No description available.

RfaH is a unique bacterial protein that enhances transcription of a select group of long operons in many Gram-negative bacteria. Operons regulated by RfaH possess an upstream operon polarity suppressor sequence, which recruits the RfaH protein to the RNA polymerase during transcription of genes, most of which are involved in the synthesis of cell-surface features. These include synthesis of the lipopolysaccharide (LPS) core and O-antigen in Salmonella and Escherichia coli, as well as F-plasmid conjugation pilus and capsule in E. coli. LPS is an important virulence factor in many Gram-negative bacteria, and protects Y. pseudotuberculosis against host antimicrobial chemokines. Recently published high-throughput transposon mutant screens have also suggested a role for RfaH in the ability of Y. pseudotuberculosis to colonize mice. However, the role of RfaH in Y. pseudotuberculosis and its descendent Yersinia pestis has not been carefully examined. In these studies we investigated the effect RfaH has on the structure of the LPS in both species at different temperatures. We also identified LPS-synthesis related genes that are regulated by RfaH. We determined the effect of RfaH on bacterial resistance to host defense peptides, and the ability of Y. pseudotuberculosis to colonize mice. We found that the loss of the rfaH gene had different effects in Y. pseudotuberculosis and Y. pestis. Loss of rfaH caused a truncation in the core region in Y. pseudotuberculosis strain IP32953 at both 21°C and 37°C, but only at 37°C in Y. pestis strain KIM6+. Similarly, we found that transcription of individual genes that are predicted to function in core or O-antigen synthesis were downregulated in the rfaH mutant strains in both species, but the impact of rfaH deletion was greater in Y. pseudotuberculosis. When tested for their ability to survive in the presence of antimicrobial peptides, the Y. pseudotuberculosis rfaH deficient bacteria were much more susceptible than wild-type to killing by polymyxin and by the antimicrobial chemokine CCL28. However, the Y. pestis rfaH mutant strain was equally susceptible to CCL28 as the wild-type strain. Infection of mice with Y. pseudotuberculosis show that rfaH deficient bacteria were able to survive as effectively as the wild-type following oral or intravenous inoculation, with or without the pYV virulence plasmid. Overall, our results show that while RfaH controls LPS gene expression in both Y. pseudotuberculosis and Y. pestis, its impact is much greater in Y. pseudotuberculosis. Furthermore, although loss of rfaH greatly reduces the ability of Y. pseudotuberculosis to resist antimicrobial peptides, it is not required for virulence in this species.

No description available.

Multifunctional proteins that undergo major structural changes to perform different functions are known as “Transformer Proteins”, which is a recently identified class of proteins. One such protein that shows a remarkable structural plasticity and has two distinct functions is the transcription antiterminator, RfaH. Depending on the interactions between its N-terminal domain and its C-terminal domain, the RfaH CTD exists as either an all-α-helix bundle or all-β-barrel structure. Another example of a transformer protein is the Ebola virus protein VP40 (eVP40), which exists in different conformations and oligomeric states (dimer, hexamer, and octamer), depending on the required function.I performed Molecular Dynamics (MD) computations to investigate the structural conversion of RfaH-CTD from its all-a to all-b form. I used various structural and statistical mechanics tools to identify important residues involved in controlling the conformational changes. In the full-length RfaH, the interdomain interactions were found to present the major barrier in the structural conversion of RfaH-CTD from all-a to all-b form. I mapped the energy landscape for the conformational changes by calculating the potential of mean force using the Adaptive Biasing Force and Jarzynski Equality methods. Similarly, the interdomain salt-bridges in the eVP40 protomer were found to play a critical role in domain association and plasma membrane (PM) assembly. This molecular dynamic simulation study is supported by virus like particle budding assays investigated by using live cell imaging that highlighted the important role of these saltbridges. I also investigated the plasma membrane association of the eVP40 dimer in various PM compositions and found that the eVP40 dimer readily associates with the PM containing POPS and PIP2 lipids. Also, the CTD helices were observed to be important in stabilizing the dimer-membrane complex. Coarse-grained MD simulations of the eVP40 hexamer and PM system revealed that the hexamer enhances the PIP2 lipid clustering at the lower leaflet of the PM. These results provide insight on the critical steps in the Ebola virus life cycle.