PKCε controls the transport of endocytosed β1‐integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCε target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCε dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCε/integrin positive vesicles. Finally, introduction of ectopic wild‐type vimentin is shown to promote cell motility in a PKCε‐dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCε null cells. Our results indicate that PKC‐mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.

The adaptive and further evolutionary responses of Staphylococcus aureus to selection pressure with the antibiotic rifampin have not been explored in detail. We now present a detailed analysis of these systems. The use of rifampin for the chemotherapy of infections caused by S. aureus has resulted in the selection of mutants with alterations within the ␤ subunit of the target enzyme, RNA polymerase. Using a new collection of strains, we have identified numerous novel mutations in the ␤ subunits of both clinical and in vitro-derived resistant strains and established that additional, undefined mechanisms contribute to expression of rifampin resistance in clinical isolates of S. aureus. The fitness costs associated with rifampin resistance genotypes were found to have a significant influence on their clinical prevalence, with the most common clinical genotype (H 481 N, S 529 L) exhibiting no fitness cost in vitro. Intragenic mutations which compensate for the fitness costs associated with rifampin resistance in clinical strains of S. aureus were identified for the first time. Structural explanations for rifampin resistance and the loss of fitness were obtained by molecular modeling of mutated RNA polymerase enzymes.Resistance to antibiotics arising from point mutations in bacterial genes that encode drug targets is a well-recognized phenomenon (38), and expression of these mechanisms often confers a fitness cost that results from the decreased physiological activity of the altered target (1, 37). Nevertheless, increasing evidence obtained both from laboratory and from epidemiological studies indicates that intragenic compensatory mutations often act to maintain the long-term persistence of resistant bacteria by eliminating or reducing the fitness costs associated with the development of target-based resistance (27). Consequently, bacterial products that are the targets of antibiotic action present interesting systems for the study of structure-function relationships from the perspectives of resistance, fitness, and compensatory evolution (15,20). Furthermore, fitness costs and compensatory evolution are factors that can influence the prevalence of specific antibiotic resistance genotypes in the clinical setting (5,19,20,30,43).We have recently explored the genetic and structural basis of mupirocin resistance and fitness in Staphylococcus aureus and related this to the incidence of mupirocin resistant isoleucyltRNA synthetase genotypes arising in the clinic (19,20). A similar opportunity to examine these paradigms in relation to rifampin resistance now arises.Mutations that confer resistance to rifampin (Fig. 1) arise in the ␤ subunit (encoded by rpoB) of the target enzyme RNA polymerase (RNAP) and have been mapped to this location in all bacteria examined so far, including Escherichia coli (21), Mycobacterium tuberculosis (5, 36), and S. aureus (32,43). The fitness burdens and compensatory evolution associated with mutations in rpoB that confer resistance to rifampin have been studied in E. coli (37). However, the ...

In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.

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