Background: The 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. Results: Subtractive 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. Conclusion: A 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.

Summary: The capacity of a bacterial pathogen to produce a disease in a treated host depends on the former's 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.

We have found conditions for saturation mutagenesis by restriction enzyme mediated integration that result in plasmid tagging of disrupted genes. Using this method we selected for mutations in genes that act at checkpoints downstream of the intercellular signalling system that controls encapsulation in Dictyostelium discoideum. One of these genes, mkcA, is a member of the mitogen-activating protein kinase cascade family while the other, regA is a novel bipartite gene homologous to response regulators in one part and to cyclic nucleotide phosphodiesterases in the other part. Disruption of either of these genes results in partial suppression of the block to spore formation resulting from the loss of the prestalk genes, tagB and tagC. The products of the tag genes have conserved domains of serine protease attached to ATP-driven transporters, suggesting that they process and export peptide signals. Together, these genes outline an intercellular communication system that coordinates organismal shape with cellular differentiation during development.

Deficient autophagy causes a distinct phenotype in Dictyostelium discoideum, characterized by the formation of multitips at the mound stage. This led us to analyze autophagy in a number of multitipped mutants described previously (tipA⁻, tipB⁻, tipC⁻, and tipD⁻). We found a clear autophagic dysfunction in tipC⁻ and tipD⁻ while the others showed no defects. tipD codes for a homolog of Atg16, which confirms the role of this protein in Dictyostelium autophagy and validates our approach. The tipC-encoded protein is highly similar to human VPS13A (also known as chorein), whose mutations cause the chorea-acanthocytosis syndrome. No member of the VPS13 protein family has been previously related to autophagy despite the presence of a region of similarity to Atg2 at the C terminus. This region also contains the conserved domain of unknown function DUF1162. Of interest, the expression of the TipC C-terminal coding sequence containing these 2 motifs largely complemented the mutant phenotype. Dictyostelium cells lacking TipC displayed a reduced number of autophagosomes visualized with the markers GFP-Atg18 and GFP-Atg8 and an impaired autophagic degradation as determined by a proteolytic cleavage assay. Downregulation of human VPS13A in HeLa cells by RNA interference confirmed the participation of the human protein in autophagy. VPS13A-depleted cells showed accumulation of autophagic markers and impaired autophagic flux.

The use of antibiotics for the treatment of infectious diseases has led to important changes in the structure of pathogenic bacterial populations. However, these changes could be buffered if the expression of antibiotic resistance genes were to lead to the counter-selection of antibiotic-resistant strains in antibiotic-free environments. To test the effect of antibiotic resistance on bacterial fitness, we analysed the effect of the overproduction of the multidrug efflux pump SmeDEF on the physiology of Stenotrophomonas maltophilia. SmeDEF confers resistance to antibiotics belonging to different structural families, and its overexpression is associated with an antibiotic resistance phenotype in clinical isolates of S. maltophilia. Two S. maltophilia isogenic strains were analysed: the wild-type strain D457 and strain D457R, which is a SmeDEF overproducer. In co-culture experiments, under non-selective pressure the wild-type strain displaced the mutant strain D457R. Metabolic profiling showed that SmeDEF overproduction leads to several changes in S. maltophilia metabolism. Using a Dictyostelium discoideum model of bacterial virulence, we found overexpression of SmeDEF to be associated with a reduction in S. maltophilia virulence. Together, these data indicate that overexpression of the multidrug efflux pump SmeDEF causes a metabolic burden for S. maltophilia.

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 th...