Predicting the ligand-binding properties of Borrelia burgdorferi ss Bmp proteins in light of the conserved features of related Borrelia proteins

Bacteria of the genus Borrelia cause vector-borne infections like the most important hard tick-borne disease in the northern hemisphere, Lyme borreliosis (LB), and soft tick or louse transmitted relapsing fevers (RF), prevalent in temperate and tropical areas. Borrelia burgdorferi sensu lato (s.l.) includes several genospecies and causes LB in humans. In infected patients, Borrelia burgdorferi sensu stricto (s.s.) expresses the BmpA, BmpB, BmpC and BmpD proteins. The role of these proteins in the pathogenesis of LB remains incompletely characterized, but they are, however, closely related to Treponema pallidum PnrA (Purine nucleoside receptor A), a substrate-binding lipoprotein of the ATP-binding cassette (ABC) transporter family preferentially binding purine nucleosides. Based on 3D homology modeling, the Bmp proteins share the typical fold of the substrate-binding protein family and the ligand-binding properties of BmpA, BmpB and BmpD are highly similar, whereas those of BmpC differ markedly. Nevertheless, these residues are highly conserved within the genus Borrelia and the inferred phylogenetic tree also reveals that the RF Borrelia lack BmpB proteins but has an additional Bmp protein (BmpA2) missing in LB-causing Borrelia burgdorferi s.l. Our results indicate that the Bmp proteins could bind nucleosides, although BmpC might have a different ligand-binding specificity and, therefore, a distinct function. Furthermore, the work provides a means for classifying the Bmp proteins and supports further elucidation of the roles of these proteins

Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans 4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH 7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs

Conserved lysine residues in decorin binding proteins of Borrelia garinii are critical in adhesion to human brain microvascular endothelial cells

Lyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato spirochetes (Lyme borreliae). When the disease affects the central nervous system, it is referred to as neuroborreliosis. In Europe, neuroborreliosis is most often caused by Borrelia garinii. Although it is known that in the host Lyme borreliae spread from the tick bite site to distant tissues via the blood vasculature, the adherence of Lyme borreliae to human brain microvascular endothelial cells has not been studied before. Decorin binding proteins are adhesins expressed on Lyme borreliae. They mediate the adhesion of Lyme borreliae to decorin and biglycan, and the lysine residues located in the binding site of decorin binding proteins are important to the binding activity. In this study, we show that lysine residues located in the canonical binding site can also be found in decorin binding proteins of Borrelia garinii, and that these lysines contribute to biglycan and decorin binding. Most importantly, we show that the lysine residues are crucial for the binding of Lyme borreliae to decorin and biglycan expressing human brain microvascular endothelial cells, which in turn suggests that they are involved in the pathogenesis of neuroborreliosis.</p

The role of Tal2 and Tal1 in the differentiation of midbrain GABAergic neuron precursors.

Peer reviewe

Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition

Virulence and survival mechanisms in Borrelia and Klebsiella infections – a structural bioinformatics perspective

Bacteria use a wide variety of mechanisms to establish an infection in their host and to compete with other microorganisms for space and nutrients. These virulence and survival mechanisms mainly rely on the functions of proteins, which perform a multitude of highly specialized tasks to ensure bacterial colonization and survival in the host, e.g., through adhesion to host tissues, nutrient import, immune evasion mechanisms and toxin delivery. As the function of a protein is governed by its structure, determining its three dimensional structure makes it possible to gain knowledge also about its function. This thesis is focused on virulence factors in Borrelia and Klebsiella bacteria and I have used structural bioinformatics methods to provide a detailed understanding of how structural features govern the function of a protein. The BmpA, BmpB, BmpC and BmpD proteins of the Lyme disease-causing bacteria Borrelia burgdorferi are an example of how bacteria can survive and reproduce successfully in a host despite a very limited biosynthetic capacity. By analysing 3D structural models of the Bmp proteins, we showed that they are involved in the purine salvage pathway, which is used by the Borrelia bacteria to obtain vital purine nucleotides even though they lack the enzymes needed to synthesize them. BmpD was shown experimentally to bind to the purines adenosine and inosine. Our structural comparison showed a high similarity between BmpA, BmpB and BmpD, while BmpC differed significantly from the others, indicating a preference for a different ligand. The evolutionary relationships between the Borrelia Bmp proteins were studied in a phylogenetic analysis, which provided an improved classification of the Bmp proteins and revealed significant differences between the Lyme disease-causing and the relapsing fever-causing Borrelia. To successfully colonize a host, Borrelia bacteria also need to adhere to host tissues, and this is done through specialized surface-located proteins that bind to receptors on host cells or in the extracellular matrix. Based on our mutational studies, the Borrelia garinii DbpA and DbpB proteins are essential for adhesion to cells in the nervous system and mediate adherence by binding to the proteoglycans decorin and biglycan on the surface of HBMECs (human brain microvascular endothelial cells). Negatively charged glycosaminoglycan (GAG) chains on the proteoglycans interact with positively charged lysine residues on the surface of the Dbp proteins and these interactions were inhibited by mutating the lysines. Our structural analysis showed significant changes in surface potential for the mutated proteins, which explained the loss of electrostatic interactions, and highlighted the importance of individual lysine residues for proteoglycan binding. The highly antibiotic resistant bacterium Klebsiella pneumoniae uses secretion systems to inject toxic effectors into host cells or rival microorganisms. The effectors of the type VI secretion system (T6SS) can suppress the host immune system and VgrG proteins play a key role in the function of this system. Our experimental studies showed that K. pneumoniae VgrG4 causes a ROS-mediated toxic effect in the host and that its C-terminal DUF2345-containing part is sufficient for ROS induction. Structural modelling predicted that this region has a beta-helical fold, which is characteristic for VgrG spikes. The immunity protein Sel1E prevents the toxic effects of VgrG4 from harming the bacteria itself, and by combining a structural analysis with a study of its evolutionary conservation patterns we highlighted residues possibly involved in interactions with VgrG4. In summary, this work deepens our understanding of the structure and function of bacterial proteins essential for virulence and survival. Insights into the detailed molecular mechanisms of a protein’s function can greatly aid the development of highly targeted novel treatment methods. ---------- Bakterier använder sig av en mängd olika mekanismer för att infektera sin värdorganism och för att konkurrera om utrymme och näringsämnen med andra mikroorganismer. Dessa virulens- och överlevnadsmekanismer baserar sig främst på funktionen hos olika proteiner som utför en mängd mycket specialiserade uppgifter för att säkerställa kolonisering och överlevnad i värdorganismen, t.ex. genom adhesion till värdens vävnader, genom upptag av näringsämnen, undvikande av immunförsvaret och leverans av toxiska substanser. Eftersom ett proteins funktion avgörs av dess struktur kan man få fram information om proteinets funktion genom att bestämma dess tredimensionella struktur. Den här avhandlingen är fokuserad på virulensfaktorer i Borrelia- och Klebsiella-bakterier och strukturbioinformatikmetoder har använts för att ge en detaljerad förståelse för hur strukturella egenskaper styr ett proteins funktion. BmpA, BmpB, BmpC och BmpD proteinerna i Borrelia burgdorferi, som orsakar borrelios, är exempel på hur bakterier kan överleva och föröka sig framgångsrikt i en värdorganism trots en mycket begränsad biosyntetisk kapacitet. Genom att analysera 3D-strukturmodeller för Bmp proteinerna visade vi att de är involverade i den så kallade purine salvage-rutten, som bakterierna använder för att få tillgång till livsnödvändiga purin-nukleotider, trots att de saknar de enzym som behövs för att syntetisera dem. Experimentella studier visade att BmpD binder till purinerna adenosin och inosin. Vår strukturella jämförelse visade att det finns en stor likhet mellan BmpA, BmpB och BmpD medan BmpC tydligt skiljer sig från de andra, vilket indikerar att den binder till en annorlunda ligand. De evolutionära förhållandena mellan Borrelias Bmp proteiner studerades genom en fylogenetisk analys, som resulterade i en förbättrad klassificering av Bmp proteinerna och visade på tydliga skillnader mellan de Borrelia-bakterier som orsakar borrelios och de som orsakar återfallsfeber. För att framgångsrikt kolonisera en värdorganism behöver Borrelia-bakterier också fästa sig vid värdens vävnader, och detta görs genom specialiserade ytproteiner som binder till receptorer på värdcellens yta eller i det extracellulära matrixet. Baserat på våra mutationstudier är DbpA och DbpB proteinerna i Borrelia garinii väsentliga för adhesion till celler i nervsystemet och förmedlar detta genom att binda till proteoglykanerna decorin och biglykan på ytan av mikrovaskulära endotelceller i människans hjärna (human brain microvascular endothelial cells, HBMECs). Negativt laddade glykosaminoglykankedjor (GAG) på proteoglykanerna interagerar med positivt laddade lysiner på ytan av Dbp proteinerna, och dessa interaktioner inhiberas när lysinerna muteras. Vår strukturella analys visade tydliga skillnader i ytpotential för de muterade proteinerna, vilket förklarar förlusten av de elektrostatiska interaktionerna, och betonar vikten av individuella lysiner för proteoglykanbindning. Den antibiotikaresistenta bakterien Klebsiella pneumoniae använder sig av speciella utsöndringssystem för att överföra toxiska effektormolekyler till värdceller eller konkurrerande mikroorganismer. Effektormolekylerna i utsöndringssystem VI (type six secretion system, T6SS) kan hämma värdorganismens immunförsvar och VgrG proteinerna utgör en viktig del i systemet. Våra experimentella studier visade att VgrG4 från K. pneumoniae orsakar en toxisk effekt i värdcellen och att detta styrs genom inducering av oxidativ stress. Proteinets C-terminala DUF2345-innehållande del visade sig vara tillräcklig för detta och genom strukturell modellering visade vi att denna region har en beta-helikal struktur, vilket är typiskt för spike-delen hos VgrG proteiner. Immunitetsproteinet Sel1E förhindrar den toxiska effekten av VgrG4 att skada bakterien själv, och genom att kombinera strukturella analyser med studier av proteinets evolutionära konservationsmönster kunde vi peka ut aminosyror som potentiellt interagerar med VgrG4. Detta arbete bidrar till en fördjupad kunskap om strukturen och funktionen hos bakteriella proteiner som är nödvändiga för virulens och överlevnad. Insikter i de detaljerade molekylära mekanismerna för ett proteins funktion kan bidra med viktig information vid utvecklingen av nya och specifikt målinriktade behandlingsmetoder

Näkökulmia päihdeongelmaisten läheisten kohtaamis- ja auttamistyöhön

Opinnäytetyössäni tarkasteltiin läheistä päihdeongelmaisen rinnalla palveluiden kohteena ja käyttäjänä. Tarkoituksena oli lisätä tiedon lisäksi tietoisuutta siitä, että myös päihdeongelmaisen läheinen on avun tarvitsija joskus vuosiakin kestävässä tapahtumakierteessä. Työn tavoite oli kaksijakoinen. Ensiksi haluttiin selvittää, millaista apua sosiaali- ja terveysalan ammattilaiset voisivat tarjota läheisille. Toiseksi haluttiin palvelumuotoilua apuna käyttäen havainnollistaa, kuinka opinnäytetyönä syntyvä tieto leviäisi ammattilaisten käyttöön. Työn teoreettinen osuus keskittyi tarkastelemaan päihdeongelmaisen läheistä Marja Holmilan luoman ja Outi Holman mukaileman suhdeurateorian kautta. Suhdeurateorian mukaan läheinen joutuu jatkuvasti määrittelemään, kuinka suhtautuu päihdeongelmaan ja päihdeongelmaiseen. Päihdepoliittisesta katsauksesta kävi ilmi, että yhteiskuntamme käsittelee päihdeongelmaa indivualistisena asiana, joka heijastuu päihdepalveluihin niin, että päihdeongelmainen on palveluiden keskiössä. Läheisistä tarvitaan lisää tietoa ja läheiset tarvitsevat lisää tietoa palveluista. Opinnäytetyön tuloksena syntynyt opas vastaa tähän haasteeseen kertomalla päihdeongelmasta, läheisen eri vaiheista ja näkökulmista, joiden avulla on helpompi ymmärtää ja auttaa päihdeongelmaisen läheistä. Palvelupolun avulla pystyttiin havainnollistamaan, miten asiakkaan tietoa palveluiden sisällöstä voi parantaa ja oppaan avulla taas pystyttiin vaikuttamaan ammattilaisten asiakastiedon lisäämiseen.This bachelor’s thesis discusses the role of substance abusers’ family members and friends as service users and target group members. The aim was to increase knowledge of their needs to receive services in a vortex of crises that can often last years. The main objective of this thesis is twofold: first to find out what kind of help social and health professionals could offer to substance abusers’ family members and close ones. The second objective is to demonstrate how knowledge gathered in a thesis could be disseminated to professionals. To answer the first objective, the theoretical part of this thesis is focused on encountering substance abusers’ family and close ones through Marja Holmila’s and Outi Holma’s versions of near relationship and relationship career models. A closer look on Finland’s intoxicant policies showed that most services have been designed for the substance user and more information about substance users’s family and close ones is needed. The conceptual process of this thesis is built around service design. To answer the second objective, a poster of available services and a guide book for social and health service professionals on how to encounter substance abusers’ family and close ones were created

Regulation of Sticholysin II-Induced Pore Formation by Lipid Bilayer Composition, Phase State, and Interfacial Properties

Sticholysin II (StnII) is a pore-forming toxin that uses sphingomyelin (SM) as the recognition molecule in targeting membranes. After StnII monomers bind to SM, several toxin monomers act in concert to oligomerize into a functional pore. The regulation of StnII binding to SM, and the subsequent pore-formation process, is not fully understood. In this study, we examined how the biophysical properties of bilayers, originating from variations in the SM structure, from the presence of sterol species, or from the presence of increasingly polyunsaturated glycerophospholipids, affected StnII-induced pore formation. StnII-induced pore formation, as determined from calcein permeabilization, was fastest in the pure unsaturated SM bilayers. In 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC)/saturated SM bilayers (4:1 molar ratio), pore formation became slower as the chain length of the saturated SMs increased from 14 up to 24 carbons. In the POPC/palmitoyl-SM (16:0-SM) 4:1 bilayers, SM could not support pore formation by StnII if dimyristoyl-PC was included at 1:1 stoichiometry with 16:0-SM, suggesting that free clusters of SM were required for toxin binding and/or pore formation. Cholesterol and other sterols facilitated StnII-induced pore formation markedly, but the efficiency did not appear to correlate with the sterol structure. Benzyl alcohol was more efficient than sterols in enhancing the pore-formation process, suggesting that the effect on pore formation originated from alcohol-induced alteration of the hydrogen-bonding network in the SM-containing bilayers. Finally, we observed that pore formation by StnII was enhanced in the PC/16:0-SM 4:1 bilayers, in which the PC was increasingly unsaturated. We conclude that the physical state of bilayer lipids greatly affected pore formation by StnII. Phase boundaries were not required for pore formation, although SM in a gel state attenuated pore formation

Semi-conservative treatment versus radical surgery in abdominal aortic graft and endograft infections

Objective: Abdominal aortic graft and endograft infections (AGIs) are rare complications following aortic surgery. Radical surgery (RS) with resection of the infected graft and reconstruction with extra-anatomical bypass or in situ reconstruction is the preferred therapy. For patients unfit for RS, a semi-conservative (SC), graft preserving strategy is possible. This paper aimed to compare survival and infection outcomes between RS and SC treatment for AGI in a nationwide cohort. Methods: Patients with abdominal AGI related surgery in Sweden between January 1995 and May 2017 were identified. The Management of Aortic Graft Infection Collaboration (MAGIC) criteria were used for the definition of AGI. Multivariable regression was performed to identify factors associated with mortality. Results: One hundred and sixty-nine patients with surgically treated abdominal AGI were identified, comprising 43 SC (14 endografts; 53% with a graft enteric fistula [GEF] in total) and 126 RS (26 endografts; 50% with a GEF in total). The SC cohort was older and had a higher frequency of cardiac comorbidities. There was a non-significant trend towards lower Kaplan–Meier estimated five year survival for SC vs. RS (30.2% vs. 48.4%; p = .066). A non-significant trend was identified towards worse Kaplan–Meier estimated five year survival for SC patients with a GEF vs. without a GEF (21.7% vs. 40.1%; p = .097). There were significantly more recurrent graft infections comparing SC with RS (45.4% vs. 19.3%; p &lt; .001). In a Cox regression model adjusting for confounders, there was no difference in five year survival comparing SC vs. RS (HR 1.0, 95% CI 0.6 – 1.5). Conclusion: In this national AGI cohort, there was no mortality difference comparing SC and RS for AGI when adjusting for comorbidities. Presence of GEF probably negatively impacts survival outcomes of SC patients. Rates of recurrent infection remain high for SC treated patients

Selective and Bioavailable HDAC6 2‑(Difluoromethyl)-1,3,4-oxadiazole Substrate Inhibitors and Modeling of Their Bioactivation Mechanism

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family mainly targeting cytosolic non­histone substrates, such as α-tubulin, cortactin, and heat shock protein 90 to regulate cell proliferation, metastasis, invasion, and mitosis in tumors. We describe the identification and characterization of a series of 2-(difluoromethyl)-1,3,4-oxadiazoles (DFMOs) as selective nonhydroxamic acid HDAC6 inhibitors. By comparing structure–activity relationships and performing quantum mechanical calculations of the HDAC6 catalytic mechanism, we show that potent oxadiazoles are electrophilic substrates of HDAC6 and propose a mechanism for the bioactivation. We also observe that the inherent electrophilicity of the oxadiazoles makes them prone to degradation in water solution and the generation of potentially toxic products cannot be ruled out, limiting the developability for chronic diseases. However, the oxadiazoles demonstrate high oral bioavailability and low in vivo clearance and are excellent tools for studying the role of HDAC6 in vitro and in vivo in rats and mice