Identification and characterization of periplasmic proteins implicated in the adaptation of Helicobacter pylori to the human gastric niche

Helicobacter pylori è un batterio Gram-negativo in grado di colonizzare la mucosa gastrica umana e persistere per l'intero arco della vita dell'ospite. E' associato a patologie gastrointestinali, quali gastrite cronica, ulcere gastriche e duodenali, adenocarcinomi e linfomi gastrici. Si tratta di uno dei patogeni più diffusi, presente in circa metà della popolazione mondiale, e il solo che si è adattato a vivere nell'ambiente ostile dello stomaco umano. Molteplici sono i fattori di virulenza che permettono al batterio la colonizzazione della nicchia gastrica e contribuiscono, anche attraverso l' induzione di una risposta infiammatoria, a profonde modificazioni dell' omeostasi gastrica. Queste ultime si associano, ad esempio, all'iperproduzione di fattori proinfiammatori, ad alterazioni sia della regolazione della secrezione acida gastrica sia del ciclo cellulare e della morte cellulare programmata (apoptosi) delle cellule epiteliali gastriche, a disordini nel metabolismo del ferro e a carenze di elementi essenziali. Studi sulla diversità genetica di H. pylori osservata in ceppi isolati da varie regioni del mondo, dimostrano che tale batterio ha avuto una coevoluzione col genere umano attraverso la storia, ed è verosimile che H. pylori sia stato un costituente del microbiota gastrico per almeno 50.000 anni. Scopo della tesi è stato quello di identificare e caratterizzare proteine importanti per la colonizzazione e l'adattamento di H. pylori alla nicchia gastrica. In particolare gli sforzi si sono concentrati su due proteine periplasmatiche, la prima coinvolta nella difesa antiossidante (l'enzima catalasi-like, HP0485), e la seconda nel trasporto di nutrienti presenti nell'ambiente dello stomaco all'interno della cellula (la componente solubile di un ABC transporter, HP0298). La strategia utilizzata prevede un'analisi bioinformatica preliminare, l'ottenimento del gene per amplificazione, mediante PCR, dal genoma dell'organismo, la costruzione di un vettore per il clonaggio, l'espressione eterologa in E. coli e la successiva purificazione. La proteina così ottenuta viene caratterizzata mediante diverse tecniche, quali spettroscopia UV, dicroismo circolare, gel filtrazione analitica, spettrometria di massa. Il capitolo 1 contiene un'introduzione generale sul batterio, il capitolo 2 e il capitolo 3 descrivono gli studi relativi alle due proteine e sono entrambi suddivisi in un abstract iniziale, un'introduzione, la presentazione dei risultati, la discussione di questi ultimi, i materiali e i metodi utilizzati. La catalasi-like (HP0485) è una proteina periplasmatica con struttura monomerica, appartenente ad una famiglia di enzimi a funzione per la maggior parte sconosciuta, ma evolutivamente correlati alla ben nota catalasi, attore fondamentale nella difesa di H. pylori, grazie alla sua azione specifica di rimozione dell'acqua ossigenata. HP0485, pur conservando il fold catalasico e il legame al cofattore eme, non può compiere la reazione di dismutazione dell'acqua ossigenata; possiede invece un'attività perossidasica ad ampio spettro, essendo in grado di accoppiare la riduzione del perossido di idrogeno all'ossidazione di diversi substrati. Come la catalasi, lavora ad alte concentrazioni di aqua ossigenata e non arriva a saturazione a concentrazioni molto elevate di questo substrato (200 mM); la velocità di reazione catalizzata rimane lineare anche a questi valori, aspetto che la differenzia dalle perossidasi che vengono in genere inattivate da concentrazioni di perossido di idrogeno superiori a 10-50 mM. Queste caratteristiche di versatilità e robustezza suggeriscono che la catalasi-like abbia un ruolo di scavenger dell'acqua ossigenata e probabilmente anche un'altra funzione connessa al suo secondo substrato, ossia l'ossidazione di composti nello spazio periplasmatico cellulare. Oltre alla caratterizzazione dell'attività è descritta anche la presenza di un ponte disolfuro, conservato nelle catalasi-like periplasmatiche, con un ruolo nell'assemblaggio dell'eme per ottenere un enzima attivo e funzionale. La proteina periplasmatica HP0298, componente di un sistema di trasporto ABC, è classificata come trasportatore di dipeptidi e appartiene a una famiglia di proteine in grado di legare diversi substrati, tra cui di- e oligopeptidi, nichel, eme, glutatione. Benchè tutte associate a trasportatori di membrana batterici, queste proteine presentano un dominio di legame al substrato che risulta essere conservato nei domini extracellulari di recettori specifici di mammifero e uomo. Un esempio sono i recettori ionotropici e metabotropici del sistema nervoso. Per caratterizzare questa proteina è stato messo a punto un protocollo di ligand-fishing accoppiato alla spettrometria di massa. La proteina purificata, avente un tag di istidine, è stata incubata con un estratto cellulare di H. pylori per poter interagire con il suo substrato specifico all'interno dell'ambiente naturale in cui avviene il legame. Il complesso proteina-ligando è stato poi purificato per cromatografia di affinità e analizzato mediante HPLC-MS. L'identificazione dei picchi differenziali tra campioni con la proteina e 5 campioni di controllo ha portato alla caratterizzazione di pentapeptidi particolarmente ricchi in aminoacidi idrofobici e con almeno un residuo carico negativamente. Considerando che H. pylori necessita di alcuni aminoacidi essenziali, per la maggior parte idrofobici, e che lo stomaco umano è particolarmente ricco di peptidi prodotti dalla digestione delle proteine introdotte con il cibo, il ruolo fisiologico di HP0298 potrebbe essere l'internalizzazione di peptidi, con caratteristiche specifiche di lunghezza e composizione, che sono naturalmente presenti nella nicchia gastrica.

Exploring several nonconventional interactions of ice-binding proteins

Traditionally, ice-binding proteins (IBPs), also known as antifreeze proteins (AFPs), have been defined by two universal activities: ice recrystallization inhibition and thermal hysteresis. However, there remains the possibility IBPs have other complementary functions given the diversity found within this protein group. This thesis explores some of these in both natural and applied settings, in the hopes of furthering our understanding of this remarkable group of proteins. Plant IBPs could function as part of a defensive strategy against ice nucleators produced by certain pathogens. To assess this hypothesis, recombinant IBPs from perennial ryegrass and purple false brome were combined with the ice nucleation protein (INP) from the plant pathogen, Pseudomonas syringae. Strikingly, the plant proteins depressed the freezing point of the bacterial INP, while a fish AFP could not, nor did the INPs have any effect on IBP activity. Thus, the interaction between these two different proteins suggests a role in plant defensive strategies against pathogenic bacteria as another IBP function. In addition, the potential use of hyperactive insect IBPs in organ preservation was investigated. Current kidney preservation techniques involve storing the organ at 4 °C for a maximum of 24 h prior to transplantation. Extending this “safe” time would have profound effects on renal transplants, however, ischemic injury is prevalent when storage periods are prolonged. Experiments described here allowed subzero preservation for 72 h with the addition of a beetle IBP to CryoStasis® solution. Kidneys stored using the traditional technique for 24 h and the method developed here for 72 h showed similar levels of biomarker enzymes, underscoring the potential utility of insect IBPs for future transplant purposes. Finally, IBP function in the freeze-tolerant gall fly, Eurosta solidaginis, was examined. Larvae representing the mid-autumn stage displayed ice-binding activity, suggesting an IBP is being expressed, possibly as a protective measure against freezing damage when fall temperatures can unpredictably drop. IBP activity was also observed in the larvae’s host plant, Solidago spp. Mass spectrometry analysis of ice-affinity purified plant extracts provided three candidate pathogenesis-related proteins that could be responsible for the detected activity, further demonstrating additional functions of IBPs.

Afffinity tag excision strategies for Car9-tagged proteins

Thesis (Master's)--University of Washington, 2016-06

Surfactant-free purification of membrane proteins with intact native membrane environment

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.

Mass spectrometry-based methods for identifying oxidized proteins in disease:advances and challenges

Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease.

Efficient purification of chromatin architectural proteins:histones, HMGB proteins and FKBP3 (FKBP25) immunophilin

A two-step process of high ionic strength lysis of chicken erythrocyte cell nuclei followed by cation-exchange chromatography has separated at very high yield all the histone and HMGB (high-mobility group B) nuclear proteins, except the less-soluble histone tetramers. Surprisingly high yields of the nuclear immunophilin FKBP3 (FKBP25) and Hsp70 (heat-shock protein 70) co-fractionate with HMGB1 and HMGB3. Furthermore, these proteins can be separated by anion-exchange chromatography. The purified nuclear proteins retain their native, post-translational modification (PTM) marks, including those associated with chromatin-fibre remodelling. These marks are intimately associated with the control of the cell cycle. The methods herein are therefore of value for targeting these and other nuclear proteins for future proteomic studies in healthy and diseased cells. This journal is © 2012 The Royal Society of Chemistry.

A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids.

A systematic analysis of the role of GGDEF-EAL domain proteins in virulence and motility in Xanthomonas oryzae pv. oryzicola

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ΔXOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ΔXOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ΔXOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.

Exploring several nonconventional interactions of ice-binding proteins

Traditionally, ice-binding proteins (IBPs), also known as antifreeze proteins (AFPs), have been defined by two universal activities: ice recrystallization inhibition and thermal hysteresis. However, there remains the possibility IBPs have other complementary functions given the diversity found within this protein group. This thesis explores some of these in both natural and applied settings, in the hopes of furthering our understanding of this remarkable group of proteins. Plant IBPs could function as part of a defensive strategy against ice nucleators produced by certain pathogens. To assess this hypothesis, recombinant IBPs from perennial ryegrass and purple false brome were combined with the ice nucleation protein (INP) from the plant pathogen, Pseudomonas syringae. Strikingly, the plant proteins depressed the freezing point of the bacterial INP, while a fish AFP could not, nor did the INPs have any effect on IBP activity. Thus, the interaction between these two different proteins suggests a role in plant defensive strategies against pathogenic bacteria as another IBP function. In addition, the potential use of hyperactive insect IBPs in organ preservation was investigated. Current kidney preservation techniques involve storing the organ at 4 °C for a maximum of 24 h prior to transplantation. Extending this “safe” time would have profound effects on renal transplants, however, ischemic injury is prevalent when storage periods are prolonged. Experiments described here allowed subzero preservation for 72 h with the addition of a beetle IBP to CryoStasis® solution. Kidneys stored using the traditional technique for 24 h and the method developed here for 72 h showed similar levels of biomarker enzymes, underscoring the potential utility of insect IBPs for future transplant purposes. Finally, IBP function in the freeze-tolerant gall fly, Eurosta solidaginis, was examined. Larvae representing the mid-autumn stage displayed ice-binding activity, suggesting an IBP is being expressed, possibly as a protective measure against freezing damage when fall temperatures can unpredictably drop. IBP activity was also observed in the larvae’s host plant, Solidago spp. Mass spectrometry analysis of ice-affinity purified plant extracts provided three candidate pathogenesis-related proteins that could be responsible for the detected activity, further demonstrating additional functions of IBPs.

Effects of Purified Saccharomyces cerevisiae (1→3)-β-Glucan on Venous Ulcer Healing

Water-insoluble glucan was isolated from the baker’s yeast Saccharomyces cerevisiae. The yeast cells were treated with alkali and the residue then with acid. Chemical and NMR (1D and 2D) analyses showed that a linear (1→3)-β-glucan was purified that was not contaminated with other carbohydrates, proteins or phenolic compounds. The effects of the glucan on wound healing were assessed in human venous ulcers by histopathological analysis after 30 days of topical treatment. (1→3)-β-glucan enhanced ulcer healing and increased epithelial hyperplasia, as well as increased inflammatory cells, angiogenesis and fibroblast proliferation. In one patient who had an ulcer that would not heal for over 15 years, glucan treatment caused a 67.8% decrease in the area of the ulcer. This is the first study to investigate the effects of (1→3)-β-glucan on venous ulcer healing in humans; our findings suggest that this glucan is a potential natural biological response modifier in wound healing

Effects of Purified Saccharomyces cerevisiae (1→3)-β-Glucan on Venous Ulcer Healing

Water-insoluble glucan was isolated from the baker’s yeast Saccharomyces cerevisiae. The yeast cells were treated with alkali and the residue then with acid. Chemical and NMR (1D and 2D) analyses showed that a linear (1→3)-β-glucan was purified that was not contaminated with other carbohydrates, proteins or phenolic compounds. The effects of the glucan on wound healing were assessed in human venous ulcers by histopathological analysis after 30 days of topical treatment. (1→3)-β-glucan enhanced ulcer healing and increased epithelial hyperplasia, as well as increased inflammatory cells, angiogenesis and fibroblast proliferation. In one patient who had an ulcer that would not heal for over 15 years, glucan treatment caused a 67.8% decrease in the area of the ulcer. This is the first study to investigate the effects of (1→3)-β-glucan on venous ulcer healing in humans; our findings suggest that this glucan is a potential natural biological response modifier in wound healing

Effects of Purified Saccharomyces cerevisiae (1→3)-β-Glucan on Venous Ulcer Healing

Water-insoluble glucan was isolated from the baker’s yeast Saccharomyces cerevisiae. The yeast cells were treated with alkali and the residue then with acid. Chemical and NMR (1D and 2D) analyses showed that a linear (1→3)-β-glucan was purified that was not contaminated with other carbohydrates, proteins or phenolic compounds. The effects of the glucan on wound healing were assessed in human venous ulcers by histopathological analysis after 30 days of topical treatment. (1→3)-β-glucan enhanced ulcer healing and increased epithelial hyperplasia, as well as increased inflammatory cells, angiogenesis and fibroblast proliferation. In one patient who had an ulcer that would not heal for over 15 years, glucan treatment caused a 67.8% decrease in the area of the ulcer. This is the first study to investigate the effects of (1→3)-β-glucan on venous ulcer healing in humans; our findings suggest that this glucan is a potential natural biological response modifier in wound healing

Effects of Purified Saccharomyces cerevisiae (1→3)-β-Glucan on Venous Ulcer Healing

Water-insoluble glucan was isolated from the baker’s yeast Saccharomyces cerevisiae. The yeast cells were treated with alkali and the residue then with acid. Chemical and NMR (1D and 2D) analyses showed that a linear (1→3)-β-glucan was purified that was not contaminated with other carbohydrates, proteins or phenolic compounds. The effects of the glucan on wound healing were assessed in human venous ulcers by histopathological analysis after 30 days of topical treatment. (1→3)-β-glucan enhanced ulcer healing and increased epithelial hyperplasia, as well as increased inflammatory cells, angiogenesis and fibroblast proliferation. In one patient who had an ulcer that would not heal for over 15 years, glucan treatment caused a 67.8% decrease in the area of the ulcer. This is the first study to investigate the effects of (1→3)-β-glucan on venous ulcer healing in humans; our findings suggest that this glucan is a potential natural biological response modifier in wound healing

Efeito inseticida de vicilinas isoladas de sementes de erythrina velutina em condições de semi-campo para moscas das frutas (ceratitis capitata)

Chitin-binding vicilins from legume seeds (Erythrina velutina. Canavalia ensiformes and Phaseolus vulgares) were isolated by ammonium sulfate followed by affinity chromatography on a chitin column. Effect of these vicilins on female adults of Ceratitis capitata was examined by bioassay and in a semi-field assay model. Mechanism of action of the vicilins was determined by in vivo digestibility and chitin affinity. Among the tested vicilins, E. velutina when added to diet caused strong effect on mortality at 10% dose. This insecticidal property was tested in a semi-field assay which showed the same effect observed in laboratory conditions, where doses of 10% and 15% were lethal to female adults of C. capitata. These deleterious effects were not only associated to the binding to chitin structures present in peritrophic membrane, but principally to its low digestibility in the C. capitata digestive tract. This fact was confirmed because chiting binding proteins as WGA and the other tested vicilins were not toxic to female adults of C. capitata due susceptibility of these proteins to digestive enzymes of the insects. By other side EvV was more resistant to digestive enzymes, causing deleterious effects on female adults of C. capitata. These results showed that EvV may be part of the pest management programs or an alternative in plant improvement program in the population control of this fruticulture pest

KINESIN MOTOR PROTEINS ARE ESSENTIAL FOR MALE GAMETOPHYTE DEVELOPMENT IN MARSILEA VESTITA

The male gametophyte of the semi-aquatic fern, Marsilea vestita, produces multiciliated spermatozoids in a rapid developmental sequence that is controlled post-transcriptionally when dry microspores are placed in water. Development can be divided into two phases, mitosis and differentiation. During the mitotic phase, a series of nine successive division cycles produce 7 sterile cells and 32 spermatids in 4.5-5 hours. During the next 5-6 hours, each spermatid differentiates into a corkscrew-shaped motile spermatozoid with ~140 cilia. This document focuses on the role of motor proteins in the regulation of male gametophyte development and during ciliogenesis. In order to study the mechanisms that regulate spermatogenesis, RNAseq was used to generate a reference transcriptome that allowed us to assess the abundance of transcripts at different stages of development. Over 120 kinesin-like sequences were identified in the transcriptome that represent 56 unique kinesin transcripts. Members of the kinesin-2, -4, -5, -7, -8, -9, -12, -13, and -14 families, in addition to several plant specific and ‘orphan’ kinesins are present. Most (91%) of these kinesin transcripts change in abundance throughout gametophyte development, with 52% of kinesin mRNAs enriched during the mitotic phase and 39% enriched during differentiation. Functional analyses show that the temporal regulation of kinesin transcripts during gametogenesis directly correlates with kinesin protein function. Specifically, Marsilea makes one kinesin-2 (MvKinesin-2) and two kinesin-9 (MvKinesin-9A and MvKinesin-9B) transcripts, which are present during spermatid differentiation and ciliogenesis. Silencing experiments showed that MvKinesin-2 and MvKinesin-9A are required for ciliogenesis and motility in the Marsilea male gametophyte; however, these kinesins display atypical roles during these processes. In contrast, spermatozoids produced after the silencing of MvKinesin-9B exhibit normal morphology. MvKinesin-2 is necessary for cytokinesis as well as for regulating ciliary length and MvKinesin-9A is needed for the correct orientation of basal bodies, events not typically associated with these proteins. In addition, Marsilea makes motile, ciliated gametophytes without the help of IFT dynein, outer arm dynein, or the BBsome. These results are the first to investigate the kinesin-linked mechanisms that regulate ciliogenesis in a land plant.