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Inflammation is characterised by the infiltration of leukocytes from the circulation and into the inflamed area. Leukocytes are guided throughout this process by chemokines. These are basic proteins which interact with leukocytes to initiate their activation and extravasation via chemokines receptors. This is enabled through chemokine immobilisation by glycosaminoglycans (GAGs) at the luminal endothelial surface of blood vessels. A specific stretch of basic amino acids on the chemokine, often at the C-terminus, interacts with the negatively charged GAGs which is considered an essential interaction for the chemokine function. Short chain peptides based on this GAG binding region of the chemokines CCL5, CXCL8 and CXCL12γ were synthesised using standard Fmoc chemistry. These peptides were found to bind to GAGs with high affinity which translated into a reduction of leukocyte migration across a cultured endothelial monolayer in response to chemokines. The leukocyte migration was inhibited upon removal of heparan sulphate (HS) from the endothelial surface and was found to reduce the ability of the chemokine and peptide to bind to endothelial cells in binding assays and to human rheumatoid arthritis (RA) tissue. Furthermore, control peptides lacking the basic amino acids showed reduced interaction with HS and no anti-chemotactic ability for leukocytes across an endothelial monolayer. The data suggest that the peptide competes with the wildtype (WT) chemokine for binding to GAGs such as HS and thereby reduces chemokine presentation and subsequent leukocyte migration. Furthermore, the lead peptide based on CXCL8 could reduce the disease severity and serum levels of the pro-inflammatory cytokine TNFα in a murine antigen induced arthritis model. Taken together, evidence is provided for interfering with the chemokine – GAG interaction as a relevant therapeutic approach. The use of site specific sequences of chemokines to target GAGs and compete with WT chemokines is a novel and promising avenue for the field.

BipA is a novel member of the ribosome binding GTPase superfamily and is widely distributed in bacteria and plants. We report here that it regulates -multiple cell surface- and virulence-associated -components in the enteropathogenic Escherichia coli (EPEC) strain E2348/69. The regulated components include bacterial flagella, the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE). BipA positively regulated the espC and LEE gene clusters through transcriptional control of the LEE-encoded regulator, Ler. Additionally, it affected the pattern of proteolysis of intimin, a key LEE-encoded adhesin specified by the LEE. BipA control of the LEE operated independently of the previously characterized regulators Per, integration host factor and H-NS. In contrast, it negatively regulated the flagella-mediated motility of EPEC and in a Ler-independent manner. Our results indicate that the BipA GTPase functions high up in diverse regulatory cascades to co-ordinate the expression of key pathogenicity islands and other virulence-associated factors in E. coli.

Salmonella adaptation to low pH is a critical survival response and essential for virulence. Here, we show that another key virulence-associated process, flagellamediated cell motility, is co-regulated by low pH via the PhoPQ signal transduction system. Using a proteomic approach, we found that phase 1 and phase 2 flagellin were specifically down-regulated when acid-adapted (pH 5.0) Salmonella SL1344 cells were exposed to pH 3.0. Decreased flagellin expression and cell motility was dependent on activation of the PhoPQ pathway, which directly or indirectly negatively regulated transcription of the flagellin gene fliC. In contrast, the general stress sigma factor RpoS (S) positively regulated flagellar gene expression. Low external pH had no effect on the level of H-NS protein, a further regulator of flagellar gene expression. We suggest that flagellar repression at low pH conserves ATP for survival processes and helps to limit the influx of protons into the cytosol. These results highlight the power of proteomics to reveal unanticipated links between relatively well-characterised regulatory systems in bacteria.

Microbes present special opportunities for proteomic analysis that are not yet available for other types of organisms, due mainly to the relative abundance of information on their genomes, their low levels of functional redundancy and their experimental tractability. They are also being used to develop and validate powerful new experimental approaches that surmount some important current limitations in this field. The review surveys the different proteomic procedures that are available and considers the advantages and disadvantages of different experimental strategies. The ways in which microbiologists - and others - can exploit proteomic data are also discussed.

We report the functional characterization of BipA, a GTPase that undergoes tyrosine phosphorylation in an enteropathogenic Escherichia coli (EPEC) strain. BipA mutants adhere to cultured epithelial cells but fail to trigger the characteristic cytoskeletal rearrangements found in cells infected with wild-type EPEC. In contrast, increased expression of BipA enhances actin remodelling and results in the hyperformation of pseudopods. BipA appears to be the first example of a new class of virulence regulator, as it also controls flagella-mediated cell motility and resistance to the antibacterial effects of a human host defence protein. Its striking sequence similarity to ribosome-binding elongation factors suggests that it uses a novel mechanism to modulate gene expression.

Data from permeability profiling using the parallel artificial membrane permeability assay (PAMPA) and cell monolayer (Caco-2 and MDR1-MDCKII) methods were compared for two published compound sets and one in-house set. A majority of compounds in each set correlated (R(2) = 0.76-0.92), indicating the predominance of passive diffusion in the permeation of these compounds. Compounds that did not correlate grouped into two subsets. One subset had higher PAMPA permeability than cell monolayer permeability and consisted of compounds that are subject to secretory mechanisms: efflux or reduced passive diffusion of bases under Caco-2 when run under a pH gradient. The other subset had higher cell monolayer permeability than PAMPA permeability and consisted of compounds that are subject to absorptive mechanisms: paracellular, active transport, or increased passive diffusion of acids under Caco-2 when run under a pH gradient. Given the characteristics of the two methods, these studies suggest how PAMPA and Caco-2 can be synergistically applied for efficient and rapid investigation of permeation mechanisms in drug discovery. During early discovery, all compounds can be rapidly screened using PAMPA at low pH and neutral pH to assess passive diffusion permeability to indicate potential for gastrointestinal and cell assay permeation. During intermediate discovery, selected compounds can be additionally assayed by apical-to-basolateral Caco-2, which, in combination with PAMPA data, indicates susceptibility to additional permeation mechanisms (secretory and absorptive). During mid-to-late discovery, selected candidates can be examined in detail via multiple directional Caco-2 experiments and with transporter inhibitors for complete characterization of permeation mechanisms.

Publisher Summary This chapter describes some current protocols used in proteome studies of bacterial pathogens together with some relevant applications. Proteome analysis experiments can be divided into five stages: (1) sample preparation, (2) fractionation, (3) spot detection, (4) spot identification, and (5) protein microsequencing. The proteomes of bacterial pathogens are intimately linked with the use of 2D-GE, which is the only technique capable of resolving the vast majority of expressed proteins, and giving direct information on their patterns of expression. The proteome concept, however, is more general than this and has emerged because of the need to study the ways, in which cellular responses are integrated and to define the roles of the many novel proteins currently being discovered. Just as the initial methods for sequencing genomes are being replaced by ever more rapid procedures that require little human intervention, it is probable that 2D-GE eventually be superseded by methods that combine greater separating power with increased accuracy, speed, and sensitivity of protein detection.

Salmonella typhimurium (official designation Salmonella enterica serovar Typhimurium) is an enteric pathogen and a principal cause of gastroenteritis in humans. A comprehensive description of the proteins of Salmonella and their patterns of expression under different environmental conditions would greatly increase our understanding of the virulence of this organism at the molecular level and provide insights into many other aspects of Salmonella biology. While a variety of two-dimensional studies of Salmonella have been previously carried out to address specific questions, little systematic information is available at the protein level on the numbers of Salmonella polypeptides that have homologues in other organisms, their abundance, and the frequency of post-translational modifications. To test the feasibility of determining the proteome of Salmonella, the identities of 53 randomly sequenced cell envelope proteins have been determined by N-terminal sequencing of spots from two-dimensional gels. In addition to confirming the existence of previously hypothetical proteins predicted from genomic sequencing projects, we found that approximately 20% of the proteins had no matches in sequence databases. The results suggest that proteome analysis is an efficient way to identify novel proteins from prokaryotes and that the analysis provides a useful approach to the study of Salmonella virulence.