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A vast number of studies have been completed on the virulence determinants of Yersinia spp.; however, the focus of many of these studies has been on the virulence plasmid and the plasmid-encoded Type three secretion system. Nevertheless, many chromosomal genes whose products are directly involved in virulence have also been identified. Some of these critical virulence determinants are outer membrane proteins. Outer membrane proteins of Gram-negative bacteria often have important physiological roles; however, some have also been found to be important for pathogenesis. In this thesis, we investigated two Yersinia. pestis outer membrane proteins, Ail and OmpA, and their roles in virulence. We provide evidence that Y. pestis Ail is a highly expressed outer membrane protein that is absolutely essential for Y. pestis to resist the killing action of the complement system present in human blood and tissues, as well as the blood and tissues of other mammalian hosts. Furthermore, Ail was important for virulence in a Y. pestis-Canorhabditis elegans model of infection.The work in this thesis also provided the first evidence that another surface-exposed outer membrane protein, termed OmpA, is required for both Yersinia pseudotuberculosis and Y. pestis to survive and proliferate intracellularly in macrophages. Finally, we provide evidence that Y. pestis has a functional small RNA MicA that controls the expression of OmpA. This is the first demonstration of sRNA-mediated regulation of a Yersinia virulence factor. This work has paved the way for future studies on the role of outer membrane proteins in virulence, particularly the role of Ail and OmpA.

The energetic mechanism of preprotein export in Escherichia coli has been a source of controversy for many years. In vitro studies of translocation reactions that use purified soluble and membrane components have now clarified the main features of this mechanism. Translocation occurs through consecutive steps which each have distinct energy requirements. Initiation of translocation requires ATP and the SecA protein. Most of the further steps can be driven by the protonmotive force (DELTA-p).

In bacteria, what __drives__ the process of cell division is unknown. Possibly, forces generated by an internal protein ring (termed __the Z-ring__) are responsible for the division process, but direct evidence is lacking. Here we describe the development of a method to measure forces in a single dividing bacterium. Using optical tweezers, forces can be measured on optically trapped micron-sized beads. To attach such a bead to a living bacterium, one of its outer membrane proteins is engineered to present a binding epitope on the cell surface. We show that it is possible to attach a bead to this __molecular handle__ via a DNA-molecule, and characterize the strength of this molecular construct. Finally, we show that genetic fusion of the surface exposed protein domain to an internal domain with mid-cell affinity can alter the localization pattern of the exposed domain. Our findings suggest that it is possible to create a fusion protein that exposes a binding epitope and localizes specifically to the division site.

The field of membrane protein folding is relatively new compared to soluble protein folding. This thesis describes spectroscopy investigations of the refolding and dynamics of a β-barrel membrane protein. The amphiphilic, β-barrel outer membrane protein A (OmpA) refolds and inserts directly into a lipid vesicle or micelle from a denatured state in aqueous urea solution. Spectroscopic probes used to study this system are native tryptophans located at positions 7, 15, 57, 102, and 143. Steady-state and time-resolved fluorescence measurements were performed using single tryptophan mutants of full-length OmpA (325 residues) and the truncated, transmembrane domain (176 residues). Both full-length and truncated mutants exhibit similar tryptophan emission lifetimes, suggesting that the transmembrane microenvironment is not greatly perturbed by the presence of the C-terminus. While the microenvironments of folded full-length and truncated OmpA appear similar, the dynamics of refolding at each tryptophan position exhibit subtle differences when the C-terminus is present. Specifically, we observe that tryptophan-102, which faces the pore interior, inserts and folds the fastest while tryptophan-7, which does not cross the bilayer, is the slowest. Fluorescence anisotropy decays also indicate that tryptophan-7 is the most flexible residue compared to the other tryptophans. Temperature studies below the lipid gel-liquid transition temperature have also been performed. In the lipid gel phase, OmpA adsorbs to the surface of the vesicles but contains immediate β-sheet structure upon folding as well as very hydrophobic tryptophan environments. It is still uncertain from ensemble measurements whether this species is a true intermediate. Fluorescence energy transfer kinetics have successfully determined the intramolecular distance between tryptophan-7 and cysteine-175 labeled with a dansyl fluorophore. These results reveal that the barrel ends of OmpA come into contact early in the refolding process and remain close together up to the final assembly of the barrel. We also have evidence that the adsorbed species at low temperatures is not an intermediate in the folding pathway since no energy transfer is observed for this species. These spectroscopic investigations have provided the foundation for further fundamental studies to dissect the molecular mechanism of the folding pathway of OmpA as well as other integral membrane proteins.

My work focussed on several significant aspects of the folding mechanism of outer membrane protein A of Escherichia coli, which is composed of a 155 residue periplasmic domain and of a 170 residue transmembrane (TM) domain that forms an 8-stranded TM β-barrel.First, I investigated the folding kinetics of OmpA into model membranes containing the main components of the inner leaflet of the OM: phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). The results obtained showed that model membranes mimicking the composition of the outer membrane (containing PE and PG at a molar ratio of 80 and 20) resulted in very low OmpA folding efficiency possibly due to the strong surface-dehydration caused by intermolecular hydrogen-bonds between the ammonium- and the phosphate- parts of PE headgroups. In contrast model membranes where PE was excluded or partially replaced with PC (which forms bilayers and contains a charged trimethyl ammonium group which cannot participate in hydrogen bonding) resulted in high folding yields. When PG was included in moderate amounts (20-30%) into PC/PG membranes, the folding kinetics of OmpA were stimulated significantly because the repulsion between negatively charged PG molecules and the increased hydration shell of this headgroup leads to more water in the headgroup region of negatively charged PG compared to PC.My next two projects sought to explore possible chaperone assisted folding pathways of OmpA that may exist in bacteria. In bacteria, outer membrane proteins like OmpA are synthesized in the cytosol. They are then translocated across the cytoplasmic (inner) membrane into the periplasm in unfolded form. Periplasmic chaperones like SurA or Skp bind to the polypeptide chains after they emerge from the translocon.I demonstrated that OmpA binds ~ 3 molecules of Skp and forms a soluble complex in which OmpA is kept largely unfolded. This complex then binds a small number of n = 2-7 LPS per OmpA in solution to form a folding and insertion component form of OmpA that is bound to Skp and LPS. In this second complex, OmpA develops no or only very small amounts of secondary structure. When this insertion competent form of OmpA was reacted with preformed phospholipid bilayers, OmpA rapidly inserted and folded to its native state. The results indicated that OmpA that is bound to Skp and LPS does not fold in absence of lipid bilayers, but folds into lipid bilayers with accelerated folding kinetics. In contrast, the kinetics of OmpA folding into lipid bilayers from a state bound to Skp in absence of LPS or from a denatured state in 8 M urea were both slower.The SurA assisted folding pathway of OmpA is markedly different in comparison with the folding pathway described above. According to my results, LPS is not required and the presence of SurA prevents the interaction of OmpA with LPS. The chaperone effect of SurA was manifested in experiments where OmpA was incubated in aqueous buffer either in absence or in presence of SurA. In absence of SurA, preincubation in aqueous solution led to aggregated forms of OmpA that did not fold upon subsequent addition of lipid bilayers. In presence of SurA, the formation such aggregated forms was suppressed. SurA therefore appeared to prevent aggregation of OmpA, which still folded into lipid bilayers in high yields. In contrast to experiments with Skp and LPS, for which folding was stimulated compared to simple refolding experiments of urea-denatured OmpA into lipid bilayers, the effect of SurA on unfolded OmpA was small.The last chapter of my thesis presents relevant new data concerning the mechanism of formation and membrane insertion of transmembrane β-barrel domain of OmpA. The goal of my study was to investigate the formation of the transmembrane β-barrel domain of OmpA (residues 1 to 171) on the level of individual β-strands and to relate their association to the insertion of OmpA into the lipid membrane.A new method was developed to monitor the association of individual β-strands in pairs during the formation of the β-barrel domain. I used a series of single Trp, single Cys mutants of OmpA. The Trp and Cys residues of each mutant were located on neighbouring β-strands. The Cysteine-residue was labelled with a nitroxyl spin-label that functions as a short-range fluorescence quencher. Association of the neighboring β-strands during OmpA folding triggers the quenching of Trp fluorescence signal by the spin-labelled Cys residue.The results of the last chapter of the present thesis led to the following conclusions: (i) the assembly of individual strands in pairs during the OmpA barrel formation is a correlated, highly concerted process (and not a sequential one), (ii) the association of individual β-strands in pairs and the sealing of the 8 stranded β-barrel between strands 1 and 8 take place simultaneously (iii) the mechanism of formation and insertion of OmpA β-barrel domain into the lipid bilayers is concerted.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded β-barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the β-barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of β-sheet secondary structure and β-barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i. e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of β-barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates.

One hundred avian Pasteurella multocida isolates recovered from cases of fowl cholera and related infections in England and Wales over a 13-year period were characterised by capsular PCR typing and analysis of outer membrane protein (OMP) profiles. Sixty-eight percent of the strains were of capsular type A, 14% were type F, 5% were type D, 4% were type B and 9% were untypable. Nineteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. Fifty-six percent of the isolates were represented by 15 OMP-types, whereas 44% of the isolates were associated with four OMP-types. The extensive molecular mass heterogeneity of the OmpA and OmpH proteins supports previous findings that avian P. multocida strains are very diverse. Furthermore, the isolates studied were associated with different clinical symptoms and were recovered from a wide range of lesions and tissues. The high degree of strain diversity together with the wide variety of clinical symptoms suggest that certain avian strains of P. multocida are opportunistic pathogens of relatively low virulence. Strains of capsular types B, D and F, as well as the untypable isolates, were associated exclusively with specific OMP-types and represent distinct and widely disseminated clonal groups. These observations support the view that avian strains of P. multocida have a clonal population structure. Based on previous studies, the molecular mass heterogeneity of the OmpA and OmpH proteins might provide a selective advantage to P. multocida by generating antigenic variation.

Background It is widely believed that integral outer membrane (OM) proteins in bacteria are able to diffuse laterally in the OM. However, stable, immobile proteins have been identified in the OM of Escherichia coli. In explaining the observations, a hypothesized interaction of the immobilized OM proteins with the underlying peptidoglycan (PG) cell wall played a prominent role. Results OmpA is an abundant outer membrane protein in E. coli containing a PG-binding domain. We use FRAP to investigate whether OmpA is able to diffuse laterally over long-range (> ~100 nm) distances in the OM. First, we show that OmpA, containing a PG binding domain, does not exhibit long-range lateral diffusion in the OM. Then, to test whether PG interaction was required for this immobilization, we genetically removed the PG binding domain and repeated the FRAP experiment. To our surprise, this did not increase the mobility of the protein in the OM. Conclusions OmpA exhibits an absence of long-range (> ~100 nm) diffusion in the OM that is not caused by its PG binding domain. Therefore, other mechanisms are needed to explain this observation, such as the presence of physical barriers in the OM, or strong interactions with other elements in the cell envelope.

The energetic mechanism of preprotein export in Escherichia coli has been a source of controversy for many years. In vitro studies of translocation reactions that use purified soluble and membrane components have now clarified the main features of this mechanism. Translocation occurs through consecutive steps which each have distinct energy requirements. Initiation of translocation requires ATP and the SecA protein. Most of the further steps can be driven by the protonmotive force (DELTA-p).

The folding reaction of a β-barrel membrane protein, outer membrane protein A (OmpA), is probed with Förster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. This article is part of a Special Issue entitled: Membrane protein structure and function.