clone pArATNa136 codes for the biochemically defined alpha 1-isoform of the Na,K-ATPase alpha-subunit and reinforce the importance of this isoform in osmoregulation at the three larval stages studied. The alpha 2-isoform may also be involved in osmoregulation during the first stages of larval development.

Vmp1 Regulates PtdIns3P Signaling During Autophagosome Formation in Dictyostelium discoideum

Generation and turnover of phosphatidylinositol 3‐phosphate (PtdIns3P) signaling is essential for autophagosome formation and other membrane traffic processes. In both Dictyostelium discoideum and mammalian cells, autophagosomes are formed from specialized regions of the endoplasmic reticulum (ER), called omegasomes, which are enriched in the signaling lipid PtdIns3P. Vacuole membrane protein 1 (Vmp1) is a multispanning membrane protein localized at the ER that is required for autophagosome formation. There are conflicting reports in the literature as to whether Vmp1 is strictly required or not for autophagy‐related PtdIns3P signaling and its hierarchical relationship with Atg1 and PI3K. We have now addressed these questions in the Dictyostelium model. We show that Dictyostelium cells lacking Vmp1 have elevated and aberrant PtdIns3P signaling on the ER, resulting in an increased and persistent recruitment of Atg18 and other autophagic proteins. This indicates that Vmp1 is not strictly essential for the generation of PtdIns3P signaling but rather suggests a role in the correct turnover or modulation of this signaling. Of interest, these PtdIns3P‐enriched regions of the ER surround ubiquitinated protein aggregates but are unable to form functional autophagosomes. vmp1 null cells also have additional defects in macropinocytosis and growth, which are not shared by other autophagy mutants. Remarkably, we show that these defects and also the aberrant PtdIns3P distribution are largely suppressed by the concomitant loss of Atg1, indicating that aberrant autophagic signaling on the ER inhibits macropinocytosis. These results suggest that Atg1 functions upstream of Vmp1 in this signaling pathway and demonstrates a previously unappreciated link between abnormal autophagy signaling and macropinocytosis.