Proteins Involved in Host-Pathogen Recognition

MASARYK UNIVERSITY Faculty of Science

National Centre for Biomolecular Research

PROTEINS INVOLVED IN HOST-PATHOGEN RECOGNITION

EVA FUJDIAROVÁ

Ph.D. Thesis

Supervisor: Prof. RNDr. Michaela Wimmerová, Ph.D.

Brno 2020

BIBLIOGRAFICKÝ ZÁZNAM

Autor: MVDr. Eva Fujdiarová Masarykova univerzita, Přírodovědecká fakulta Národní centrum pro výzkum biomolekul,

Název práce: Proteiny zapojené do rozpoznávání patogenu hostitelem Studijní program: Biomolekulární chemie a bioinformatika

Vedoucí práce: Prof. RNDr. Michaela Wimmerová, Ph.D. Přírodovědecká fakulta, Masarykova univerzita Ústav biochemie a Národní centrum pro výzkum biomolekul - NCBR, Středoevropský technologický institut – CEITEC, Masarykova univerzita

Akademický rok: 2019/2020

Počet stran: 138+165

Klíčová slova: Lektiny, sacharidy, Photorhabuds laumondii, reaktivní formy kyslíku, fenoloxidáza, multivalence

BIBLIOGRAPHIC ENTRY

Author: MVDr. Eva Fujdiarová National Centre for Biomolecular Research, Faculty of Science, Masaryk University

Title of Thesis: Proteins involved in host-pathogen interaction

Degree programme: Biomolecular chemistry and bioinformatics

Field of Study: Biomolecular chemistry and bioinformatics

Supervisor: Prof. RNDr. Michaela Wimmerová, Ph.D. Masaryk University, Faculty of Science, Department of Biochemistry and National Centre for Biomolecular Research Central European Institute of Technology, Masaryk University

Academic year: 2019/2020

Number of Pages: 138+165

Keywords: Lectins, saccharides, Photorhabdus laumondii, reactive oxygen species, phenoloxidase, multivalency

ACKNOWLEDGMENT

My journey to biomolecular chemistry was not straightforward. I studied to be a veterinarian. Although I did enjoy the biochemistry classes, I threw away all my notes shortly after graduation thinking I would never need them again. I remember myself during all those lab practical seminaries thinking – I am not studying such a demanding field only to be pipetting in a lab. I wanted to be a surgeon, to save animals and gain people’s respect – as all of us did. And look at me now 10 years later – happily pipetting. 

In this place, I would like to thank everyone who has been there for me in this chapter of my life. I thank my parents and family for supporting my crazy idea to move to Brno and switch fields, for hiding their fears and doubts from me and telling only: you can do it, and there is always a place for you here. Their belief in me and the feeling of having a safe place to return helped me to face all challenges that doctoral study brings. I thank my supervisor professor Wimmerová for allowing me to learn and grow independently in her lab while watching my progress from distance, letting me go through all the necessary mistakes one needs to do in order to learn. But I thank her also for stepping forward whenever necessary, providing support, counsel, and insight I did not have, not letting me to walk in circles for too long. I thank Janča Vičanová and my shifu Dou Wanchun for English corrections of this text. I thank Pepa, Lenka, Janča, Jitka, Majka and all members of the Glyco group for creating the environment where it is not a shame to ask stupid questions and where co-workers become friends. I thank my friends for simply being. For traditions we have created during the years that I love so much – the movie club, Travný expeditions, TMOU!, boat trips… A special thanks to Hanka for our Dominion sessions, Andula for many hiking memories, and all people connected to “Mycelium” for making me feel I belong somewhere. Thank you all for reminding me that there are more ways to see the world and that it is important to keep things in perspective, especially when you are digging in proteins on an atomic level. And one special note for my little nephew: MITO MITO!

I hereby declare that the thesis “Proteins involved in host-pathogen recognition“, was written by me under the guidance of the thesis supervisor and with the use of literature sources.

Eva Fujdiarová

ABSTRAKT

Lektiny jsou skupina proteinů neimunitního původu, které rozeznávají sacharidy s neobyčejně vysokou specifitou. Díky této vlastnosti jsou lektiny ideálním nástrojem pro čtení “cukerného kódu”, který se nachází na povrchu všech buněk zapsán do struktury specifických cukerných epitopů. Lektiny zprostředkovávají buněčnou komunikaci na molekulární úrovni a jsou zapojeny do mnoha fyziologických i patofyziologických procesů. Patogenní bakterie a viry využívají lektiny k přichycení na hostitelskou tkáň, což je jeden z předpokladů pro rozvoj infekce. Blokace adheze patogenů specifickými lektinovými inhibitory je základem anti-adhezivní terapie, alternativního přistupu k léčbě infekcí způsobených multirezistentními bakteriálními kmeny. Tato dizertační práce je zaměřena převážně na studium lektinů z bakterie Photorhabdus laumondii. Tato bakterie má komplexní životní cyklus, který zahrnuje fázi mutualismu s mikroskopickou hlísticí rodu Heterorhabditis a také fázi patogenity vůči hmyzu. Bakterie ve svém genomu kóduje lektiny, jejichž přesná role v životním cyklu bakterie není dosud známá. Detailní charakterizace lektinů, jejich struktury a vazebných vlastností, stejně tak i studium delečních bakteriálních mutantů, může na tuto otázku odpovědět. Obsahem dizertační práce je také testování účinnosti multivalentních cukerných inhibitorů, zacílených na blokaci lidských patogenů jako Pseudomonas aeruginosa, Burkholderia cenocepacia a Photorhabdus asymbiotica. Text dizertační práce je rozdělen na teoretický úvod a praktickou část. Teoretický úvod je zaměřený na problematiku lektinů, antiadhezivní terapie a rodu Photorhabdus. V praktické části práce jsou popsány tři projekty, do kterých jsem byla v rámci studia zapojena a jsou v ní shrnuty dosažené výsledky.

ABSTRACT

Lectins are a group of proteins of non-immune origin that recognize carbohydrates with extremely high specificity. Due to this property, lectins are the ideal tool for reading a glyco code which is found on the surface of every cell and is encoded in the structure of specific sugar epitopes. Lectins mediate cellular communication at the molecular level and are involved in many physiological and pathophysiological processes. Pathogenic bacteria and viruses use lectins to attach to host tissues, which is one of the prerequisites for infection development. Blocking the adhesion of pathogens by specific lectin inhibitors is the basis of anti-adhesion therapy, an alternative approach to the treatment of infections caused by multi-resistant bacterial strains. This thesis is focused mainly on the study of lectins from Photorhabdus laumondii. This bacterium has a complex life cycle that includes the phase of mutualism with the microscopic nematode of the genus Heterorhabditis and also the phase of pathogenicity towards insects. The bacterium encodes lectins in its genome. Their exact role in the bacterial life cycle is not yet known. The detailed characterization of lectins, their structure, and binding properties, as well as the study of deletion bacterial mutants, may answer this question. The content of this thesis is also testing the effectiveness of multivalent sugar inhibitors aimed at blocking human pathogens such as Pseudomonas aeruginosa, Burkholderia ceanocepatia, and Photorhabdus asymbiotica. The text of the thesis is divided into a theoretical introduction and a practical part. The theoretical introduction is focused on lectins, anti-adhesion therapy, and Photorhabdus genus. In the practical part of the thesis, there are three projects, which I was involved in during the study, described and the results are summarized.

ABBREVIATIONS

3OMG 3-O-methyl-D-glucose AFP alpha-fetoprotein AUC analytical ultracentrifugation CA15-3 carbohydrate antigen associated with breast cancer CRDs carbohydrate-binding domains CRP C-reactive protein CuAAC copper-catalyzed azide-alkyne cycloaddition DMSO dimethyl sulfoxide EPNs entomopathogenic nematodes fMLF N-formyl-L-methionyl-L-leucyl-L-phenylalanine HA hemagglutinin HBSS Hank`s balanced salt solution HIA hemagglutination inhibition assay IJ infective juvenile IPTG isopropyl-β-D-1-thiogalactoside ITC isothermal titration calorimetry LCA Lens culinaris agglutinin MIC minimal inhibitory concentration MBL mannose-binding lectin MGMR 3,6-O-Me2-D-Glcβ1-4(2,3-O-Me2)-L-Rhaα MRSA methicillin-resistant Staphylococcus aureus RNA ribonucleic acid ROS reactive oxygen species SAP serum amyloid P SNR signal to noise ratio sp2 linker -O-(p-C6H4)-O-CH2CH2NH2 SPR surface plasmon resonance STD NMR saturation transfer difference nuclear magnetic resonance P1 primary variant of Photorhabdus (phase one) P2 secondary variant of Photorhabdus (phase two) PCR polymerase chain reaction PHA Phaseolus vulgaris agglutinin PMA phorbol 12-myristate 13-acetate PRSA penicillin-resistant Staphylococcus aureus PO phenoloxidase pPO prophenoloxidase UTIs urinary tract infections VRSA vancomycin-resistant Staphylococcus aureus WGA wheat germ agglutinin

TABLE OF CONTENT

BIBLIOGRAFICKÝ ZÁZNAM ...... 3 BIBLIOGRAPHIC ENTRY ...... 5 ACKNOWLEDGMENT ...... 7 ABSTRAKT ...... 11 ABSTRACT ...... 13 ABBREVIATIONS ...... 15 TABLE OF CONTENT ...... 17 1. LECTINS ...... 21 1.1. History ...... 21 1.2. Basic characterization and classification ...... 23 1.2.1. Microbial and viral lectins ...... 25 1.2.2. Plant lectins ...... 28 1.2.3. Animal lectins ...... 29 1.3. Lectin applications ...... 30 1.3.1. Research ...... 30 1.3.2. Agriculture ...... 31 1.3.3. Medicine ...... 32 2. ANTI-ADHESION THERAPY ...... 34 2.1. Introduction ...... 34 2.2. Approaches to anti-adhesion therapy ...... 35 2.2.1. Inhibition of pilus assembly ...... 36 2.2.2. Usage of adhesin analogs ...... 36 2.2.3. Dietary supplements ...... 36 2.2.4. Carbohydrates as lectin inhibitors ...... 37 2.2.5. Other approaches ...... 39 2.3. Perspectives of anti-adhesion therapy ...... 40 3. PHOTORHABDUS GENUS ...... 41 3.1. History and taxonomy ...... 41 3.2. Basic characterization ...... 43 3.3. Life cycle ...... 44 3.4. Lectins from Photorhabdus spp...... 45 3.5. Utilization of entomopathogenic complex ...... 47 THE AIM OF THE THESIS ...... 48 4. CHARACTERIZATION OF LECTINS FROM PHOTORHABDUS LAUMONDII ...... Chyba! Záložka není definována. 4.1. Project overview ...... Chyba! Záložka není definována. 4.2 Novel lectins from P. laumondii ...... Chyba! Záložka není definována. 4.3. Materials and methods ...... Chyba! Záložka není definována. 4.3.1. Protein production ...... Chyba! Záložka není definována. 4.3.2. Protein purification ...... Chyba! Záložka není definována. 4.3.3. Glycan microarray ...... Chyba! Záložka není definována. 4.3.4. Isothermal titration calorimetry (ITC) ... Chyba! Záložka není definována. 4.3.5. Surface plasmon resonance (SPR) ...... Chyba! Záložka není definována. 4.3.6. Analytical ultracentrifugation (AUC) .... Chyba! Záložka není definována. 4.3.7. Crystallization ...... Chyba! Záložka není definována. 4.3.8. Data collection and structure determination ...... Chyba! Záložka není definována. 4.3.9 Reactive oxygen species (ROS) production in human blood ...... Chyba! Záložka není definována. 4.3.10. Phenoloxidase (PO) activity in insect haemolymph Chyba! Záložka není definována. 4.4. Results and discussion ...... Chyba! Záložka není definována. 4.4.1. Protein production and purification ...... Chyba! Záložka není definována. 4.4.2. Glycan microarray ...... Chyba! Záložka není definována. 4.4.3. Isothermal titration calorimetry (ITC) ... Chyba! Záložka není definována. 4.4.4. Surface plasmon resonance (SPR) ...... Chyba! Záložka není definována. 4.4.5. Analytical Ultracentrifugation (AUC) ... Chyba! Záložka není definována. 4.4.6. Crystallization and X-ray structure determination..... Chyba! Záložka není definována. 4.4.7. Reactive oxygen species (ROS) production in human blood ...... Chyba! Záložka není definována. 4.4.8. Phenoloxidase (PO) activity in insect haemolymph ... Chyba! Záložka není definována. 4.5. Conclusion ...... Chyba! Záložka není definována. 5. PREPARATION OF KNOCK-OUT MUTANTS OF PHOTORHABDUS LAUMONDII ...... Chyba! Záložka není definována. 5.1. Project overview ...... Chyba! Záložka není definována. 5.2. Materials and methods ...... Chyba! Záložka není definována. 5.2.1. Design of vector sequences and primers Chyba! Záložka není definována. 5.2.2. Transformation of E. coli XL1 cells ...... Chyba! Záložka není definována. 5.2.3. Molecular cloning ...... Chyba! Záložka není definována. 5.2.4. Transformation of E. coli S17-1 cells ... Chyba! Záložka není definována. 5.2.5. Gene knock-out via bacterial conjugation ...... Chyba! Záložka není definována. 5.2.6. Identification of knock-out mutants ...... Chyba! Záložka není definována. 5.2.7. Phenotype variant identification ...... Chyba! Záložka není definována. 5.3. Results and discussion ...... Chyba! Záložka není definována. 5.3.1. Design of vector sequences and primers Chyba! Záložka není definována. 5.3.1. Molecular cloning ...... Chyba! Záložka není definována. 5.3.2. Gene knock-out via bacterial conjugation ...... Chyba! Záložka není definována. 5.3.3. Colony PCR...... Chyba! Záložka není definována. 5.3.4. Phenotype variant identification ...... Chyba! Záložka není definována. 6. DEVELOPMENT OF SYNTHETIC MULTIVALENT INHIBITORS OF LECTINS ...... Chyba! Záložka není definována. 6.1. Project overview ...... Chyba! Záložka není definována.

6.2. Synthesis of α-L-fucoside-presenting glycoclusters and investigation of their interaction with Photorhabdus asymbiotica lectin (PHL) ...... Chyba! Záložka není definována. 6.3. Selectivity of original C-hexopyranosyl calix[4]arene conjugates towards lectins of different origin ...... Chyba! Záložka není definována. 6.4. Synthesis of β-D-galactopyranoside-presenting glycoclusters, investigation of their interactions with Pseudomonas aeruginosa lectin A (PA-IL) and evaluation of their anti-adhesion potential ...... Chyba! Záložka není definována. 7. OTHER PROJECT ...... Chyba! Záložka není definována. SCIENTIFIC PUBLICATIONS ...... 49 REFERENCES ...... 52 CURRICULUM VITAE ...... 71 APPENDIX ...... 76

1. LECTINS

1.1. History

Lectins are a group of saccharide recognizing proteins with a characteristic ability to agglutinate cells (e.g. erythrocytes). Although the term ”lectin” first appeared in the mid 20th century [1], the research on this topic started several decades earlier. The first mention of hemagglutination activity appeared in 1860 when Silas Weil Mitchel from the University of Philadelphia observed agglutination of the pigeon blood after mixing it with rattlesnake venom [2]. Nevertheless, the generally acknowledged start of “lectinology” was almost 30 years later, in 1888 at the University of Dorpat (now Tartu, Estonia). In his doctoral study (Fig. 1), Peter Hermann Stillmark isolated and described the first hemagglutinin – ricin, a toxic substance in the crude extract of the castor bean seeds (Ricinus communis) [3]. Because of its toxicity, there were some attempts to use ricin as a weapon during World War II. Fortunately, the ricin bomb tested by the British military had never been finished [4]. Subsequently, H. Helin, also from the Tartu University, described another toxic hemagglutinin called abrin found in the extract of the jequirity bean (Abrus precatorius) [4]. In the light of their hemagglutinating activity, these Figure 1: Frontispiece of the doctoral proteins were called hemagglutinins, or thesis of Peter Hermann Stillmark phytohemagglutinins, because they were found mostly in plants. The first pure hemagglutinin, concanavalin A, was isolated from jack bean (Canavalia ensiformis) by James B. Sumner at Cornell University, New York in 1919 [5]. Concanavalin A was reported to agglutinate erythrocytes and yeasts and it also precipitated glycogen from the solution. In 1936, James B. Sumner and Howell showed that the activity of concanavalin A can

21 be inhibited by sucrose [6]. This experiment was the first demonstration of the sugar specificity of hemagglutinins. The hemagglutination activity was thoroughly studied in the 1950s. Olavi Mäkelä, a doctoral student at the University of Helsinki, examined seed extracts from 743 plant species from Leguminosae family. He detected hemagglutination activity in more than one-third of them, and almost 10% showed blood type specificity [7]. Hemagglutinins played a crucial role in the investigation of ABO blood group antigens. Walter J. T. Morgan and Winifried M. Watkins at the Lister Institute in London found that hemagglutination of different blood groups can be inhibited by different monosaccharides [8], thus concluded the blood group antigens are determined by distinct sugar epitopes. This work was one of the first evidences for the presence of the saccharides on the cell surface and their potential role as the identity markers. The interaction of hemagglutinins with other cell types was also studied in the 1960s. Peter C. Nowell discovered that the lymphocyte mitosis can be stimulated upon binding of phytohemagglutinin (PHA) from red kidney bean (Phaseolus vulgaris) [9]. Several other lectins were proven to have mitogenic activity in a short time, among them also concanavalin A. An inhibition of the mitogenic activity of concanavalin A by monosaccharides, e.g. D-mannose, was of special importance [10]. This experiment proved that lymphocytes are stimulated to mitosis by the binding of agglutinins to the cell surface of glycans. This was one of the earliest demonstrations of a biological role of cell surface glycans. The name ”lectin” was proposed by Boyd and Shapleigh in a short article published in Science, 1954 [1]. The name originates from Latin legere, “to pick out” or “to choose”. It is based on the ability of plant agglutinins to distinguish among different blood groups. The name was later generalized to embrace all saccharide specific proteins of non-immune origin without catalytic activity. With the advances of the recombinant techniques in the 1970s, the studies of lectins were intensified. The major contribution to the development of the modern age of ”lectinology” was a work of Nathan Sharon and Halina Lis who purified multiple lectins [4]. Nathan Sharon devoted his work to lectin research and contributed tremendously to the current state of the art. The first lectin with a determined primary sequence and also the first lectin with a 3D structure solved by x-ray crystallography was concanavalin A in 1972 [11,12]

(Fig. 2A). The number of identified lectins has been continuously growing to this day. Today there are 536 distinct lectin sequences with solved 3D structure (Fig. 2B) [13]. Lectins have grown into a heterogeneous group of proteins with a ubiquitous prevalence in nature and with various roles in organisms, mediating the cell-cell communication at a molecular level. The progress in whole genome sequencing and modern bioinformatic tools allows us to accelerate searching for new potential lectins. [14]

Figure 2: A) The overall structure of concanavalin A (PDB: 3CNA). Individual monomers are color coded (green, red, orange and cyan). Divalent cations are depicted as spheres – Ca2+ green and Mn2+ violet. B) Distribution of solved lectin 3D structures in the nature according to Glyco3D database [14], last update 06.12.2019.

1.2. Basic characterization and classification

As was mentioned before, lectins are a group of proteins capable of binding saccharides. Lectins differ from other groups of carbohydrate recognizing proteins, e.g. carbohydrate-specific antibodies and enzymes, in several important aspects. In contrast to antibodies, lectins are not a product of the immune system and are present also in organisms incapable of conducting immune reaction (e.g. microorganisms). Lectins are structurally diverse, whereas antibodies are structurally similar. The high specificity of lectins makes them akin to enzymes, but in contrast to enzymes, lectins lack catalytic activity and they leave the ligand unaffected [15].

The lectin-saccharide interaction is mainly mediated via the formation of hydrogen bonds with sugar hydroxyl groups. However, other binding forces can be involved too. Hydrophobic interactions and van der Waals interactions often contribute to the binding [16]. The presence of divalent cation, usually Ca2+ or Mn2+, is required for saccharide binding for the whole group of leguminous lectins [17], animal C-type lectins [15] and in exceptional cases for bacterial lectins [18,19]. Recently, the importance of CH-π interactions between aromatic amino acids and the apolar part of carbohydrate molecules is becoming the center of interest [20–22]. Due to their exceptionally high saccharide specificity, lectins are a perfect tool to interpret the glyco code. The information about organism type, cell line and intracellular processes of the individual cell is reflected in the structure of oligosaccharides present on the surface of every cell [23]. The coding capacity of saccharides derives from variability in multiple sugar properties and their combination: a number and type of different monosaccharides, a monosaccharide linkage (usually three to four hydroxyls are available for covalent bond per monosaccharide), an anomeric position (α or β anomers) and a ring size (furanose or pyranose). Potential covalent modification (e.g. methylation, sulphation, phosphorylation) and the possibility of creating branched chains increase the diversity of possible formed molecules even further (Fig. 3) [17,24–26]. As a result, 3.55 x 104 unique tetrasaccharides can be created from four different monosaccharides, whereas only 24 distinct tetranucleotides can be formed from four bases. Classification of lectins has been a challenge and it has been difficult to find a mutual agreement. Therefore, lectins are divided according to multiple aspects. On the bases of their specificity, lectins can be sorted into 5 groups according to the monosaccharide they preferentially bind: D-mannose, L-fucose, D-galactose/N-acetyl-D-galactosamine, N-acetylneuraminic acid, N-acetyl-D-glucosamine [15,17]. Among all numerous saccharides present in nature, those five monosaccharides are typical constituents of eukaryotic cell surface glycosylation. However, this classification masks the fact that lectins often prefer oligosaccharides as their ligands. Lectin-monosaccharide interaction has usually weak affinity, with the dissociation constants in the millimolar range. However, the affinity towards oligosaccharides can be as much as 1000-fold higher. Moreover, lectins from the same

Figure 3: The structural variation of glycans is a result of differences in (a) anomeric stereochemistry; (b) variability of linkages; (c) ring size; and (d) covalent modification of hydroxyl groups [26]. monosaccharide specificity group may differ markedly in their affinities towards oligosaccharides [17]. According to their structural properties, lectins can be sorted into three classes: 1. simple, 2. mosaic (or multidomain) and 3. macromolecular assemblies. The simple lectins are composed of small numbers of subunits. This group covers almost all known plant lectins as well as animal galectins. The mosaic lectins are diverse in molecular weight and are composed of several kinds of domains, but only one of them includes a sugar-binding site. This group includes viral hemagglutinins as well as C-, P- and I- type animal lectins. Lectins from the macromolecular assemblies group are common in bacteria, where they form bacterial fimbriae or pili [17]. The most general classification of lectins is according to their origin and they are described in this chapter following this classification.

1.2.1. Microbial and viral lectins Microbial lectins are often considered to be the virulence factors of pathogenic microorganisms. They facilitate the pathogen adhesion on the host tissues (Fig. 4), which is one of the prerequisites of infection initiation [27]. Lectins involved in the microbe attachment to the host tissue can be present on the

25 surface of the bacterial cells in a form of fimbriae or pili. A typical fimbria consists of a major subunit forming the stem (shaft) and a minor subunit responsible for binding activity and sugar specificity. The fimbrial carbohydrate specificity varies among and also within bacterial species [28]. The most studied fimbriae are from the family Enterobacteriaceae. The mannose-specific type 1 fimbria with lectin FimH is produced by Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and others [29]. Escherichia coli uses the type 1 fimbriae for adhesion to the urinary tract tissues and subsequently causes urinary tract infections. (UTIs) [30].

Figure 4: Schematic representation of lectin-mediated adhesion. Bacteria (ovals) and viruses (crystals) are adhering to the host cell via interaction with surface sugar epitopes (black dots).

In addition to fimbrial lectins, bacterial lectins can be located directly on the bacterial surface. Soluble surface lectins are transported to the cell surface by an often unknown mechanism but they are not actively excreted to the environment [31]. Examples of this type of lectins are BabA and SabA lectins from Helicobacter pylori. This bacterium is the causative agent of peptic ulcer and the lectins serve as adhesins to the gastric tissues. BabA is more involved in the initial stage of the infection, whereas SabA mediates the adhesion to the inflamed mucosa in the later stages of the infection [32–34]. Lectins PA-IL and PA-IIL from Pseudomonas aeruginosa [35,36], the human pathogen associated with cystic fibrosis, are responsible for

26 the bacterial attachment to the lung tissues and are involved in the creation of the biofilm [37,38]. The intriguing saccharide specificity of pathogen adhesins directly influences the host-pathogen specificity. Examples are the influenza viruses hemagglutinins (HA). HA of all the influenza strains recognize sialic acid but are extremely specific towards the linkage between sialic acid and galactose. Human strains of influenza recognize α2-6 linkage (Neu5Acα2–6Gal) that is expressed in human trachea. Avian and equine strains of influenza specifically recognize α2-3 linkage (Neu5Acα2–3Gal) which is present in horse trachea and avian intestine. Finally, porcine influenza strains are able to recognize both linkages and pig trachea expresses both saccharides [31,39]. Influenza HA is also the major immunogenic antigen and changes in this antigen contribute to the outbreaks of viral epidemics [31]. Another example of lectins facilitating the host specificity of pathogenic microbe is a bacterial strain Escherichia coli K99. This strain binds glycolipids containing N-glycolylneuraminic acid (Neu5Gc) which is expressed in piglet’s intestine. Therefore, the bacteria can cause lethal diarrhea to piglets, but they are not able to infect humans who lack N-glycolylneuraminic acid (Neu5Ac). Humans express N-acetylneuraminic acid, and even though the difference is only in one acyl group, Escherichia coli K99 is not able to recognize this glycan [17]. Another group of microbial lectins are secreted toxins. These lectins lack catalytic activity, but they form domains or subunits in the complex with enzymes involved in pathogenesis and they serve as recognition and targeting agents. A typical example of these types of proteins are members of the AB5 family. Proteins consist of one A subunit responsible for the toxic effect and five B subunits with the lectin activity. B subunit is responsible for binding to the cell surface receptors and the transport of the toxic A subunit to the cell [40]. AB5 toxins are employed by major bacterial pathogens causing several lethal human diseases such as whooping cough (pertussis toxin from Bordetella pertussis) [41], cholera (cholera toxin from Vibrio cholerae) [42] and copious watery diarrhea (heat-labile toxin from enterotoxigenic Escherichia coli) [43].

1.2.2. Plant lectins Plant lectins form a well-studied group consisting of several hundreds of lectins. Plant lectins are usually located in seeds, but they were isolated also from other plant tissues such as leaves, roots, fruits, and stems [17]. The function of plant lectins is intensively studied, but not yet thoroughly understood. The general theory proposes that plant lectins act as defense agents against phytopathogens (e.g. fungi, insects or invertebrates) [44,45]. The second theory assumes that lectins facilitate the symbioses between leguminous plants and nitrogen-fixing bacteria (mostly rhizobia) [45,46]. Recently it has been discovered that rhizobium-plant specificity is mediated by the interaction between rhizobial lipo-chitin oligosaccharides (Nod-factors) and LysM receptor kinase in the leguminous plant [47]. Nevertheless, gain-of- function experiments show that lectins are involved in the symbioses facilitating the bacterial attachment rather than directing the symbioses [45]. The largest family of plant lectins are lectins from leguminous plants. Leguminous lectins are mostly found in seeds. They are all structurally similar, with approx. 20 % of amino acids conserved in the sequence [17]. Residues involved in sugar binding are conserved (an asparagine, an aspartic acid, and an aromatic amino acid) and they occupy an identical spatial position in all these lectins. However, the sugar specificity of leguminous lectins differ. Discrimination between closely related saccharide ligands is achieved by structural variability of the unconserved amino acids lining and surrounding the binding pocket. The presence of Ca2+ or Mn2+ ion is essential for sugar binding [15]. Typical leguminous lectins are concanavalin A from jack bean (Canavalia ensiformis) and phytohemagglutinin (PHA) from red kidney bean (Phaseolus vulgaris). Another group of plant lectins is that from cereals. In contrast to leguminous lectins, lectins from cereals are exceptionally rich in cysteine and they do not require cations for saccharide binding. Cereal lectins are all specific to N-acetyl-D-glucosamine or N-acetylneuraminic acid. A typical example is a wheat germ agglutinin (WGA) [15]. Another plant lectin worth mentioning is ricin (Fig. 5) from beans of castor bean (Ricinus communis). Ricin is one of the deadliest poisons known. It consists of two domains linked by the S-S bridge. Globular domain B, specific to galactose and lactose, mediate ricin attachment to the cell surface.

Domain A possesses the cytotoxic activity by enzymatically inactivating the ribonucleic acid (RNA) involved in protein synthesis. According to some estimates, a single ricin molecule is enough to kill a cell [15].

Figure 5: Structure of ricin (PDB 2AAI). Chain A responsible for toxicity is colored green, chain B with lectin activity is colored red.

1.2.3. Animal lectins Animal lectins, including the human lectins, are a wide group of proteins with various functions in organisms. They are classified in several groups, each characterized by its own carbohydrate-binding domain (CRD) with highly conserved amino acids involved in saccharide binding. Galectins are a family of soluble lectins, specific to β-galactosides such as lactose or N-acetyllactosamine [15]. Interestingly, the tertiary structure of galectins may exhibit the jelly-roll fold typical for legume lectins without any significant sequence similarity between the two lectin families [48]. Galectins are found inside the cytoplasm and in the nucleus, occasionally on the cell surface. Their expression is regulated and they are believed to be essential for normal tissue development [17]. They also play a role in protein trafficking [49], inflammation [50], tumor metastasis [51] and apoptosis [52]. C-type lectins are the most widespread animal lectins with a broad range of biological functions. They require Ca2+ ions for binding. They are mosaic molecules in which CRD is attached to a variable number of protein domains.

The C-type lectins are divided into sixteen groups based on their structure and phylogenetic relationship [53]. The two most studied families are selectins and collectins. The selectins mediate the selective contact between leucocytes and endothelial cells through sialyl Lewis X recognition, thus mediating the migration of leukocytes to the site of infection [15]. The collectins are soluble lectins present in blood serum of mammals and birds. An important member of collectins is MBL – mannose-binding lectin which can activate the complement cascade – an important part of the innate immune system [54,55]. Pentraxins are evolutionary highly conserved proteins characterized by a multimeric (usually pentameric) structure. The classic examples are C-reactive protein (CRP) and serum amyloid P component (SAP). They are acute-phase proteins in humans produced by the liver in response to proinflammatory signals, most prominently interleukin 6 (IL-6) [56,57].

1.3. Lectin applications

Lectins stand out as important tools in various areas of research, medicine, and agriculture where their saccharide binding abilities and biological properties are utilized [15,58].

1.3.1. Research

Lectins are employed in detection, isolation, and characterization of glycoconjugates in the tissue sections, and on the cells and the subcellular organelles. For these purposes, lectins can be derivatized with a fluorescent dye, gold nano-particles or enzymes [15]. A novel platform emerging in recent years is lectin microarray (Fig. 6). A series of lectins (or carbohydrate specific antibodies) immobilized on a glass slide greatly facilitate the analysis of an extensive range of glycoconjugates present on the cell surface. Various procedures for rapid glycan profiling have been developed for glycan-related biomarkers connected to cancer or chronic diseases [59,60]. Lectins covalently bound to resin are used for purification and isolation of glycoproteins, glycopeptides, and oligosaccharides by affinity chromatography [15].

Figure 6: A basic concept of lectin microarray. Multiple lectins are immobilized on the glass slide, organized in a grid where one spot contains one lectin. Prelabelled glycoproteins or cells are then allowed to interact with lectins. Positive spots are identified by illumination under an appropriate scanner. Modified from [59].

1.3.2. Agriculture Numerous lectins are tested as tools for the development of the integrated insect pest control strategies due to their insecticidal activity. The insecticidal lectins are often used in the form of sprayings. The coding genes for such lectins can also be engineered into a variety of crops (e.g. wheat, rice, tobacco, and potatoes) to enhance the plant resistance against insect pests [61]. The example of lectins successfully used this way are Bt toxins from Bacillus thuringiensis. They act by binding to glycolipid receptors conserved among nematodes and insects, but absent in vertebrates, and generate pores to the host cell membrane [31]. Another example of lectin tested for these purposes is Orysata, a mannose-specific lectin found in rice seedlings (Orysum sativum) [62]. It was successfully expressed in the transgenic tobacco and its insecticidal activity against important pest insect was studied. The larvae of the beet armyworm (Spodoptera exigua) and the green peach aphid (Myzus persicae) fed on the transformed plants showed higher mortality compared to the larvae fed on the wild type plants [63]. Introducing transgenic plants does not seem to have any negative effect on the environment. Moreover, it is beneficial to the environment due to the lower consumption of chemical insecticides and herbicides [64]. Unfortunately, the occurrence of insects resistant against Bt-crops has been reported already [65].

1.3.3. Medicine The usage of lectins in medicine is heavily investigated and several strategies based on lectin properties have been developed. However, lectins are more frequently used in medical research and in vitro studies than in clinical practice. Numerous lectins display an anti-tumor activity and are tested as potential anti- tumor drugs. In in vitro studies, multiple lectins showed antiproliferative activity on the cancer cells or they elicit the cancer cell apoptosis [66]. From numerous examples we can mention the lectin from dark red kidney bean (Phaseolus vulgaris) with an antiproliferative activity towards leukemia L1210 cells [67], the lectin from Sophora flavescens which induces apoptosis in HeLa cell line (cervical cancer) [68] or BIL, a galactose-binding C-type lectin from Bothrops leucurus snake venom which induces death of melanoma cells [69]. Lectins are also tested for cancer therapy in vivo. A lectin from mistletoe (Viscum album) has been used to improve the quality of life in breast cancer patients [70], however, the results of clinical trials are highly inconsistent [71]. In order to improve the mistletoe lectin efficacy and use in clinical practice, an improved delivery system is being developed [72]. Lectins have been investigated as detection agents in cancer diagnosis due to their extremely high saccharide specificity. It is known that altered glycosylation occurs in cancer development and aberrantly expressed proteins are used as biomarkers of cancer development [60,73,74]. The diagnosis of hepatocellular carcinoma (HCC) is based on the detection of alpha-fetoprotein (AFP) in blood serum combined with ultrasonography. Elevation of AFP levels is associated not only with HCC but also with other types of cancer (stomach, pancreas or biliary tree), chronic liver disease or pregnancy [75]. In cases where the characteristic AFP blood levels overlap, the microheterogeneity of protein glycosylation is targeted to improve the diagnoses. The AFP-L3 isoform with the increased core fucosylation is related to hepatocellular carcinoma. This isoform is targeted by Lens culinaris agglutinin (LCA) isolated from the lentil seeds [74]. A commercial kit for hepatocellular carcinoma diagnosis based on LCA was developed [76] and it is widely used in the USA as a valuable alternative to more expensive diagnostics methods [77]. However, only two proteins, previously mentioned AFP and antigen CA15-3 specific for breast cancer, are clinically monitored for their glycan changes. All other cancer biomarkers are monitored for their total protein

32 levels. Recently, lectin microarrays are employed in the search for the new glycan-based cancer markers [60]. There have been attempts to develop a lectin-based drug delivery system over the past 40 years [78,79]. The rationale of the system is again based on the altered glycosylation of the diseased cells and the lectin ability to specifically target these unusual glycosylations. Lectins are used as carriers of the drug and allow a tissue-specific effect of the treatment. A tomato lectin was investigated as a potential intestinal bioadhesive agent which could slow down the intestinal transit of the oral drugs and increase their bioavailability. The tomato lectin was also proven to cross the intestinal mucosa in vitro [80]. The concept of lectin-mediated drug absorption enhancement can be applied also to other biological barriers, such as nasal mucosa, lungs or the blood-brain barrier [78]. Problems with toxicity and immunogenicity still need to be addressed, nevertheless, the lectin delivery system represents a promising way of drug administration. Several other clinical applications of lectins could be mentioned. The usage of a soybean agglutinin for the purging of human bone marrow for transplantation is one of them. Mature T cells present in the marrow are selectively agglutinated to prevent the lethal graft-versus-host reaction [15]. Mitogenic stimulation by lectins (e.g. concanavalin A or PHA) provides means to improve the immunocompetence of the immunocompromised patients, such as patients with AIDS or patients with immunosuppressive therapy [81]. Blood typing can be performed with the L-fucose specific lectins from Lotus tetragonolobus and Ulex europaeus which are able to select blood type-O cells [15]. Last but not least, the usage of lectins as a target in anti-adhesion therapy should be mentioned. This novel therapeutic approach focused on infections caused by the antibiotic-multiresistant bacterial strains will be discussed in a more detailed way in the next chapter.

2. ANTI-ADHESION THERAPY

2.1. Introduction

Since the discovery of penicillin in 1928 [82] and its usage in clinical practice since the 1940s, antibiotics have been the main way how to fight bacterial infections [83]. Unfortunately, the golden era of antibiotics is slowly coming to an end. Bacteria have found a way how to adapt to the new conditions and resistant bacterial strains are appearing and rapidly spreading. These strains are usually present in an environment with a frequent antibiotic administration, especially in hospitals, and nosocomial infections are becoming a serious problem of the 21st century. The situation can be demonstrated on the example of Staphylococcus aureus, an important human pathogen. S. aureus rapidly acquired resistance against novel types of antibiotics straight after they were introduced to the market (Fig. 7). Nowadays, methicillin-resistant strains of S. aureus (MRSA) are spread worldwide [84] and MRSA caused an estimated 80 400 cases of infection in the USA in 2011 [85]. For the MRSA strains, one effective antibiotic, glycopeptide vancomycin, remained. Despite careful administration of vancomycin as the antibiotic of the last choice. Clinical isolates of S. aureus with complete vancomycin resistance have already been reported [84,86,87].

Figure 7: Timeline of the advent of the antibiotic therapy and emergence of resistant strains of Staphylococcus aureus. PRSA = penicilin resistant Staphylococcus aureus, MRSA = methicillin resistant Staphylococcus aureus, VRSA = vancomycin resistant Staphlycoccus aureus. Modified from [84].

Due to the rise of the antibiotic-resistant bacterial strains, alternative approaches to treat bacterial infections are needed. The anti-adhesion therapy represents one of the promising ways how to deal with the multi-resistant bacterial strains.

2.2. Approaches to anti-adhesion therapy

The anti-adhesion therapy targets the attachment of the pathogen to the host cells or tissues, which is one of the first steps during the bacterial infection. Pathogens unable to adhere to the host tissues are restricted in growth and the formation of a protective biofilm is also prevented. This helps the natural clearance mechanisms of the body (such as urine flow in the urinary tract, airflow and mucociliary removal in the airways or shedding of upper epithelial cell layers) and the host immune system to eliminate the pathogen (Fig. 8) [88,89]. Another advantage is that the anti-adhesion therapy does not kill the pathogen, therefore a selective pressure is not created and the resistance to the anti-adhesives is not developed [90]. The most important adhesins expressed by numerous bacteria are lectins, present on the bacterial surface in the form of fimbriae/pili or as soluble surface lectins (see chapter 1.2.1.). Several strategies on how to interfere with lectin- mediated pathogen adhesion are being developed.

Figure 8: Schematic representation of the principle of the anti-adhesion therapy. Bacteria are adhering to the host cells via interaction with surface sugar epitopes (black dots). In the presence of inhibitor (red dots) bacteria lack free lectins and the adhesion is prevented.

2.2.1. Inhibition of pilus assembly The assembly of pili in Gram-negative bacteria is coordinated by the chaperone-usher pathway, a very well conserved assembly and secretion system [91]. Small organic molecules interfering with this system – so-called pilicides – are being developed [92]. The pilicide compounds were shown to bind on chaperons [93], strongly bind to uropathogenic Escherichia coli [94] and interfere with the pili assembly of the uropathogenic Escherichia coli strains in a dose-dependent manner [95].

2.2.2. Usage of adhesin analogs This strategy is based on the assumption that an isolated pathogen adhesin or its fragment binds to the host receptor and blocks the bacterial adhesion competitively. This approach needs to deal with potential toxicity and immunogenicity of the chosen adhesin [89]. Nevertheless, partial successes in experiments with Streptococcus mutans, the main cause of dental caries, were achieved. A synthetic 20-residue peptide based on the S. mutans adhesin was able to inhibit the S. mutans adhesion to immobilized salivary receptors in in vitro studies [96]. The re-colonization of the peptide pre-treated teeth with S. mutans was retarded. Nevertheless, this promising result needs to be treated carefully, because the adhesion of S. mutans may be mediated also by other adhesins [89].

2.2.3. Dietary supplements Empirical observations suggested that certain food may have a beneficial effect in the protection against bacterial infections. The well-known example of such nutrient is human maternal milk. The maternal milk is rich in oligosaccharides capable of the inhibition of various surface lectins of enteric bacteria and it was proven to protect breast-fed infants against diarrhoea [97–99]. Special attention is now paid to the fucosylated oligosaccharides (e.g. fucosyllactose) that are inhibitors of Campylobacter jejuni, the major cause of the diarrhoea worldwide [100]. Also, lactoferrin, a glycoprotein in breast milk, is able to inhibit the adhesion of several bacterial species (e.g. Actinobacillus actinomycetemcomitans, Prevotella intermedia or Escherichia coli) and thus contribute to the infant protection [101–103].

Another nutritive that is known to have an anti-adhesive effect is cranberry (Vaccinium macrocarpon). The beneficiary effect of drinking cranberry juice in the therapy of the urinary tract infections (UTIs) is long known folk wisdom. The scientific research on this topic dates to the 1960s [104,105]. Nowadays, the inhibition of biofilm formation and adhesion of uropathogenic Escherichia coli by urine of the person treated with cranberry juice was shown in vitro [106] and reduction of bacteriuria after drinking cranberry juice was proven in several small double-blinded placebo-controlled clinical studies [107,108]. The main active substances are proposed to be proanthocyanidins [109], which are now available in the synthetic form as a nutrition supplement to prevent the urinary tract infection. Despite some positive results, meta-analyses of data from 24 studies did not show any significant effect of the cranberry juice on the urinary tract infection compared to placebo. Only the cranberry products (pills) with a high content of active substances might have a positive effect, although further study is needed [110].

2.2.4. Carbohydrates as lectin inhibitors Usage of saccharides to block the lectin-mediated bacterial adhesion is inspired by the protective effect of oligosaccharides present in human maternal milk. There were some experiments with monosaccharides conducted and some of their results were promising. The positive effect of D-mannose on the therapy of the UTIs has been known since 1979 when the co-administration of methyl α-D-mannoside together with type 1 fimbriated Escherichia coli into the mice urinary bladder was tested. Methyl α-D-mannoside reduced the rate of mice urinary tract infections by two thirds, while methyl α-glucoside had no effect [111]. A more recent pilot study conducted in Rome in 2014 suggests that D-mannose can be effective support at acute UTIs in women [112]. The effect of other saccharides on ongoing infections was explored as well. A study with rhesus monkeys infected by Helicobacter pylori suggests that sialyl-3- lactose could be a safe anti-adhesive agent against H. pylori, albeit the study included only a small number of animals [113]. Also, a mixture of three monosaccharides (D-mannose, L-rhamnose, and D-galactose) showed a promising result in preventing the adhesion of Pseudomonas aeruginosa on canine corneocytes [114]. The last study mentioned was supported by a pharmaceutic company Virbac which included this patented saccharide

37 mixture in a commercial product Epi-otic III, used in veterinary medicine as prevention of otitis externa in dogs and cats. Despite the optimistic results of in vitro and in vivo studies, the usage of monosaccharides in the therapy of bacterial infections remains less effective compared to other active substances (e.g. antibiotics). This might be caused by the nature of lectin-monosaccharide interaction which is generally in the low- affinity range. An effective anti-adhesion therapy requires a high-affinity ligand. Therefore synthetic multivalent glycosides or glycomimetics are being developed where several copies of a monosaccharide are bound together on a synthetic scaffold [115]. Large series of multivalent glycoconjugates were designed in the last decades with different valencies and topological properties (Fig. 9). They can be divided into two distinct groups:

• Glycoclusters and glycodendrimers are limited in a number of epitopes (usually under 20), but they provide a systematic control over the number of sugar epitopes [115,116]. A large number of scaffolds have been developed as a synthetic core, such as fullerenes [117,118], calixarenes [119–121], porphyrins [122], cyclodextrins [123] or functionalized oligoethylene glycols [124]. On this core, a number of linker arms with one or more monosaccharides on the top is attached. Individual glycocompounds differ in type and number of bound monosaccharides, length of linker arms and the inner flexibility of the compound.

• Glycopolymers or glyconanoparticles can include a large number of sugar-epitopes, but with less control over their number [115,116]. Carbon nanotubes [125,126] or poly(p-phenylene ethylene) [127] can be used as the core of the linear glycopolymers. Compared to the linear polymers, glyconanoparticles display a globular shape. Besides a large number of sugar epitopes, they provide also variability in ligand density on the surface. Glyconanoparticles are in rapid development nowadays and encouraging results have been reported recently [128,129]. The examples of glyconanoparticle types are gold glyconanoparticles [130], silver glyconanoparticles [131], diamond glyconanoparticles [132], semiconductor glyco-quantum dots [133] and magnetic glyconanoparticles [134].

Figure 9: The example of different synthetic multivalent glycoconjugates [115].

2.2.5. Other approaches For the sake of completeness, other anti-adhesive therapy approaches that are not directly associated with lectins or carbohydrates should be mentioned. Somewhat controversial is the administration of sublethal doses of antibiotics. Some studies show this strategy reduces pathogen adherence to various substrates [135,136], whereas other studies report an increase in pathogen adhesion instead [137,138]. Nevertheless, this issue is important to study, because there is a high likelihood of periods of sublethal antibiotic concentration in the system of the antibiotic-treated patients due to the poor compliance in drug administration. The development of adhesin-based vaccines is a relatively new approach that uses a conserved region of adhesin as a vaccine agent [139,140]. The active means of anti-adhesin immunization faces the challenging need of increasing the mucosal IgA-mediated immunity, where active immunization, in general, promotes preferably systemic IgG- mediated immunity. Although the active immunization with adhesins in animal

39 models provides both IgG- and IgA-mediated protection (reviewed in [137]), the passive means of immunization may be more useful. The administration of antibodies in milk is known to protect suckling piglets and calves against diarrhea [141]. The use of probiotics as anti-adhesive agents is convenient and inexpensive. The probiotic bacterial strains compete with the pathogens for nutrients and life-space [142]. Healthy microflora is a natural body protection against infection, and it can be supported by healthy nutrition and administration of probiotic supplements.

2.3. Perspectives of anti-adhesion therapy

The prevalence of the antibiotic-resistant bacterial strains is rapidly increasing and the need for the alternative therapeutic approaches is more and more acute. An anti-adhesion therapy represents one of the promising strategies in how to cope with this threat. Despite the potential advantages and partial successes of the anti-adhesion strategy, it still serves the purpose of mostly being only a support for the treatment with other active substances. The major challenge in the improvement of the anti-adhesion therapy lies in the search for an effective agent to block bacterial adhesion. Such an agent needs to have high affinity towards a variety of bacterial adhesins with different specificities and it cannot be immunogenic, nor toxic. A better understanding of bacterial adhesins, their properties, specificities, and receptor-ligand interactions is needed to develop such compounds. Once these compounds become available, they may become an optional drug to treat multiple infectious diseases.

3. PHOTORHABDUS GENUS

3.1. History and taxonomy

The genus Photorhabdus embraces Gram-negative bacteria symbiotic with entomopathogenic nematodes (EPNs) from the family Heterorhabditidae. The first described bacterium living in symbiosis with (EPNs) was large, Gram-negative rod-shaped bacterium Achromobacter nematophilus, discovered by Gerard Thomas and Gorge Poinar in 1965 [143]. 14 years later the bacterium was transferred to a newly established genus Xenorhabdus in the family Enterobacteriaceae and it was renamed to Xenorhabdus nematophilus. Another member of Xenorhabdus genus was Xenorhabdus luminescens, a bacterium with a unique ability of bioluminescence [144]. Distinct differences between two bacteria, not only the bioluminescence but also the catalase activity, pigment production, DNA sequence and type of symbiont, led later to the establishment of a new genus, Photorhabdus (the name means “glowing rod”) [145]. More bacterial species symbiotic with Heterorhabditidae nematodes were assigned to the genus Photorhabdus, whereas bacteria symbiotic with the EPNs from the family Steirnernematidae belong to the genus Xenorhabdus. Recent comparative genomic analyses of the members of the order Enterobacteriales and phylogenic reconstruction of this order led to the establishment of six new families, and the genus Photorhabdus was assigned to the novel family Morganellaceae [146]. The genus Photorhabdus consisted of four species with numerous subspecies [147–149] until the revisit of Photorhabdus phylogeny based on the whole- genome sequencing resulted in the elevation of numerous subspecies to the species level in 2018 [150]. Now, the genus consists of 19 species, which are listed in Table 1.

Table 1: Taxonomy of the genus Photorhabdus (ncbi.nlm.nih.gov). Four “original” species described before 2018 are highlighted in bold.

Superkingdom Bacteria Phylum Proteobacteria Class Gammaproteobacteria Order Enterobacteriales Family Morganellaceae Genus Photorhabdus Species Photorhabdus arkhustii Photorhabdus asymbiotica Photorhabdus australis Photorhabdus bodei Photorhabdus caribbeanensis Photorhabdus cinerea Photorhabdus hainanensis Photorhabdus heterorhabditis Photorhabdus kayaii Photorhabdus khanii Photorhabdus kleinei Photorhabdus laumondii Photorhabdus luminescens Photorhabdus namnaonensis Photorhabdus noenieputensis Photorhabdus stackebrandtii Photorhabdus tasmaniensis Photorhabdus temperata Photorhabdus thracensis

3.2. Basic characterization

The genus Photorhabdus is formed by Gram-negative, highly motile, rod-shaped, facultatively anaerobic bacteria with the lengths from 2 to 10 µm [146]. All species of the Photorhabdus genus are pathogenic towards insect [151] and two species, P. asymbiotica, and P. australis, are also emerging human pathogens [152]. Bacteria cannot be found in the environment, as they cannot survive in soil on their own. Their natural habitat is the intestine of host nematode, or the tissues of the host during the infection stage of the life cycle (see chapter 3.3.). Photorhabdus spp. bacteria and Heterorhabditis spp. nematode form a highly effective entomopathogenic complex with each other. Two phenotypic variations of Photorhabdus bacteria have been described [153]. The primary variant (P1) is naturally present in the EPNs and can be isolated from them. The secondary variant (P2) can be generated by in vitro cultivation in low-nutrient media. The P2 variant differs from the P1 variant in metabolite production, it is still highly infective towards insect but it no longer supports the symbiosis with the nematode [151,154]. The interesting ability of bioluminescence is possible thanks to the lux operon. Around 30 other bacterial species are capable of bioluminescence, but they are all marine species, making the genus Photorhabdus the only known terrestrial bioluminescent bacteria [155]. Visible blue-green light (wavelength ~490 nm) is produced in a reaction catalyzed by the enzyme luciferase (Fig. 10) [155,156]. The biological purpose of the bioluminescence is still not clear. The three general hypotheses are: (i) it represents a signal within bacterial population or from bacteria to nematode to synchronize the symbiosis; (ii) it is a visual warning for nocturnal scavengers to avoid a cadaver, thus saving the tissues for Photorhabdus and the symbiotic nematode; or (iii) it attracts more insect to the site of the cadaver, and thus provides further prey. It could also serve as a sink for molecular oxygen, which could cause starvation of the host immune cells [151].

Figure 10: A scheme of the bacterial luciferase reaction. A long-chain fatty aldehyde (RCHO) is converted into the corresponding fatty acid (RCOOH) in the presence of

reduced flavin mononucleotide (FMNH2) and molecular oxygen (O2). Besides

RCOOH, water (H2O), oxidized flavin mononucleotide (FMN) and the light (hν) are being produced.

Photorhabdus spp. is highly pathogenic towards insect hosts. Only 50-200 bacterial cells are sufficient to cause lethal septicemia in insect prey [151]. This fact was supported by the whole genome sequencing of P. laumonidii subsp. laumnodii TT01, where more toxin genes than in any other sequenced bacterial genome were predicted [157]. Produced toxins and other metabolites (e.g. adhesins, hemolysins, proteases and antibiotics) serve the bacteria as virulence factors to overcome the host defense during the infection and also prevent other bacterial species from proliferating in the host tissues, thus saving the tissues for the symbiotic nematode [158].

3.3. Life cycle

The life cycle of the entomopathogenic Photorhabdus-Heterorhabditis complex (Fig. 11) is described very well [151,158–160]. In the soil bacteria can be found only inside the intestine of an infective juvenile (IJ), a specialized larval stage of the nematode. This larval stage has a non-functional digestive tract and it does not ingest. IJs actively search for the insect prey and when they find it, they penetrate the host body through natural openings (mouth, anus or spiracles). Unlike other nematodes, IJs of Heterorhabditis bacteriophora can enter the host body also actively, thanks to a tooth-like cuticle protuberance that can be used to disrupt the insect cuticle [161]. After entering the insect’s body, IJs regurgitate the bacteria into the insect hemocoel. The bacteria rapidly proliferate, produce toxins and kill the prey within 48 hours [151]. Meanwhile, the IJs undergo the process of recovery and starts to evolve into the adult [162]. The insect cadaver is bioconverted into the nutrients and protected from other scavengers by Photorhabdus, while Heterorhabditis nematodes feast on the

Figure 11: Schematic representation of the life cycle of the entomopathogenic Photorhabdus-Heterorhabditis complex. L1-L4 stand for larval stages of the nematode, IJ stands for infective juvenile. insect tissues as well as bacterial biomass and undergo one to three cycles of sexual reproduction. Once the host tissues are depleted, the sexual reproduction of the nematode is disrupted and new infective juveniles are produced inside the maternal uterus during a process called endotokia matricida [163]. IJs are re-associated with the bacteria and leave the cadaver to search for new insect prey.

3.4. Lectins from Photorhabdus spp.

Lectins, in general, are involved in various processes including cell to cell communication [15,27]. Lectins produced by entomopathogens with a complex life cycle like the genus Photorhabdus could play a role in three basic processes: (i) the interaction with host tissues, (ii) the interaction with nematode symbionts and (iii) the interactions within the bacterial population. Several lectins were identified and described in the genome of Photorhabdus.

The first described lectin from the genus Photorhabdus was lectin PLL from Photorhabdus luminescens [164] (now P. laumondii). Its close homolog PHL was described shortly after in Photorhabdus asymbiotica [165]. Both lectins recognize L-fucose and unusual O-methylated disaccharide 3,6-O-Me2-D-Glcβ1-4(2,3-O-Me2)-L-Rhaα present in the Mycobacterium leprae glycolipid PGL-1 [166,167]. Proteins share the monomeric fold, a seven-bladed β-propeller, but they differ in the oligomeric state, PLL being a tetramer [164] and PHL a dimer [165]. 7 potential fucose-binding sites were described in PLL, 3 of them were occupied in the crystal structure of PLL in the complex with L-fucose [164]. Besides the set of 7 potential fucose- binding sites, another set of binding sites was discovered in PHL. This second set of binding sites recognized D-galactose [165]. The proposed function of these lectins is to help the bacteria to overcome the insect defense by interfering with the insect immune system. Both proteins were shown to bind insect hemocytes and, moreover, PHL affected the production of reactive oxygen species and phenoloxidase activity – two ways of insect primary defense against pathogens [164,165]. Both bacterial species, P. laumondii and P. asymbiotica, code other potential lectins homologous to the PLL and PHL which are being investigated right now. The characterization of lectin homologs of PLL is included in the practical part of this thesis. The reason for having multiple homologous proteins in the genome as well as the possibility of lectin cooperation with each other remains unclear. Another lectin described in P. laumondii is PllA [168]. This protein is not structurally related to PLL nor PHL, but it is a homolog of the PA-IL (LecA) lectin from Pseudomonas aeruginosa with a 37% identity. The lectin was shown to recognize α-galactoside terminated glycocompounds. This property can be used for the identification of the α-Gal epitopes on the porcine cells, responsible for hyperacute rejection of pig to primate organ xenotransplant [169]. The biological function of this lectin in Photorhabdus life cycle remains unclear, too.

3.5. Utilization of entomopathogenic complex

The high toxicity for insects makes EPNs a promising agent in the biological control of insect pests in agriculture [170]. Mostly, the two genera (Steinernematidae and Heterorhabditidae) with their symbiotic bacteria (Xenorhabdus and Photorhabdus species, respectively) are being studied for this purpose. The long-term perspective is to use EPNs as a replacement of pesticides in agriculture. Unfortunately, promising results from the laboratory do not always translate into success in the field [171,172] and there are still several barriers preventing expansion to massive agricultural use. The main challenges represent the high cost of EPNs production, difficult optimization of the EPNs mass production and sub-optimal efficacy of transmission to the insect pest [173,174]. Prior to the intensive introduction of EPN to the environment, its host specificity also needs to be considered. To avoid unintentional damage in the population of beneficial soil insects, nematode species with a narrow range of insect hosts are being selected. Nevertheless, EPNs were successfully produced in smaller amounts and commercial products are already available worldwide [170,175,176]. These products achieve great success in the protection of smaller areas such as gardens or greenhouses.

THE AIM OF THE THESIS

This thesis is composed of three projects. It deals with bacterial lectins, their functional and structural characterization (project 1); deciphering their role in the bacterial life cycle (projects 1 and 2); and development of their inhibitors (project 3). The main focus of the presented work is on the novel lectins discovered in the bacterium Photorhabdus laumondii, an effective insect pathogen. The goals of the individual projects, in brief, are:

Project 1: Characterization of lectins from Photorhabdus laumondii. Characterization of newly identified hypothetical lectins P. laumondii – PLL2, PLL3, PLL4, and PLL5, which includes:

• Production of lectins in a recombinant form. • Functional analysis - specificity and affinity (glycan microarray, surface plasmon resonance, and isothermal titration calorimetry). • Determining the structure (analytical ultracentrifugation and X-ray crystallography) • Revealing lectins influence on the host innate immune system (phenoloxidase activity, production of reactive oxygen species)

Project 2: Preparation of knock-out mutants of Photorhabdus laumondii

• Preparation of a library of P. laumondii knock-out mutants with deleted genes for selected lectins – PLL, PLL2, PLL3, PLL4, PLL5, and PLU1 - in various combinations.

Project 3: Development of synthetic multivalent inhibitors of lectins

• Test the ability of selected multivalent synthetic glycocompounds to interact with a complex environment of the bacterial surface.

SCIENTIFIC PUBLICATIONS

Manuscript 1 (Appendix I)

Eva Fujdiarová, Josef Houser, Pavel Dobeš, Gita Paulíková, Nikolay Kondakov, Leonid Kononov, Pavel Hyršl, Michaela Wimmerová. Heptabladded β-propeller lectins PLL2 and PHL from Photorhabdus spp. recognize O-methylated sugars and influence the host immune system. Submitted to FEBS Journal, after 1st review, in revision. Author’s contribution Manuscript writing, investigation (protein expression and purification, glycan microarray, ITC, SPR, crystallization).

Article 1 (Appendix II)

Lukáš Faltinek*, Eva Fujdiarová*, Filip Melicher, Josef Houser, Martina Kašáková, Nikolay Kondakov, Leonid Kononov, Kamil Parkan, Sébastien Vidal, Michaela Wimmerová. Lectin PLL3, a novel monomeric member of seven-bladed β-propeller lectin family. * These authors contributed equally Molecules, 2019, 24, 4540 DOI: 10.3390/molecules24244540 Author’s contribution Manuscript writing, investigation (protein expression and purification, glycan microarray, ITC, SPR).

Article 2 (Appendix III)

Gita Jančaříková, Mihály Herczeg, Eva Fujdiarová, Josef Houser, Katalin E. Köver, Anikó Borbás, Michaela Wimmerová, and Magdolna Csávás.

Synthesis of α-L-fucoside-presenting glycoclusters and investigation of their interaction with Photorhabdus asymbiotica lectin (PHL). Chemistry: A European Journal, 2018, 24(16), 4055-4068 DOI: 10.1002/chem.201705853 Author’s contribution Investigation (P. asymbiotica cross-linking experiments), manuscript writing (P. asymbiotica growth, cell aggregation assay).

Article 3 (Appendix IV)

Martina Kašáková, Lenka Malinovská, Tomáš Klejch, Martina Hlaváčová, Hana Dvořáková, Eva Fujdiarová, Zdeňka Rottnerová, Olga Maťátková, Pavel Lhoták, Michaela Wimmerová, Jitka Moravcová. Selectivity of original C-hexopyranosyl calix[4]arene conjugates towards lectins of different origin. Carbohydrate Research, 2018, 469, 60-72. DOI: 10.1016/j.carres.2018.08.012 Author’s contribution Investigation (B. cenocepacia cross-linking experiments).

Article 4 (Appendix V)

Lenka Malinovská, Son Thai Le, Mihály Herczeg, Michaela Vašková, Josef Houser, Eva Fujdiarová, Jan Komárek, Petr Hodek, Anikó Borbás, Michaela Wimmerová, Magdolna Csávás.

Synthesis of β-D-galactopyranoside-presenting glycoclusters, investigation of their interactions with Pseudomonas aeruginosa lectin A (PA-IL) and evaluation of their anti-adhesion potential. Biomolecules, 2019, 9(11). DOI: 10.3390/biom9110686. Author’s contribution Investigation (P. aeruginosa cross-linking experiments).

Article 5 (Appendix VI)

Petra Sýkorová, Jitka Novotná, Gabriel Demo, Guillaume Pompidor, Eva Dubská, Jan Komárek, Eva Fujdiarová, Josef Houser, Lucia Hároníková, Annabelle Varrot, Nadezhda Shilova, Anne Imberty, Nicolai Bovin, Martina Pokorná, Michaela Wimmerová. Characterization of novel lectins from Burkholderia pseudomallei and Chromobacterium violaceum with seven-bladed β-propeller fold. International Journal of Biological Macromolecules, 2019, in press. DOI: 10.1016/j.ijbiomac.2019.10.200 Author’s contribution Investigation (glycan microarray experiments), manuscript writing (P. aeruginosa cross-linking assay).

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CURRICULUM VITAE

Personal details Name: Eva Fujdiarová Date of birth: 14.12.1983 Nationality: Czech Contact: [email protected]

Employment 2015-present Researcher at Glycobiochemistry group, CEITEC, and NCBR, Masaryk University, Brno, Czech Republic 2012-2013 Veterinary inspector in veterinary hygiene department, Regional Veterinary Administration, Nový Jičín 2009-2012 Veterinarian, Veterinary clinics for small animals (dogs, cats, and small rodents) Čáslav, Brno, and Příbor

Education since 9/2014 Doctoral study of Biomolecular chemistry and bioinformatics, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno 2003-2009 Master study of Veterinary medicine, University of Veterinary and Pharmaceutical Sciences, Brno

Research experience Glycobiology with the focus on bacterial lectins, their interaction with carbohydrates and structural properties. Experience with following methods: bioinformatics, molecular biology methods (gene cloning, PCR, colony PCR, gene knockout, CRISPR), protein expression and purification, biophysical methods related to biomolecular interaction measurements (ITC, SPR, MST), methods for macromolecular structure determination (DLS, AUC), protein crystallography.

Teaching activities Co-supervisor of two bachelor theses Lukáš Faltinek, Agglutination of Photorhabdus luminescens cells using multivalent saccharides compounds (2015-2017) Šárka Hanáková, Purification of lectins from Photorhabdus luminescens (2015-2017) Consultant of two diploma theses Filip Melicher, Structural studies of lectins from Photorhabdus luminescens (2017-2018) Lukáš Faltinek, Characterization of lectins from Photorhabdus spp. (2018- 2019)

Publications Lukáš Faltinek*, Eva Fujdiarová*, Filip Melicher, Josef Houser, Martina Kašáková, Nikolay Kondakov, Leonid Kononov, Kamil Parkan, Sébastien Vidal, Michaela Wimmerová: Lectin PLL3, a novel monomeric member of seven-bladed β-propeller lectin family. Molecules, 2019, 24, 4540, doi.org/10.3390/molecules24244540 Lenka Malinovská, Son Thai Le, Mihály Herczeg, Michaela Vašková, Josef Houser, Eva Fujdiarová, Jan Komárek, Petr Hodek, Anikó Borbás, Michaela Wimmerová, Magdolna Csávás. Synthesis of β-D-galactopyranoside- presenting glycoclusters, investigation of their interactions with Pseudomonas aeruginosa lectin A (PA-IL) and evaluation of their

72 anti-adhesion potential. Biomolecules, 2019, 9(11), DOI: 10.3390/biom9110686. Petra Sýkorová, Jitka Novotná, Gabriel Demo, Guillaume Pompidor, Eva Dubská, Jan Komárek, Eva Fujdiarová, Josef Houser, Lucia Hároníková, Annabelle Varrot, Nadezhda Shilova, Anne Imberty, Nicolai Bovin, Martina Pokorná, Michaela Wimmerová. Characterization of novel lectins from Burkholderia pseudomallei and Chromobacterium violaceum with seven- bladed β-propeller fold. International Journal of Biological Macromolecules, 2019, in press, DOI: 10.1016/j.ijbiomac.2019.10.200. Martina Kašáková, Lenka Malinovská, Tomaš Klejch, Martina Hlavačková, Hana Dvořaková, Eva Fujdiarová, Zdeňka Rottnerová, Olga Maťatková, Pavel Lhoták, Michaela Wimmerová, Jitka Moravcová. Selectivity of original C- hexopyranosyl calix[4]arene conjugates towards lectins of different origin. Carbohydrate research, 2018, ISSN 1873-426X, doi: 10.1016/j.carres.2018.08.012. Gita Jančaříková, Mihály Herczeg, Eva Fujdiarová, Josef Houser, Katalin E. Kövér, Anikó Borbás, Michaela Wimmerová, and Magdolna Csávás. Synthesis of a-Lfucopyranoside-presenting glycoclusters and investigation of their interaction with Photorhabdus asymbiotica lectin (PHL). Chemistry: A European Journal, 2018, ISSN 1521-3765, DOI: 10.1002/chem.201705853.

International fellowships Department of Microbiology, University College Cork, Ireland. 08 - 21 February 2016. I was trained in the laboratory of microbiology of professor David Clark. During the fellowship, I was introduced to the theory of bacterial genome modification. On the example of Photorhabdus laumondii, I became familiar with the method of gene knock-out using the bacteriophage derived vector pDS132, transferred by the bacterial conjugation process. I also independently conducted experiments leading to the construction of the desired P. laumondii mutant.

Active conference participation ▪ XVI Discussions in Structural Molecular Biology, 21 – 23 March 2019, Nové Hrady. Influence of glucose 3-O-methylation on binding properties towards PLL2 lectin (poster presentation) ▪ 29th International Carbohydrate Symposium, 15 – 19 July 2018, Lisbon. Glycoclusters as a tool for inhibition of lectin-mediated bacterial adhesion (poster presentation) ▪ The 43rd FEBS Congress, 07 – 13 July 2018, Prague. Study of lectins from Photorhabdus luminescens bacterium (speed talk + poster presentation) ▪ XVIII Meeting of Biochemists and Molecular Biologists, 14 – 15 November 2017, Brno. Study of Photorhabdus luminescens lectins to reveal their function in the entomopathogenic life cycle (student talk) ▪ XXV Biochemical Congress, 13 - 16 September 2016, Praha. Characterization of lectins from Photorhabdus luminescens to reveal their function in an entomopathogenic complex with Heterorhabditis bacteriophora (student talk)

Selected workshops and courses Macromolecular X-ray crystallography ▪ Advanced diffraction techniques for biology. 19-22 November 2019, Institute de Biologie Structurale, Grenoble, France. ▪ Workshop on data collection and structure solving in macromolecular X-ray diffraction. 12-16 July 2019, Jerzy Haber Institute of Catalysis and Surface Chemistry, Krakow, Poland. ▪ HERCULES European school. 25 February – 03 March 2018, European Synchrotron Radiation Facility (ESRF), Grenoble, France. ▪ Biomacromolecular crystallization workshop. 23 – 26 October 2017, CEITEC, Masaryk University, Brno. ▪ Tutorial in Molecular Crystallography. 06 – 10 March 2017, Institute de Biologie Structurale (IBS), Grenoble, France. ▪ Structural glycoscience – workshop. 28 June – 1 July 2016, Université Grenoble Alpes, Grenoble, France.

Protein interaction and characterization ▪ SPR Workshop Application in life-science. 15 - 16 November 2018, CEITEC, Masaryk University, Brno. ▪ How to characterize your sample and check its quality. 23 - 25 May 2018, CEITEC, Masaryk University, Brno. ▪ Differential Scanning Calorimetry. 03 – 04 October 2017, CEITEC, Masaryk University, Brno. ▪ Workshop on Surface Plasmon Resonance – theory and hands-on. 22 – 24 May 2017, CEITEC, Masaryk University, Brno. ▪ Microscale thermophoresis and differential scanning fluorimetry workshop. 12 – 13 November 2015, CEITEC, Masaryk University, Brno.

Molecular biology ▪ Modern Techniques in Molecular Biology and Genome Engineering Workshop. 17 – 20 July 2017, Vienna biocentre Core Facility, Vienna, Austria.

APPENDIX

APENDIX I

APENDIX II

Lukáš Faltinek*, Eva Fujdiarová*, Filip Melicher, Josef Houser, Martina Kašáková, Nikolay Kondakov, Leonid Kononov, Kamil Parkan, Sébastien Vidal, Michaela Wimmerová. Lectin PLL3, a novel monomeric member of seven-bladded β-propeller lectin family. * These authors contributed equally Molecules, 2019, 24, 4540 doi.org/10.3390/molecules24244540

APENDIX III

Gita Jančaříková, Mihály Herczeg, Eva Fujdiarová, Josef Houser, Katalin E. Köver, Anikó Borbás, Michaela Wimmerová, and Magdolna Csávás.

Synthesis of α-L-fucoside-presenting glycoclusters and investigation of their interaction with Photorhabdus asymbiotica lectin (PHL). Chemistry: A European Journal, 2018, 24(16), 4055-4068 DOI: 10.1002/chem.201705853 Full text is used with permission of John Wiley & Sons in printed and electronic version (Licence number: 4334690785003)

APENDIX IV

APENDIX V

Lenka Malinovská, Son Thai Le, Mihály Herczeg, Michaela Vašková, Josef Houser, Eva Fujdiarová, Jan Komárek, Petr Hodek, Anikó Borbás, Michaela Wimmerová, Magdolna Csávás.

APENDIX VI