Foodborne diseases caused by non-typhoidal Salmonella represent an important public health problem worldwide (Zhao et al., 2003). The transmission of Salmonella between animals and humans has been well established in epidemiological studies. In the case of complicated illness caused by Salmonella where antibiotics need to be administered, treatment can be compromised if the infecting organism is resistant to the prescribed antimicrobial agent. This study and earlier studies have shown that many Salmonella carry temperate bacteriophages as lysogens. Many of these bacteriophages are capable of mediating generalised transduction (Schicklmaier and Schmieger, 1995; Schicklmaier et al., 1998; Mmolawa et al., 2002). Schmieger and Schicklmaier (1999) demonstrated that bacteriophages ES18 and PDT17 are capable of transduction of antibiotic resistance genes from DT104. Phage-mediated transduction of antibiotic resistance genes has been largely neglected in the study of genetic transfer of antibiotic resistance in bacteria. This study investigates whether bacteriophages exist in antibiotic resistant Salmonella isolates. Such temperate phages in antibiotic resistant isolates could play a significant role in the transfer of resistance to other species of enteric bacteria, such as E. coli. Molecular epidemiology studies of antibiotic resistance genes were undertaken with Salmonella isolates from chicken, pig and human sources that were subjected to PCR for ampicillin (blaTEM-1), tetracycline (tetA, tetB) and streptomycin (aadA1, aadA2, strA, strB) resistance genes as well as Class 1 integrons. The blaTEM-1 gene was widely detected in isolates from pigs and chickens but rarely detected in human isolates. The tetB gene was more commonly found in pig isolates, while the tetA gene was associated with tetracycline resistance in chicken isolates. The strA and strB genes were responsible for streptomycin resistance in the S. Typhimurium isolates while the aadA1 gene was commonly detected in S. Kiambu and S. Virchow isolates. The aadA2 gene was associated with streptomycin resistance in the S. Ohio isolates from pigs. Class 1 integrons were widely distributed across serovars tested from chicken, pig and human sources. Temperate bacteriophages were induced using mitomycin C from antibiotic resistant Salmonella. These phages were able to infect antibiotic-sensitive Salmonella isolates from humans. Bacteriophages induced from one S. Sofia isolate also plaqued on Shigella flexneri. Bacteriophages induced from one S.Kiambu isolate and S. Typhimurium DB21 with an inserted Tn10 transposon (S. Typhimurium DB21 Tn10) were capable of transducing ampicillin and tetracycline resistance, respectively into S. Enteritidis PT1 isolates by in vitro methods. The molecular basis for resistance was established in subsequent PCR for antibiotic resistance genes in donor and recipient strains. This finding, in particular in the wild-type S. Kiambu strain, indicates that Salmonella from a natural source are able to infect and transfer antibiotic resistance by generalised transduction in controlled laboratory experiments. This current study has investigated the transfer of tetracycline and ampicillin resistance from a wild-type Salmonella strain and a laboratory strain of Salmonella to wild-type Salmonella bacteria as it occurs within the normal flora of the chicken gastrointestinal tract. It was demonstrated that the genetic transfer of tetracycline and ampicillin resistance genes as well as Class 1 integrons can occur within the chicken gastrointestinal tract. Transfer of tetracycline and ampicillin resistance could be demonstrated both in vitro and by using bacteriophage lysates obtained from in vivo studies in transduction experiments. It was clearly shown that bacteriophage isolated from chicken faeces and caeca could infect antibiotic sensitive recipient Salmonella. Interaction between phages of the administered Salmonella strains may be occuring with phages of bacteria in the normal flora allowing previously inactive phage in the indigenous flora to plaque on indicator strains. Additionally, strong evidence was presented to suggest that the environment of the chicken gastrointestinal tract could mediate phage type conversion in recipient and transductant strains. Phage typing of these recipient and transductant strains demonstrated a trend for recipient strains to become more resistant to phages in the S. Enteritidis typing panel. This led to weakened phage reactions such RDNC (reaction does not conform) and untypable. The acquisition of phages may be a way for Salmonella to enhance competitive fitness and generate new strains in order to evolve and diversify. Or the acquisition of plasmids either by transduction or conjugation may also mediate phage type conversion. MLVA typing was performed on selected recipient, donor and transductant strains. The changes to tandem repeat loci in Salmonella isolates that have passed through a chicken gastrointestinal tract have not been described before. The changes to fragment length suggest that the bacterial chromosome is undergoing rearrangement; this may be attributed to a number of factors including acquisition of phages, prophage integration into tRNA sites, slipped-strand mispairing or the adaption to changing environment, in this case the chicken gastrointestinal tract. This study has provided molecular epidemiological data on the antibiotic resistance genes and integrons present in Australian Salmonella isolates from human and animal sources. Information on the role of bacteriophages in the transfer of antibiotic resistance genes in vitro and in a chicken gastrointestinal tract has also been established. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2010

Ph.D.

With the spread of antibiotic resistance, alternative treatment methods for bacterial pathogens are needed. Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that is a common cause of healthcare associated infections and is listed as a critical priority for research and development of treatments by the Centers for Disease Control and Prevention. P. aeruginosa poses an increased risk to patients within the surgical or intensive care unit, patients with indwelling catheters, cystic fibrosis, and burn wound victims. With a paucity of antibiotics in the pipeline for Gram negative bacteria, phage therapy has reemerged as a potential treatment option. Bacteriophages were first discovered in 1917 by Felix d’Herelle, but by the end of World War II, they were all but forgotten in favor of antibiotics. Eastern European countries and the former Soviet Union continued to develop phage therapy since its discovery, but studies were not on par with today’s standards. Recently the idea of phage therapy has reemerged in the Western world due to antibiotic resistance. In vitro and in vivo studies have shown that bacteriophages are easily isolated from the environment, with P. aeruginosa specific phages commonly found in hospital waste water and in sewage. Phage therapy has shown to be very effective at treating planktonic and biofilm forms of antibiotic resistant P. aeruginosa in vitro and in vivo. In humans, clinical trials are limited but phage therapy has successfully treated chronic otitis infections caused by P. aeruginosa and other studies have demonstrated the safety of phage therapy, reporting mild, if any, adverse effects. Bacteriophages may also synergize with several antibiotics, suggesting it may be beneficial to use them in conjunction to treat difficult or chronic infections. Additionally, P. aeruginosa bacteriophages may be beneficial in prophylactic treatment as well. When phages were combined with chlorine, a significant decrease in P. aeruginosa counts in chronic biofilms was observed, while also reducing its ability to form new biofilms. Similar results were noted when phages were applied to the lumen of catheters. These early results are promising for the future, but there are many steps that must be taken before starting new clinical trials and the widespread use of phage therapy begins.

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

No description available.

Leishmania is a protozoan parasite of vertebrates transmitted by the bite of infected phlebotomine sandflies. In humans, it causes a disease called leishmaniasis, which ranks as one of the most serious neglected tropical diseases. In the vectorial part of the life cycle, the crucial moment is when the flagellate forms (promastigotes) attach to the midgut epithelium of the sandfly. For most leishmania species, little is known about which types of phlebotomine receptors and leishmania surface antigens participate in the binding. Phage display was used to screen for Leishmania mexicana peptide ligands which may play a role in such binding. By affinity selection of phages incubated with promastigote cells, 16 unique peptides were identified. Fluorescent labelling of peptide-bearing phages indicated their putative binding sites on the leishmania surface. Based on the hypothesis that the identified peptides may be a part of receptors found in the phlebotomine midgut, experiments were performed where the sandflies were infected with promastigotes whose binding sites were blocked by two different peptide-bearing phages. The extent of the infection was different between the two cases. However, no statistically significant difference from the control group was observed. Despite unsuccessful attempts to identify a...

In affinity protein biotechnology the selective bindingbetween a chosen protein and an interacting biomolecule isutilized for a variety of applications including bioseparation,detection and therapy. Traditionally, affinity proteinsrecruited for such applications have been derived from naturalproteins or immunoglobulins generated via immunization routes.More recently, advances in the construction and handling oflarge collections of proteins(denoted libraries) generated invitro have opened up for new routes for the development ofaffinity proteins with desired properties. In this study, phage display selection technology was usedfor the isolation of novel human CD28 (hCD28)-specific affinityproteins from a protein library constructed by combinatorialprotein engineering of a 58 aa protein domain (Z) derived fromstaphylococcal protein A (SPA). From selections using hCD28 asa target molecule, several hCD28-specific affinity proteins(denoted affibodies) could be identified and analysis of theisolated affibody variants revealed a high degree of sequencehomology between the different clones. The biosensor analysisshowed that all variants bound to hCD28 with micromolardissociation constants (KD) and no significant cross-reactivitytowards the structurally related T-cell receptor hCTLA-4 couldbe observed. The apparent binding affinity for hCD28 of one ofthe isolated affibodies was further improved through fusion toa human Fc fragment fusion partner, resulting in a homodimericversion of the affibody ligand showing avidity effects uponhCD28 binding. Further, a co-culture experiment involvingJurkat T-cells and CHO cell lines tranfected to express eitherhuman CD80 or LFA-3 on the cell surface showed that apreincubation of Jurkat cells with one of the affibody variantsresulted in a specific concentration-dependent inhibition ofthe CD80 induced IL-2 production. This indicates that thisaffibody binds to hCD28 and specifically interferes with theco-stimulation signal mediated via hCD28 and hCD80. ACD28-specific binding protein could have potential as an agentfor various immunotherapy applications. In a second study, anaffinity protein-based strategy was investigated forsite-specific anchoring of proteins onto cellulose for woodfiber engineering purposes. Here, affinity proteins derivedfrom different sources were used for the assembly of acellulosome-like complex for specific and reversible anchoringof affinity domain-tagged reporter proteins to acellulose-anchored fusion protein. A fusion protein between acellulose binding module (Cel6A CBM1) derived from the fungalTrichoderma reesei and a five-domain staphylococcal protein A(SPA) moiety was constructed to serve as a platform for thedocking of reporter proteins produced as fusion to two copiesof a SPA-binding affibody affinity protein (denoted ZSPA-1),selected by phage display technology from a Z domain basedprotein library. In a series of experiments, involving repeatedwashing and low pH elutions, affinity tagged Enhanced GreenFluorescent Protein (EGFP) and Fusarium solani pisi lipasecutinase reporter proteins were both found to be specificallydirected from solution to a region of a cellulose-based filterpaper where the SPA-CBM fusion protein previously had beenpositioned. This showed that the cellulose-anchored SPA-Cel6ACBM1 fusion protein had been stably anchored to the surfacewith retained binding activity and that the interaction betweenSPA and the ZSPA-1 affibody domain was selective. phage display, combinatorial, selection, CD28, cellulosome,cellulose, affibody / NR 20140805

Bacteriophages, or simply "phages," are the most abundant biological entities on the planet and are thought to be the largest untapped reservoir of available genetic information. They are also important contributors to both soil health and nutrient recycling and have significantly influenced our current understanding of molecular biology. Bacteria in the genus Streptomyces are also known to be important contributors to soil health, as well as producing a number of useful antibiotics. The genetic diversity of large (> 30) groups of other actinobacteriophages, i.e. phages infecting a few close relatives of the Streptomycetes, has been explored, but this is the first formal effort for Streptomyces-infecting phages. Described here are a group of 45 phages, isolated from soil using a single Streptomycete host, Streptomyces griseus ATCC 10137. All 45 phages are tailed phages with double-stranded DNA. Siphoviruses predominate, six of the phages are podoviruses, and no myoviruses were observed. Notably present are seven phages with prolate icosahedral capsids. Genome lengths and genome termini vary considerably, and the distributions of each are in line with findings among other groups of studied actinobacteriophages. Interestingly, the average G+C among the 45 phages is around 11% lower than that of the isolation host, a larger disparity than reported for other groups of actinobacteriophages. Eighteen of the phages carry between 17 and 45 tRNAs and 12 of those carry a single tmRNA. Forty-three phages were grouped into seven clusters and two subclusters based on dot plot analysis, average nucleotide identities, and gene content similarities. Two phages were not clustered with other phages in this dataset. A total of 5250 predicted genes were sorted into 1300 gene "phamilies," with about 8% of the total phamilies having only a single member. Analysis of gene content among the 45 phages indicates first that most clusters presented here appear to be relatively isolated from one another, with phages in any one cluster generally sharing < 10% of their genes with phages in other clusters described here. Secondly, most of the phages here are more than twice as likely to share genes with phages isolated on bacteria outside of the genus Streptomyces than they are other phages isolated using a Streptomycete as host. These observations suggest that (1) the phage clusters here have a distinct extended host range, (2) those host ranges share overlap, and (3) Streptomyces griseus is likely not the preferred natural host for all phages described.

Thesis advisor: Eranthie Weerapana / Antibiotic resistance of bacterial pathogens poses an increasing threat to the wellbeing of our society and urgently calls for new strategies for infection diagnosis and antibiotic discovery. The overuse and misuse of broad-spectrum antibiotics has contributed to the antibiotic resistance crisis. Additionally, treatment of infections with broad-spectrum antibiotics can cause disruption to the host gut microbiome. The development of narrow-spectrum antibiotics would be ideal to avoid unnecessary cultivation of antibiotic resistance and damage to the human microbiota. Bacteria present many mechanisms of resistance, including modulating their cell surface with amine functionalities. In an age where infections are no longer responding to typical antibiotic treatments, novel drugs that target the characteristics of antibiotic resistance would be beneficial to remedy these defiant infections. Herein, we describe the utility of iminoboronate formation to target the amine- presenting surface modifications on bacteria, particularly those that display antibiotic resistance. Specifically, multiple 2-acetylphenylboronic acid warheads were incorporated into a peptide scaffold to develop potent peptide probes of bacterial cells. Further, by engineering a phage display library presenting the 2-acetylphenylboronic acid moieties, we were able to perform peptide library screens against live bacterial cells to develop reversible covalent peptide probes of target strains of bacteria. These peptide probes, which were developed for clinical strains of Staphylococcus aureus and Acinetobacter baumannii which display resistance, can label the target bacterium at submicromolar concentrations in a highly specific manner and in complex biological milieu. We further show that the identified peptide probes can be readily converted to bactericidal agents that deliver generic toxins to kill the targeted bacterial strain with high specificity. It is conceivable that this phage display platform is applicable to a wide array of bacterial strains, paving the way to facile diagnosis and development of strain-specific antibiotics. Furthermore, it is intriguing to speculate that even higher potency binding could be accomplished with better designed phage libraries with dynamic covalent warheads. This work is currently underway in our laboratory. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Gp12 est une protéine qui se fixe symétriquement au centre de chacun des 60 hexamères de la capside icosaédrique du bactériophage SPP1. La protéine produite dans un système d’expression hétérologue se lie à la capside de particules virales dont le gène codant gp12 a été inactivé. Cette interaction a lieu spécifiquement avec des capsides qui ont subi le processus d’expansion et encapsulé l'ADN viral.L'analyse de la séquence de gp12 montre la présence d'un motif (GXY)n retrouvé dans des protéines de type collagène. Nous avons démontré que gp12 est un trimère allongé en solution. Ce trimère s'avère sensible à la collagénase VII qui coupe la protéine gp12 dans un site spécifique du motif (GXY)8. Le profil de dichroïsme circulaire de gp12 porte aussi la signature d'une protéine de type collagène. La fixation de gp12 sur la capside virale conduit à une augmentation de 20°C de sa stabilité thermique. Gp12 peut être dénaturée-dissociée et puis renaturée-reassociée sous l'effet de la température. Le trimer de gp12 et sa forme dénaturée se fixent à la capside de SPP1 mais avec des profils d’interaction différents. Ces propriétés permettent d’utiliser gp12 comme un ligand réversible de la capside phagique en fonction de la température. Gp12 a une organisation modulaire avec un motif collagène qui sépare les modules amino et carboxyl-terminaux. Des protéines avec une organisation similaire sont codées par des gènes adjacents à celui codant pour la protéine majoritaire de la capside dans des prophages de Bacilli, suggérant une fonction similaire à gp12. Leurs modules ont une taille variable. Une recherche de protéines procaryotes et virales avec des segments collagène a montré qu’elles sont abondantes parmi les bactéries et les virus. Le motif est rare parmi les archées et leurs virus. Ces résultats montrent l’importance des protéines avec des séquences de type collagène dans le monde non-eucaryote et du développement de leur étude biochimique et fonctionnelle. / Gp12 is a protein found distributed symmetrically at the surface of the icosahedral capsid from bacteriophage SPP1. Recombinant gp12 binds to phage particles whose gene coding for gp12 was disrupted. This interaction occurs specifically with capsids that undergone expansion and packaged DNA.The gp12 protein sequence is marked by the presence of a stretch of 8 repeats of a GXY motif, which is the sequence signature of collagen. Our results showed that gp12 is an elongated trimer in solution. The trimer is sensitive to collagenase VII that cuts the gp12 protein inside the collagen motif. Its circular dichroism profile has also the signature of a collagen-like protein. Binding of gp12 to SPP1 capsids increases its thermal stability by 20°C. Gp12 is denatured and dissociated reversibly by temperature shift. The gp12 trimer and its denatured form bind to SPP1 capsids but with a different interaction behavior. These properties allow to use gp12 as thermo-switchable SPP1 capsid binder. Gp12 has a modular organization with a central collagen motif that connects the amino and carboxyl termini. Proteins with a similar organization that are encoded by genes adjacent to the gene coding for the major capsid protein were identified in prophages of Bacilli, suggesting a function similar to gp12. Their modules have a variable length.A pangenome-wide search for collagen-like proteins in prokaryotes and viruses shows that they are abundant among bacteria and viruses. In contrast, this motif is rare is archaea and their viruses. Our analysis highlights the importance of collagen-like proteins in the non-eukaryotic world and supports the interest to develop their biochemical and structural study.