Characterization of IgG N-glycans employing a microfluidic chip that integrates glycan cleavage, sample purification, LC separation, and MS detection.

A novel polymeric microfluidic device with an on-chip enzyme reactor has been developed for the characterization of recombinant glycoproteins. The enzyme reactor chip packed with PNGase F-modified solid support material was combined with a microfluidic glycan cleanup chip and a commercially available HPLC-chip to perform glycoprotein deglycosylation, protein removal, glycan capture, glycan LC separation, and nanoelectrospray into a time-of-flight mass spectrometry (TOF-MS) system. With this integrated chip, the combined sample preparation and sample analysis time was reduced from multiple hours to less than 10 min. A once tedious and time-consuming glycan analysis workflow is now integrated into an HPLC-chip device. Glycan profiling analysis has been achieved with as little as 100 ng of monoclonal antibody. Furthermore, a single chip was shown to retain activity and perform equivalently for over 250 replicate glycan profiles from a recombinant antibody.

Interactions of bacterial effector proteins with host proteins.

Pathogenic bacteria have evolved several clever survival strategies for manipulating host cell signaling pathways to establish beneficial replicative niches within the host. Recent literature has revealed novel mechanisms adopted by bacteria to manipulate host responses. For instance, host signaling pathways that were traditionally thought to be regulated by phosphorylation events have now been shown to be irreversibly blocked by bacterially-mediated acetylation, beta-elimination, and lytic modifications. This review highlights some of the common host proteins and signaling cascades targeted by such pathogens.

Adaptor function of PapF depends on donor strand exchange in P-pilus biogenesis of Escherichia coli.

P-pilus biogenesis occurs via the highly conserved chaperone-usher pathway and involves the strict coordination of multiple subunit proteins. All nonadhesin structural P-pilus subunits possess the same topology, consisting of two domains: an incomplete immunoglobulin-like fold (pilin body) and an N-terminal extension. Pilus subunits form interactions with one another through donor strand exchange, occurring at the usher, in which the N-terminal extension of an incoming subunit completes the pilin body of the preceding subunit, allowing the incorporation of the subunit into the pilus fiber. In this study, pilus subunits in which the N-terminal extension was either deleted or swapped with that of another subunit were used to examine the role of each domain of PapF in functions involving donor strand exchange and hierarchical assembly. We found that the N-terminal extension of PapF is required to adapt the PapG adhesin to the tip of the fiber. The pilin body of PapF is required to efficiently initiate assembly of the remainder of the pilus, with the assistance of the N-terminal extension. Thus, distinct functions were assigned to each region of the PapF subunit. In conclusion, all pilin subunits possess the same overall architectural topology; however, each N-terminal extension and pilin body has specific functions in pilus biogenesis.

P pilus assembly motif necessary for activation of the CpxRA pathway by PapE in Escherichia coli.

P pilus biogenesis occurs via the highly conserved chaperone-usher pathway, and assembly is monitored by the CpxRA two-component signal transduction pathway. Structural pilus subunits consist of an N-terminal extension followed by an incomplete immunoglobulin-like fold that is missing a C-terminal seventh beta strand. In the pilus fiber, the immunoglobulin-like fold of each pilin is completed by the N-terminal extension of its neighbor. Subunits that do not get incorporated into the pilus fiber are driven "OFF-pathway." In this study, we found that PapE was the only OFF-pathway nonadhesin P pilus subunit capable of activating Cpx. Manipulation of the PapE structure by removing, relocating within the protein, or swapping its N-terminal extension with that of other subunits altered the protein's self-associative and Cpx-activating properties. The self-association properties of the new subunits were dictated by the specific N-terminal extension provided and were consistent with the order of the subunits in the pilus fiber. However, these aggregation properties did not directly correlate with Cpx induction. Cpx activation instead correlated with the presence or absence of an N-terminal extension in the PapE pilin structure. Removal of the N-terminal extension of PapE was sufficient to abolish Cpx activation. Replacement of an N-terminal extension at either the amino or carboxyl terminus restored Cpx induction. Thus, the data presented in this study argue that PapE has features inherent in its structure or during its folding that act as specific inducers of Cpx signal transduction.

Targeting virulence for antimicrobial chemotherapy.

Untreatable bacterial infections constitute a dark but valid threat, with numbers of antibiotic resistant pathogens, as well as newly emerging ones, rising quickly. To combat this dangerous prospect, growing research into antimicrobials could be aimed at targeting the virulence of pathogens. Virulence refers to an organism's ability to establish an infection and cause disease. Many steps involved in the infection process can be targeted, including adherence, invasion and host defense evasion. Identification and characterization of virulence factors that aid in bacterial pathogenicity will lead to new drugs that can be applied to a variety of pathogens.