Mechanisms of fimbrial Assembly via non-classical Chaperone-usher pathways And receptor recognition by fimbrial adhesins

The attachment of a pathogen to target cells is a critical step in establishing an infection. Most Gram-negative pathogens display specialized adhesive pili, or fimbriae, assembled via the dedicated classical, alternative and archaic chaperone-usher (CU) pathways from protein subunits. CU pathways represent attractive inhibition targets, as the suppression of bacterial adhesion will allow the natural clearance of the pathogen without the development of antibiotic resistance. This thesis explores the mechanism and biological significance of the polyvalent attachment of fimbrial adhesins to host cell receptors and elucidates the assembly mechanism of fimbriae via the archaic CU pathway. In this study, we focused on several medically important polyadhesins: AAF/I (Escherichia coli O4H104), Myf (Yersinia enterocolitica), and Psa (Y. pestis and Y. pseudotuberculosis). We present the atomic resolution crystal structures of the fiber forming subunits of these organelles and show that they are assembled into linear homo- (Myf and Psa) or heteropolymers (AAF/I) by donor strand complementation. Each protein subunit of these three fimbriae binds to host cell receptors, thereby establishing multipoint attachment. The AggA subunits of AAF/I bind to fibronectin via a unique positively charged surface at inter-subunit interfaces. The co-crystal structures of the subunits of Myf and Psa with galactose and choline, respectively, reveal the receptor binding sites and explain differences in the tissue tropism of pathogenic species of Yersinia. The high avidity of the attachment and the ability of polyadhesins to recognize several receptors represent a significant challenge for drug development and underlines the importance of multivalent inhibitors. To elucidate the fimbriae biogenesis via the archaic CU pathway, we determined the crystal structures of the CsuC-CsuA/B chaperone-subunit preassembly complex and self-complemented CsuA/B pilin subunit from the Csu fimbriae of Acinetobacter baumannii. We show that the biogenesis of archaic systems is strikingly different from that of the classical CU pathway. The CsuC chaperone utilizes a unique subunit anchoring mechanism involving both domains of the protein. Furthermore, CsuC maintains the CsuA/B subunit in a partially disordered state, which allows for a more flexible mode of donor strand exchange. The accessible core of the chaperone-bound subunit might represent a potential inhibition target for archaic pili assembly in bacterial pathogens.Taudinaiheuttajien kiinnittyminen kohdesoluihin infektion alkaessa on kriittinen vaihe. Useimmilla gramnegatiivisilla bakteereilla on tarttumiseen erikoistuneita fimbrioita, adhesiineja, jotka muodostuvat proteiinialayksiköistä klassisen, vaihtoehtoisen ja arkaaisen kaitsija-portieeri (chaperon-usher, CU) -eritysjärjestelmän kautta. CU-järjestelmät ovat houkuttelevia inhibition kohteita, koska bakteerien kiinnittymisen ehkäisy mahdollistaa taudinaiheuttajien luonnollisen poistamisen ilman, että kehittyy antibioottiresistenssiä. Tässä väitöskirjassa käsitellään polyadhesiinien kiinnittymismekanismia kohdereseptoreihin ja arkaaisen CU-järjestelmän mukaisia fimbrioiden muodostumisperiaatteita. Tutkimuksemme kohteena olivat lääketieteellisesti tärkeät polyadhesiinit AAF/I (Escherichia coli O4H104), Myf (Yersinia enterocolitica) ja Psa (Y. pestis ja Y. pseudotuberculosis). Ratkaisimme näiden alayksiköiden kiderakenteet atomin tarkkuudella, ja osoitimme, että alayksiköt ryhmittyvät lineaarisiin homo- (Myf ja PSA) tai heteropolymeereihin (AAF/I) donorisäikeen komplementaatio -periaatteen mukaisesti. Jokainen proteiinialayksikkö pystyy kiinnittymään isäntäsolun reseptoreihin, joten polyadhesiinit varmistavat monipistekiinnityksen. AAF/I:t käyttävät AggA- alayksiköiden välillä sijaitsevia ainutlaatuisia positiivisesti varautuneita pintoja kiinnittyäkseen fibronektiiniin. Myf-fimbrioiden alayksikön kiderakenne galaktoosin ja Psa-fimbrioiden alayksikön kiderakenne koliinin kanssa paljastivat reseptorien sitoutumiskohdat ja selittivät näiden patogeenisten Yersinioiden solutropismin erot. Fimbrioiden voimakas sitoutumiskyky ja kyky tunnistaa useita reseptoreita on merkittävä haaste lääkekehitykselle, ja korostaa multivalenttisten inhibiittorien käytön tärkeyttä. Arkaaisten CU-fimbrioiden biogeneesiä valottaaksemme olemme ratkaisseet CsuC-CsuA/B-kaitsija-alayksikkö -kompleksin ja itsekomplementoidun CsuA/B-alayksikön kiderakenteet Acinetobacter baumanniin Csu-fimbrioista. Osoitimme, että arkaaisten fimbrioiden biogeneesi eroaa olennaisesti klassisesta CU-eritysjärjestelmästä sekä molekyylirakenteeltaan että toimintamekanismiltaan. CsuC-kaitsijaproteiini käyttää ainutlaatuista alayksikön ankkurointimekanismia, johon proteiinin molemmat domainit osallistuvat. Lisäksi CsuC pitää CsuA/B-alayksikön vain osittain laskostettuna, mikä saattaa mahdollistaa joustavamman donori-säikeen vaihdon. Kaitsijaan sidotun alayksikön suojaton ydin on mahdollinen inhibiitiokohde, mikä saattaa auttaa kehittämään uusia mikrobilääkkeitä, joilla pystytään estämään arkaaisten CU-fimbrioiden muodostamista.Siirretty Doriast

Structural basis for Acinetobacter baumannii biofilm formation

Acinetobacter baumannii-a leading cause of nosocomial infections-has a remarkable capacity to persist in hospital environments and medical devices due to its ability to form biofilms. Biofilm formation is mediated by Csu pili, assembled via the "archaic" chaperone-usher pathway. The X-ray structure of the CsuC-CsuE chaperone-adhesin preassembly complex reveals the basis for bacterial attachment to abiotic surfaces. CsuE exposes three hydrophobic finger-like loops at the tip of the pilus. Decreasing the hydrophobicity of these abolishes bacterial attachment, suggesting that archaic pili use tip-fingers to detect and bind to hydrophobic cavities in substrates. Antitip antibody completely blocks biofilm formation, presenting a means to prevent the spread of the pathogen. The use of hydrophilic materials instead of hydrophobic plastics in medical devices may represent another simple and cheap solution to reduce pathogen spread. Phylogenetic analysis suggests that the tip-fingers binding mechanism is shared by all archaic pili carrying two-domain adhesins. The use of flexible fingers instead of classical receptor-binding cavities is presumably more advantageous for attachment to structurally variable substrates, such as abiotic surfaces

Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel Mechanism of Pilus Biogenesis

AVZ is supported by the Finnish Academy (grants 140959 and 273075; http://sciencenordic.com/partner/academy-finland) and Sigrid Juselius Foundation (grant 2014; www.sigridjuselius.fi/foundation). SMis supported by the Wellcome Trust (Senior Investigator Award 100280, Programme grant 079819; http://www.wellcome.ac.uk) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Public procurement system in Russia in the process of transition: Guidelines for EU suppliers

Significant size of the market, growing economy and increased investments in healthcare system makes Russia a promising trade area for EU-suppliers from related industries. Unfortunately, public procurement market in Russia has always represented a big challenge for foreign producers due to government bureaucracy, complex regulatory requirements and lack of transparency. The purpose of this research is to identify key factors for success of foreign suppliers in the Russian state procurement market. To achieve this goal an extended amount of literature from both Russian and foreign sources has been reviewed and 10 interviews with representatives from procuring organizations, supplying and consulting companies have been conducted. Based on interviews' data, the main problems of procurement system have been identified. They include uneven financing of state organizations throughout the year, opportunistic behaviour of suppliers, lack of planning of state purchases and low level of transparency. Introduction of new procurement legislation (44-FZ) and its impact on effectiveness of procurement and position of foreign supplier has also been extensively discussed throughout the thesis. Currently, respondents have different attitudes towards new legislation: some interviewees believe that it will improve transparency by launching integrated procurement platform and implementing effective control and planning, while some respondents assume that new law will not bring any radical changes and only increase bureaucracy. Analysis of existing pitfalls of public procurement and relevant recommendations of experienced representatives from state organizations and private companies enabled development of guidelines for foreign suppliers. Defining company strategy is a starting point for any EU-supplier entering the Russian market. Thorough marketing research has to be per-formed in order to identify local equivalents of the products offered. Findings of the study reveal the utmost importance of building relationships with customers and partners. Careful planning of tenders cannot be neglected. Tender documentation has to highlight and explain the key benefits of the offer for the Customer. Results of the study have also demonstrated that good after-sales service will add significant value to the proposal. Profound knowledge of legislation and experienced team of procurement professionals are other key factors for success in public procurement

Structural basis for Acinetobacter baumannii biofilm formation

Acinetobacter baumannii-a leading cause of nosocomial infections-has a remarkable capacity to persist in hospital environments and medical devices due to its ability to form biofilms. Biofilm formation is mediated by Csu pili, assembled via the "archaic" chaperone-usher pathway. The X-ray structure of the CsuC-CsuE chaperone-adhesin preassembly complex reveals the basis for bacterial attachment to abiotic surfaces. CsuE exposes three hydrophobic finger-like loops at the tip of the pilus. Decreasing the hydrophobicity of these abolishes bacterial attachment, suggesting that archaic pili use tip-fingers to detect and bind to hydrophobic cavities in substrates. Antitip antibody completely blocks biofilm formation, presenting a means to prevent the spread of the pathogen. The use of hydrophilic materials instead of hydrophobic plastics in medical devices may represent another simple and cheap solution to reduce pathogen spread. Phylogenetic analysis suggests that the tip-fingers binding mechanism is shared by all archaic pili carrying two-domain adhesins. The use of flexible fingers instead of classical receptor-binding cavities is presumably more advantageous for attachment to structurally variable substrates, such as abiotic surfaces

Structural comparison of alternative, archaic, and classical chaperones.

<p>(<b>A</b>) Crystal structure of EcpB (cartoon diagram). Donor, subunit C-terminal carboxylate anchoring, and cysteine residues are shown as balls-and-sticks. (<b>B</b>) Superposition of subunit-bound conformations of classical Caf1M (yellow) and archaic CsuC (cyan) chaperones and subunit-free alternative EcpB (purple) and CfaA (green) chaperones by minimizing the distance between corresponding Cα atoms of domain 1 (ribbon diagram) and superposition of C-terminal domains of these chaperones (cartoon diagram). The same color-coding is used below in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005269#ppat.1005269.g008" target="_blank">Fig 8</a>.</p

Differences in the register of donor residues and position of the subunit in CsuC:CsuA/B and complexes from classical systems.

<p>(<b>A</b>) Superposition of the archaic CsuC:CsuA/B and classical Caf1M:Caf1 chaperone-subunit complexes (stereo diagram). CsuC, CsuA/B, Caf1M, and Caf1 are painted in cyan, magenta, yellow and green, respectively. Strand G₁ is shown as cartoon diagram and donor strand segments in CsuC and Caf1M are shown in blue and orange, respectively, as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005269#ppat.1005269.g003" target="_blank">Fig 3A</a> and panel B. (<b>B</b>) Detailed alignment of G₁ donor strands in CsuC and Caf1M. Hydrophobic donor residues in positions P0-5 are shown in balls-and-sticks and labelled.</p

CsuA/B self-assembly depends on its N-terminal donor strand sequence.

<p>(<b>A</b>) Alignment of sequences of Csu pilin subunits A/B, A and B and the pilin domain of subunit CsuE. ClustalW was used to align the sequences. Residues are coded as follows: identical (pink shading); conserved character (cyan shading); pilin N-terminal residues proposed to take part in donor strand complementation in the pilus (yellow); involved in chaperone binding (circles above the residue); invariant for archaic pilin domain cysteines (stars above residues). Limits and nomenclature for secondary structure elements are shown above the sequence. Dashed line indicates unstructured sequences. Structural data were derived from the crystal structure of CsuC-CsuA/B complex (this study). (<b>B</b>) CsuA/B is capable of self-polymerisation and CsuE is not. CsuA/B and CsuE were co-expressed with His₆-tagged CsuC in <i>E</i>. <i>coli</i>, co-purified from periplasmic extracts by Ni²⁺-affinity chromatography, and analysed by SDS-PAGE. Complexes were incubated at 22 or 100°C prior to the electrophoresis. (<b>C</b>) Identification of polymerisation sequence in CsuA/B. CsuA/B mutant with 12 N-terminal amino acid residues replaced by a His₆-tag (Δds) was co-expressed with wild type CsuC. CsuA/B point mutants Leu10→Ala and Ile12→Ala and a triple mutant (T) with Val8, Leu10 and Ile12 substituted to alanines were co-expressed with His₆-tagged CsuC. CsuC-CsuA/B complexes were purified and analysed by SDS-PAGE as in (B).</p