The Yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.

Microbial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors.

Protein conformational flexibility prediction using machine learning.

Using a data set of 16 proteins, a neural network has been trained to predict backbone 15N generalized order parameters from the three-dimensional structures of proteins. The final network parameterization contains six input features. The average prediction accuracy, as measured by the Pearson's correlation coefficient between experimental and predicted values of the square of the generalized order parameter is >0.70. Predicted order parameters for non-terminal amino acid residues depends most strongly on the local packing density and the probability that the residue is located in regular secondary structure.

Conformational dynamics in loop swap mutants of homologous fibronectin type III domains.

Fibronectin type III (FN-III) domains are autonomously folded modules found in a variety of multidomain proteins. The 10th FN-III domain from fibronectin (fnFN10) and the 3rd FN-III domain from tenascin-C (tnFN3) have 27% sequence identity and the same overall fold; however, the CC' loop has a different pattern of backbone hydrogen bonds and the FG loop is longer in fnFN10 compared to tnFN3. To examine the influence of length, sequence, and context in determining dynamical properties of loops, CC' and FG loops were swapped between fnFN10 and tnFN3 to generate four mutant proteins and backbone conformational dynamics on ps-ns and mus-ms timescales were characterized by solution (15)N-NMR spin relaxation spectroscopy. The grafted loops do not strongly perturb the properties of the protein scaffold; however, specific effects of the mutations are observed for amino acids that are proximal in space to the sites of mutation. The amino acid sequence primarily dictates conformational dynamics when the wild-type and grafted loop have the same length, but both sequence and context contribute to conformational dynamics when the loop lengths differ. The results suggest that changes in conformational dynamics of mutant proteins must be considered in both theoretical studies and protein design efforts.

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation.

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.