Polarization and cell-fate decision facilitated by the adaptor Ste50p in Saccharomyces cerevisiae.

In response to pheromone, many proteins localize on the plasma membrane of yeast cell to reform it into a polarized shmoo structure. The adaptor protein Ste50p, known as a pheromone signal enhancer critical for shmoo polarization, has never been explored systematically for its localization and function in the polarization process. Time-lapse single-cell imaging and quantitation shown here characterizes Ste50p involvement in the establishment of cell polarity. We found that Ste50p patches on the cell cortex mark the point of shmoo initiation, these patches could move, and remain associated with the growing shmoo tip in a pheromone concentration time-dependent manner until shmoo maturation. A Ste50p mutant impaired in patch localization suffers a delay in polarization. By quantitative analysis we show that polarization correlates with the rising levels of Ste50p, enabling rapid cell responses to pheromone that correspond to a critical level of Ste50p at the initial G1 phase. We exploited the quantitative differences in the pattern of Ste50p expression to correlate with the cell-cell phenotypic heterogeneity, showing Ste50p involvement in the cellular differentiation choice. Taken together, these findings present Ste50p to be part of the early shmoo development phase, suggesting that Ste50p may be involved with the polarisome in the initiation of polarization, and plays a role in regulating the polarized growth of shmoo during pheromone response.

The adaptor protein Ste50 directly modulates yeast MAPK signaling specificity through differential connections of its RA domain.

Discriminating among diverse environmental stimuli is critical for organisms to ensure their proper development, homeostasis, and survival. Saccharomyces cerevisiae regulates mating, osmoregulation, and filamentous growth using three different MAPK signaling pathways that share common components and therefore must ensure specificity. The adaptor protein Ste50 activates Ste11p, the MAP3K of all three modules. Its Ras association (RA) domain acts in both hyperosmolar and filamentous growth pathways, but its connection to the mating pathway is unknown. Genetically probing the domain, we found mutants that specifically disrupted mating or HOG-signaling pathways or both. Structurally these residues clustered on the RA domain, forming distinct surfaces with a propensity for protein-protein interactions. GFP fusions of wild-type (WT) and mutant Ste50p show that WT is localized to the shmoo structure and accumulates at the growing shmoo tip. The specifically pheromone response-defective mutants are severely impaired in shmoo formation and fail to localize ste50p, suggesting a failure of association and function of Ste50 mutants in the pheromone-signaling complex. Our results suggest that yeast cells can use differential protein interactions with the Ste50p RA domain to provide specificity of signaling during MAPK pathway activation.

In vivo expression of Salmonella enterica serotype Typhi genes in the blood of patients with typhoid fever in Bangladesh.

BACKGROUND: Salmonella enterica serotype Typhi is the cause of typhoid fever. It is a human-restricted pathogen, and few data exist on S. Typhi gene expression in humans. METHODOLOGY/PRINCIPAL FINDINGS: We applied an RNA capture and amplification technique, Selective Capture of Transcribed Sequences (SCOTS), and microarray hybridization to identify S. Typhi transcripts expressed in the blood of five humans infected with S. Typhi in Bangladesh. In total, we detected the expression of mRNAs for 2,046 S. Typhi genes (44% of the S. Typhi genome) in human blood; expression of 912 genes was detected in all 5 patients, and expression of 1,100 genes was detected in 4 or more patients. Identified transcripts were associated with the virulence-associated PhoP regulon, Salmonella pathogenicity islands, the use of alternative carbon and energy sources, synthesis and transport of iron, thiamine, and biotin, and resistance to antimicrobial peptides and oxidative stress. The most highly represented group were genes currently annotated as encoding proteins designated as hypothetical, unknown, or unclassified. Of the 2,046 detected transcripts, 1,320 (29% of the S. Typhi genome) had significantly different levels of detection in human blood compared to in vitro cultures; detection of 141 transcripts was significantly different in all 5 patients, and detection of 331 transcripts varied in at least 4 patients. These mRNAs encode proteins of unknown function, those involved in energy metabolism, transport and binding, cell envelope, cellular processes, and pathogenesis. We confirmed increased expression of a subset of identified mRNAs by quantitative-PCR. CONCLUSIONS/SIGNIFICANCE: We report the first characterization of bacterial transcriptional profiles in the blood of patients with typhoid fever. S. Typhi is an important global pathogen whose restricted host range has greatly inhibited laboratory studies. Our results suggest that S. Typhi uses a largely uncharacterized genetic repertoire to survive within cells and utilize alternate energy sources during infection.