The outer membrane (OM) is a formidable barrier that has made antibiotic drug discovery in Gram-negatives exceedingly difficult. Many antibiotics which are effective against Gram-positive bacteria cannot permeate the Gram-negative OM to reach their intracellular targets. Thus, it is important to explore unconventional approaches to overcome the intrinsic resistance conferred by the OM. Herein, we used both genetic and chemical means to compromise OM integrity to potentiate the activity of large-scaffold antibiotics against Escherichia coli. First, we mapped the genetic interaction network of OM biosynthetic genes using synthetic genetic arrays (SGAs) to reveal permeability determinants of the E. coli OM. This led to the creation of a publicly accessible dataset of ~155,400 double deletion strains with growth data in the presence of the large-scaffold antibiotics rifampicin and vancomycin. Investigations of a subset of synthetic sick interactions revealed connectivity in the context of permeability between lipopolysaccharide (LPS) inner core biosynthetic genes and an enigmatic gene involved in enterobacterial common antigen (ECA) regulation. Second, we leveraged a chemical screening platform based on the observation that disruption of the E. coli OM leads to antagonism of vancomycin activity at cold temperatures to uncover molecules that potentiate Gram-positive-targeting antibiotics at 37 ºC. Two of these compounds, liproxstatin-1 and MAC-0568743, were characterized to bind to LPS and disrupt OM integrity specifically without impacting the inner membrane (IM). Third, we performed genetic and chemical screening to unearth targets capable of potentiating the activity of Gram-positive-targeting antibiotics against E. coli. This validated the OM as a valuable target for antibiotic adjuvants and led to the discovery of two membrane active compounds and an inhibitor of lipid A biosynthesis. Overall, this thesis emphasizes the importance of elucidating biological factors contributing to OM permeability and the attractiveness of the OM as a target for antibiotic potentiators. / Thesis / Doctor of Philosophy (PhD)

There is an urgent need to identify novel antibiotics for multidrug-resistant Gram-negative pathogens. These bacteria are intrinsically resistant to many antimicrobials due to a formidable outer membrane barrier. Herein we investigate the potential of perturbing the outer membrane to sensitize Gram-negative bacteria to otherwise inactive antibiotics. In chapter 2, we identify the ability of mcr-1 mediated resistance to confer protection from the lytic but not outer membrane-perturbing activity of colistin. Exploiting this sensitivity, we show that colistin and clarithromycin in combination are efficacious against mcr-1-expressing Klebsiella pneumoniae in murine infection models. This demonstrates the viability of colistin combination therapies against Gram-negative pathogens harbouring mcr-1, and points to a mechanism of mcr-1-mediated resistance extending beyond the predicted reduction in binding affinity of polymyxins to the outer membrane. We continue to investigate the potential of using outer membrane perturbants with otherwise inactive antimicrobials in chapter 3. In this work, we identify the ability of OM disruption to change the rules of Gram-negative entry, render pre-existing resistance ineffective, reduce the development of spontaneous resistance and attenuate biofilm formation. Together, these data suggest that OM disruption overcomes many traditional hurdles encountered during antibiotic treatment and is a high priority approach for further development. / Thesis / Doctor of Philosophy (PhD)

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 112-126).

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 8 uppsatser.

Aeromonas hydrophila ML09-119 is an important fish pathogen that severely affects channel catfish aquaculture. To better understand this strain’s virulence factors, outer membrane vesicles (OMVs) were isolated, and their proteome was assessed. Using transmission electron microscopy and dynamic light scattering, OMVs were shown to be monodispersed particles with an average diameter of 120.33 nm. OMV proteins were identified using mass spectrometry, and analysis of the resulting proteome of 74 proteins revealed that many originated from the cytoplasm, but there was an enrichment of outer membrane, periplasmic, and extracellular proteins compared to the total proteome. The majority of the functional classifications were associated with bacterial metabolism. Of the predicted virulence factors, several had a putative function in adherence, and there were type III secretions system proteins as well as three secreted exotoxins. Overall, our data reveal new insights into A. hydrophila OMVs and their potential roles in physiology and virulence.

No description available.

Bacteroides fragilis is an important, anaerobic commensal of the human gastro-intestinal tract. As a Gram-negative bacterium, B. fragilis produces a large number of outer membrane vesicles (OMV), spherical globules consisting of outer membrane and periplasmic material, which have a range of potential functions and which are known to be able to deliver their cargo to host dendritic cells (DCs). One of the proteins believed to be packaged into the OMV of B. fragilis is BfUbb (encoded by the ubb gene) which shares 63% homology with human ubiquitin. Ubiquitin is a small, common, eukaryotic protein modifier, which is conjugated to target proteins via a series of activating, conjugating and ligating enzymes, and which has known roles in a wide range of eukaryotic cell processes. Due to key differences between the two proteins, BfUbb has the potential to act as a suicide substrate mimic of ubiquitin. BfUbb was therefore assayed for its ability to interact with ubiquitin E2 conjugating enzymes of the ubiquitylation cascade in vitro, and was found to covalently bind the majority of available enzymes in a DTT-sensitive manner. BfUbb showed a preference for three specific E2 enzymes, all of which are involved in the degradation of mitotic check point proteins, suggesting a role for BfUbb in the inhibition of cell cycle progression and, consequently, tumorigenesis. No binding partners of BfUbb were identified outside of the ubiquitylation cascade, however BfUbb was found to form spontaneous multimers in vitro, the biological function of which is unknown. This study also describes the construction of two sets of plasmids. The first set will allow the expression of untagged and fluorescently tagged forms of BfUbb for purification and use in biochemical assays. The second set will allow the expression of his-tagged and fluorescently tagged forms of BfUbb in mammalian cells, so that the effects of BfUbb on the host epithelial cells may be studied. The proteome of the OMV of B. fragilis was solved using LTQ-Orbitrap mass spectrometry. The identified proteins indicated several putative roles for B. fragilis OMV, including nutrient acquisition and protease inhibition. The suitability of techniques used during the isolation and proteomic analysis of OMV in different studies is discussed. BfUbb-carrying B. fragilis OMV were able to inhibit growth of Salmonella enterica Typhimurium, thus indicating a role for BfUbb in the inhibition of competing, pathogenic bacteria in the gastro-intestinal tract. The conclusions of this study are that the putative roles of both BfUbb and the OMV of B. fragilis may promote both survival of the bacterium and the gastro-intestinal health of the host.

Lipopolysaccharide (LPS) is the glycolipid that comprises the outer leaflet of the Gram-negative outer membrane (OM). Because it is essential in nearly all Gram-negative species, and because it is responsible for making these bacteria impervious to many types of antibiotics, LPS biogenesis has become an important area of research. While its biosynthesis at the cytoplasmic face of the inner membrane (IM) is well studied, the process by which it is removed from the IM, transported across the aqueous periplasmic compartment, and specifically inserted into the outer leaflet of the OM is only beginning to be understood. This transport process is mediated by the essential seven-protein LPS transport (Lpt) complex, LptA/B/C/D/E/F/G. The OM portion of the exporter, LptD/E, is a unique plug-and-barrel protein complex in which LptE, a lipoprotein, sits inside of LptD, a β-barrel integral membrane protein. LptD is of particular interest, as it is the target of an antibiotic in Pseudomonas aeruginosa. Part I of this thesis investigates how the cell forms the two non-consecutive disulfide bonds that connect LptD's C-terminal β-barrel to its N-terminal soluble domain. These disulfides, one of which is almost universally conserved among Gram-negatives, are essential for cell viability. Here, we show that an intermediate oxidation state with non-native disulfide bonds accumulates in the absence of LptE and in strains defective in either LptE or LptD. We then demonstrate that this observed intermediate is on-pathway and part of the native LptD oxidative folding pathway. Using a defective mutant of DsbA, the protein that introduces disulfide bonds into LptD, we are able to identify additional intermediates in the LptD oxidative folding pathway. We ultimately demonstrate that the disulfide rearrangement that activates the LptD/E complex occurs following an exceptionally slow β-barrel assembly step and is dependent on the presence of LptE. Part II describes work towards obtaining X-ray crystal structures of the LptD N-terminal domain and LptD/E complex. Expression construct and purification optimization enabled the production of stable LptD/E in quantities that make crystallography feasible. Numerous precipitants, detergents, and additives were screened, ultimately resulting in protein crystals that diffract to a resolution of 3.85 Å. / Chemistry and Chemical Biology

Legionella spp. can cause a life threatening form of pneumonia, which is observed world-wide. Outbreaks of the disease are, unfortunately, not a rare event, despite the introduction of government regulations which enforce the mandatory testing of cooling towers to ensure that they contain levels of the organism which are regarded as being within safe limits. Therefore, cooling towers should be monitored for Legionella spp. by using a biosensor. These could potentially save the community from a great deal of morbidity and mortality due to legionellosis. This study identified and investigated novel outer membrane proteins in L. pneumophila, and analysed their potential for use in a Legionella biosensor. A combination of bioinformatics and laboratory investigations was used to identify the Omp87, an outer membrane protein of L. pneumophila which had not been previously described in this organism. Sequence analysis of the protein showed that it shares similarity with various other members of the Omp85 protein family, including the D15 antigen of Haemophilus influenzae and the Oma87 of Pseudomonas aeruginosa. The omp87 gene of L. pneumophila was amplified and cloned, and was found to encode a protein of 786 amino acids, with a molecular weight of 87 kDa. Distribution studies revealed that the gene is present in most, but not all species and serogroups of Legionella. To investigate the function of the Omp87 protein in L. pneumophila, the omp87 gene was insertionally inactivated with the use of a kanamycin resistance gene. Amplicons of this disrupted gene were then introduced into L. pneumophila, and a double-cross over event occurred, integrating the inactivated gene into the genome of the organism. This resulted in non-viable cells, indicating that the gene is essential in L. pneumophila. The expression vector pRSETA was used to express the Omp87 protein in E. coli, and four truncates of varying sizes were designed, through the use of different PCR primers. Two of the protein truncates were then expressed and purified by gravity flow chromatography using columns packed with Ni-NTA sepharose resin. Following analysis of the proteins by SDS-PAGE and Western blotting, polyclonal antibodies were raised against the truncates. Distribution studies were then performed using the antiserum with different strains and species of Legionella. This study demonstrated that most serogroups of L. pneumophila, and most other Legionella species reacted with the polyclonal anti-Omp87 L. pneumophila antisera. Cross-reactivity was also observed with most other Legionella related organisms tested. The results presented in this thesis demonstrated that the Omp87 protein or the omp87 gene can be used to construct a biosensor. In addition other novel outer membrane proteins were identified which could also serve as potential targets for a biosensor. These biosensors will be able to identify Legionella spp. in water reservoirs and in clinical samples and hopefully reduce the number of infections and deaths caused by this organism.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/01/15). Includes bibliographical references.