The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

ABSTRACT Exported proteins of bacterial pathogens function both in essential physiological processes and in virulence. Past efforts to identify exported proteins were limited by the use of bacteria growing under laboratory ( in vitro ) conditions. Thus, exported proteins that are exported only or preferentially in the context of infection may be overlooked. To solve this problem, we developed a genome-wide method, named EXIT ( ex ported i n vivo t echnology), to identify proteins that are exported by bacteria during infection and applied it to Mycobacterium tuberculosis during murine infection. Our studies validate the power of EXIT to identify proteins exported during infection on an unprecedented scale (593 proteins) and to reveal in vivo induced exported proteins (i.e., proteins exported significantly more during in vivo infection than in vitro ). Our EXIT data also provide an unmatched resource for mapping the topology of M. tuberculosis membrane proteins. As a new approach for identifying exported proteins, EXIT has potential applicability to other pathogens and experimental conditions. IMPORTANCE There is long-standing interest in identifying exported proteins of bacteria as they play critical roles in physiology and virulence and are commonly immunogenic antigens and targets of antibiotics. While significant effort has been made to identify the bacterial proteins that are exported beyond the cytoplasm to the membrane, cell wall, or host environment, current methods to identify exported proteins are limited by their use of bacteria growing under laboratory ( in vitro ) conditions. Because in vitro conditions do not mimic the complexity of the host environment, critical exported proteins that are preferentially exported in the context of infection may be overlooked. We developed a novel method to identify proteins that are exported by bacteria during host infection and applied it to identify Mycobacterium tuberculosis proteins exported in a mouse model of tuberculosis

A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

<p>Abstract</p> <p>Background</p> <p>Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new <it>Henipavirus </it>genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.</p> <p>Results</p> <p>Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.</p> <p>Conclusions</p> <p>Together, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.</p

Calcium-Release Channels in Paramecium. Genomic Expansion, Differential Positioning and Partial Transcriptional Elimination

The release of Ca2+ from internal stores is a major source of signal Ca2+ in almost all cell types. The internal Ca2+ pools are activated via two main families of intracellular Ca2+-release channels, the ryanodine and the inositol 1,4,5-trisphosphate (InsP3) receptors. Among multicellular organisms these channel types are ubiquitous, whereas in most unicellular eukaryotes the identification of orthologs is impaired probably due to evolutionary sequence divergence. However, the ciliated protozoan Paramecium allowed us to prognosticate six groups, with a total of 34 genes, encoding proteins with characteristics typical of InsP3 and ryanodine receptors by BLAST search of the Paramecium database. We here report that these Ca2+-release channels may display all or only some of the characteristics of canonical InsP3 and ryanodine receptors. In all cases, prediction methods indicate the presence of six trans-membrane regions in the C-terminal domains, thus corresponding to canonical InsP3 receptors, while a sequence homologous to the InsP3-binding domain is present only in some types. Only two types have been analyzed in detail previously. We now show, by using antibodies and eventually by green fluorescent protein labeling, that the members of all six groups localize to distinct organelles known to participate in vesicle trafficking and, thus, may provide Ca2+ for local membrane-membrane interactions. Whole genome duplication can explain radiation within the six groups. Comparative and evolutionary evaluation suggests derivation from a common ancestor of canonical InsP3 and ryanodine receptors. With one group we could ascertain, to our knowledge for the first time, aberrant splicing in one thoroughly analyzed Paramecium gene. This yields truncated forms and, thus, may indicate a way to pseudogene formation. No comparable analysis is available for any other, free-living or parasitic/pathogenic protozoan

Bionano-Interfaces through Peptide Design

The clinical success of restoring bone and tooth function through implants critically depends on the maintenance of an infection-free, integrated interface between the host tissue and the biomaterial surface. The surgical site infections, which are the infections within one year of surgery, occur in approximately 160,000-300,000 cases in the US annually. Antibiotics are the conventional treatment for the prevention of infections. They are becoming ineffective due to bacterial antibiotic-resistance from their wide-spread use. There is an urgent need both to combat bacterial drug resistance through new antimicrobial agents and to limit the spread of drug resistance by limiting their delivery to the implant site. This work aims to reduce surgical site infections from implants by designing of chimeric antimicrobial peptides to integrate a novel and effective delivery method. In recent years, antimicrobial peptides (AMPs) have attracted interest as natural sources for new antimicrobial agents. By being part of the immune system in all life forms, they are examples of antibacterial agents with successfully maintained efficacy across evolutionary time. Both natural and synthetic AMPs show significant promise for solving the antibiotic resistance problems. In this work, AMP1 and AMP2 was shown to be active against three different strains of pathogens in Chapter 4. In the literature, these peptides have been shown to be effective against multi-drug resistant bacteria. However, their effective delivery to the implantation site limits their clinical use. In recent years, different groups adapted covalent chemistry-based or non-specific physical adsorption methods for antimicrobial peptide coatings on implant surfaces. Many of these procedures use harsh chemical conditions requiring multiple reaction steps. Furthermore, none of these methods allow the orientation control of these molecules on the surfaces, which is an essential consideration for biomolecules. In the last few decades, solid binding peptides attracted high interest due to their material specificity and self-assembly properties. These peptides offer robust surface adsorption and assembly in diverse applications. In this work, a design method for chimeric antimicrobial peptides that can self-assemble and self-orient onto biomaterial surfaces was demonstrated. Three specific aims used to address this two-fold strategy of self-assembly and self-orientation are: 1) Develop classification and design methods using rough set theory and genetic algorithm search to customize antibacterial peptides; 2) Develop chimeric peptides by designing spacer sequences to improve the activity of antimicrobial peptides on titanium surfaces; 3) Verify the approach as an enabling technology by expanding the chimeric design approach to other biomaterials. In Aim 1, a peptide classification tool was developed because the selection of an antimicrobial peptide for an application was difficult among the thousands of peptide sequences available. A rule-based rough-set theory classification algorithm was developed to group antimicrobial peptides by chemical properties. This work is the first time that rough set theory has been applied to peptide activity analysis. The classification method on benchmark data sets resulted in low false discovery rates. The novel rough set theory method was combined with a novel genetic algorithm search, resulting in a method for customizing active antibacterial peptides using sequence-based relationships. Inspired by the fact that spacer sequences play critical roles between functional protein domains, in Aim 2, chimeric peptides were designed to combine solid binding functionality with antimicrobial functionality. To improve how these functions worked together in the same peptide sequence, new spacer sequences were engineered. The rough set theory method from Aim 1 was used to find structure-based relationships to discover new spacer sequences which improved the antimicrobial activity of the chimeric peptides. In Aim 3, the proposed approach is demonstrated as an enabling technology. In this work, calcium phosphate was tested and verified the modularity of the chimeric antimicrobial self-assembling peptide approach. Other chimeric peptides were designed for common biomaterials zirconia and urethane polymer. Finally, an antimicrobial peptide was engineered for a dental adhesive system toward applying spacer design concepts to optimize the antimicrobial activity

Pseudoalteromonas haloplanktis TAC125 as a cell factory for the production of recombinant proteins: strain improvement and novel engineering technologies

Pseudoalteromonas haloplanktis TAC125 (PhTAC125) represents a promising biological system for the recombinant production of high-quality proteins due to its profound differences in cellular physiochemical conditions in comparison to the commonly used mesophilic bacteria. The establishment of efficient constitutive and regulated gene expression systems, optimized culture media, mathematical metabolic models, and fermentative processes allowed the exploitation of this bacterium to produce complex eukaryotic proteins. In this scenario, this research project aimed to explore and extend the biotechnological capabilities of PhTAC125 as a cell factory. In the first part of my PhD project, I focused on the development of a mutant strain engineered to boost the performance of an IPTG-inducible expression system. The obtained strain, named KrPl lacY+, proved to be able to produce the E. coli lactose transporter and a truncated Lon protease devoid of its catalytic domain. The improvement in recombinant production derived from KrPl lacY+ was also demonstrated at low temperatures and encouraged further optimization toward cheaper and sustainable industrial processes. As described in the second chapter of this thesis, KrPl lacY+ was exploited for the recombinant production of the human partially IDP kinase CDKL5, unsuccessfully produced in other prokaryotic systems. Different strategies were applied to overcome the bottlenecks affecting the overall production yield, taking into account the translational efficiency, the optimization of the coding sequence and fusion partners, and the increase of expression plasmid copy number. The establishment of such an improved platform allowed the achievement of high production yields of CDKL5 in a full-length and active form, enabling its application for functional, structural as well as therapeutic studies. Finally, to further strengthen the exploitation of PhTAC125, an asRNA-mediated regulatory system was developed. The results described in the last chapter of the present study demonstrated the feasibility of conditional gene silencing in PhTAC125, opening new perspectives for manipulating marine psychrophilic bacteria in basic and applicative studies

Smart energy grids and renewable multi-generation systems

The current carbon-based energy system is undergoing a deep transformation, mostly aimed at reducing the energy-related emissions of carbon dioxide and other air pollutions. The evolutionary trend of such transition is toward smart energy networks, combining several energy technologies for energy production, storage and utilization, and including a plurality of energy producers, users and prosumers (i.e., producers and users at the same time). The main goal of this work is to explore a series of possible solutions addressing the development of sustainable smart energy networks, analyzing pros and cons of different layouts and technologies from energy, environmental and economic viewpoints, and providing criteria and guidelines for designers, stakeholder and policy makers. Note that the researches described within this thesis are based on researches published on peer-reviewed journals, which was coauthored by the author of this thesis. The studies are based on the use of a dynamic simulation approach. Dynamic simulations can mimic the real performance and behavior of the systems under evaluation, providing crucial information about such systems; this way, it is possible to evaluate their economic profitability and their capacity to reduce fossil energy consumptions and CO2 emissions, with respect to conventional systems. TRNSYS suite is adopted for carrying out such analyses and simulations. TRNSYS is a well-known and reliable tool, widely adopted in academic and commercial applications. Note that TRNSYS environment comes with a large library of components experimentally validated. Moreover, TRNSYS allows the user to adopt in-house and user-developed models. This tool exhibits high accuracy and reliability for the calculation of the dynamic performance of several solar systems. In addition, this software proved high accuracy and risibility in simulating building energy performance. Private mobility is the first sector analyzed in the thesis; in fact, it is currently recognized as one of the most important source of energy consumptions and related CO2 emissions. Different solutions to couple electric vehicles and renewable energy technologies were proposed and analyzed, highlighting that layouts including electric vehicles, residential buildings and renewable energies can be profitably included into smart energy networks. The second chapter is devoted to polygeneration systems. Such systems manage several energy sources, vectors and final users and energy vectors, and are therefore especially attractive for the development of smart energy networks. Polygeneration systems fed by renewables can produce several energy vectors with a very limited consumption of fossil energy. In particular, geothermal and solar energy were considered in the case studies developed and analyzed; such energy source, in fact, are largely available in Campania (South of Italy), where most of the systems evaluated are located. The production of freshwater through reverse osmosis driven by photovoltaic panels was also considered, aiming to match most needs of a given residential district. In addition, reverse osmosis can exploit the excess of photovoltaic power production, avoiding the problems related with the unbalancing of the local electric grid. Moreover, the coupling of photovoltaic energy and reverse osmosis is useful for reducing the dependence on water supply shipped by the mainland, in many isolated islands of the Mediterranean Sea. In Chapter 3, several layouts involving different energy networks were developed and analyzed, also coupling the energy demands of a residential district and that related to private mobility. A further smart energy network able to simultaneously provide thermal energy, electricity and drinkable water was assessed. Finally, the case of a micro energy network was also considered, referred to a hospital facility. Hospitals are highly energy intensive buildings and represent an important candidate for inclusion within a smart micro energy network: in fact, they are usually located nearby residential areas, and their energy facilities can be easily connected or expanded to the energy network serving such residential area. In this framework, several micro energy networks based on cogeneration internal combustion engine were analyzed. A hybrid layout based on photovoltaic and cogeneration was analyzed, too

Bioinformatics Techniques for Studying Drug Resistance In HIV and Staphylococcus Aureus

The worldwide HIV/AIDS pandemic has been partly controlled and treated by antivirals targeting HIV protease, integrase and reverse transcriptase, however, drug resistance has become a serious problem. HIV-1 drug resistance to protease inhibitors evolves by mutations in the PR gene. The resistance mutations can alter protease catalytic activity, inhibitor binding, and stability. Different machine learning algorithms (restricted boltzmann machines, clustering, etc.) have been shown to be effective machine learning tools for classification of genomic and resistance data. Application of restricted boltzmann machine produced highly accurate and robust classification of HIV protease resistance. They can also be used to compare resistance profiles of different protease inhibitors. HIV drug resistance has also been studied by enzyme kinetics and X-ray crystallography. Triple mutant HIV-1 protease with resistance mutations V32I, I47V and V82I has been used as a model for the active site of HIV-2 protease. The effects of four investigational antiviral inhibitors was measured for Triple mutant. The tested compounds had significantly worse inhibition of triple mutant with Ki values of 17-40 nM compared to 2-10 pM for wild type protease. The crystal structure of triple mutant in complex with GRL01111 was solved and showed few changes in protease interactions with inhibitor. These new inhibitors are not expected to be effective for HIV-2 protease or HIV-1 protease with changes V32I, I47V and V82I. Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that causes hospital and community-acquired infections. Antibiotic resistance occurs because of newly acquired low-affinity penicillin-binding protein (PBP2a). Transcriptome analysis was performed to determine how MuM (mutated PBP2 gene) responds to spermine and how Mu50 (wild type) responds to spermine and spermine–β-lactam synergy. Exogenous spermine and oxacillin were found to alter some significant gene expression patterns with major biochemical pathways (iron, sigB regulon) in MRSA with mutant PBP2 protein

Identifisering av RseP som reseptor for hybrid bakteriocin H1 i voksende patogen Staphylococcus haemolyticus

Staphylococcus haemolyticus is an emerging pathogen which may transfer multi-drug resistance genes to highly virulent Staphylococcus species. S. haemolyticus is a biofilm-producing bacterium and one of the most frequent isolates of nosocomial infections associated with implanted medical devices, such as catheter. To this date there are only a few therapeutic options that can stop this bacterium, including the last resort antibiotic, vancomycin and teicoplanin, for which resistance have already been reported. This study shows that an alternative antimicrobial agent has great potential to inhibit this pathogen. Hybrid bacteriocin H1 consisting of N-terminal part of EntK1 and C-terminal part of EntEJ97, exhibits strong activity against S. haemolyticus. A combination of bacteriocin H1 with garvicin KS displays greater antibacterial activity against S. haemolyticus and is promising in inhibiting resistant mutants, which appear to be the main problem in H1 application. The aim of this study was to reveal the nature that indicates the specificity of H1 toward S. haemolyticus. The Zn-dependent protease RseP in S. haemolyticus was established as a receptor for bacteriocin H1. This is based on the fact that the S. haemolyticus gene rseP was heterologously-expressed in naturally H1 resistant Lactobacillus plantarum WCFS1 and the resulting transformant became highly susceptible to H1. RseP isolated from resistant mutants were sequenced in order to verify the nature of the bacterial resistance to H1. Interestingly, obtained data showed intact rseP gene in all mutants. WGS analysis was a conclusive step in this study and indicated mutation in a putative Ecs ABC transporter. Similar discoveries from previous investigations suggest the influence of Ecs transporter on RseP activity at protein level. One might speculate that Ecs protein inhibits RseP interaction with bacteriocin by blocking its activity. However, the results of this study do not allow for specific conclusions on correlation between Ecs and RseP. Further studies of between RseP and Ecs interaction with bacteriocin H1 are required.Staphylococcus haemolyticus kan overføre gener for multi-resistansen til sterkt virulente Staphylococcus arter. S. haemolyticus er en biofilm produserende bakterie og et av de hyppigste isolatene i nosokomiale infeksjoner, og er ofte forbundet med implantert medisinsk utstyr som for eksempel kateter. Til dags dato er det bare noen få terapeutiske alternativer som kan hindre denne bakterien. Dette inkluderer også bruk av antibiotika som en «last resort», selv bakterier som er resistente mot en slik behandling allerede er rapportert. Denne studien viser at et alternativt antimikrobielmiddel har stort potensiale for å hemme dette patogenet. Hybrid bakteriosin H1 består av N-terminal part av EntK1 og C-terminal part av EntEJ97 og viser sterk aktivitet mot S. haemolyticus. En kombinasjon av de to lederløse bakteriosinene H1 og garvisin KS utviser stort antibakteriell aktivitet mot S. hamolyticus og er lovende for å hemme resistente mutanter, som ser ut til å være hovedproblemet i H1 applikasjonen. Målet med denne studien var å avsløre naturen som angir spesifisiteten til H1 mot S. haemolyticus. Den Zn-avhengige proteasen RseP i S. haemolyticus ble etablert som reseptor for bakteriosin H1. Detter er basert på det faktum at S. haemolyticus - genet rseP ble heterologt uttrykt i naturlig H1-resistent Lactobacillus plantarum WCFS1 og den resulterende transformanten ble svært sensitiv til H1. RseP isolert fra resistente mutanter, ble sekvensert for å verifisere naturen av bakteriell resistans mot H1 og variasjoner i fenotype ved stressrespons. Interessant nok, var rseP i alle mutanter. WGS analysen var et avgjørende trinn i dette arbeidet, og indikerte delesjoner i Ecs ABC transporter. Tilsvarende funn fra tidligere studier antyder en påstått effekt av Ecs transporter på RseP-aktivitet på proteinnivå. Man kan spekulere at Ecs protein hemmer RseP interaksjon med bakteriosin H1 ved å blokkere dets aktivitet. Resultatene fra denne studien tillater ikke konkrete konklusjoner om sammenheng mellom Ecs og RseP. Videre studier om interaksjonen mellom RseP og Ecs med bakteriosin H1 er nødvendigM-BIOTE

The EXIT Strategy: an Approach for Identifying Bacterial Proteins Exported during Host Infection

Exported proteins of bacterial pathogens function both in essential physiological processes and in virulence. Past efforts to identify exported proteins were limited by the use of bacteria growing under laboratory (in vitro) conditions. Thus, exported proteins that are exported only or preferentially in the context of infection may be overlooked. To solve this problem, we developed a genome-wide method, named EXIT (exported in vivotechnology), to identify proteins that are exported by bacteria during infection and applied it to Mycobacterium tuberculosis during murine infection. Our studies validate the power of EXIT to identify proteins exported during infection on an unprecedented scale (593 proteins) and to reveal in vivo induced exported proteins (i.e., proteins exported significantly more during in vivo infection than in vitro). Our EXIT data also provide an unmatched resource for mapping the topology of M. tuberculosis membrane proteins. As a new approach for identifying exported proteins, EXIT has potential applicability to other pathogens and experimental conditions