Photorhabdus
asymbiotica is a gram-negative bioluminescent
bacterium living in a symbiosis with Heterorhabditis
nematodes forming a highly entomopathogenic complex. Unlike other Photorhabdus species, P. asymbiotica can act as an emerging
human pathogen as well. In its genome, we identified a gene for a putative lectin,
and examined the corresponding recombinant protein PHL from functional and
structural point of view. It exhibited high affinity for fucosylated
carbohydrates and lower affinity to several other mono- and oligosaccharides
including saccharides from bacterial cell wall or human blood epitopes. It was further
shown to interact with all types of red blood cells and insect haemocytes,
inhibit the production of reactive oxygen species in human blood and inhibit
antimicrobial activity both in human blood, serum and insect haemolymph [1].
We succeeded in
determining structure of PHL in complex with several monosaccharides revealing
its unusual properties. It was shown that fucose and galactose occupy different
group of well-defined sites, making PHL the first confirmed case of
barrel-shape lectin with two sets of sites displaying different specificity and
arranged in two layers. As it further forms dimer, the maximal number of
potential binding sites per biological unit is 28. This arrangement lead to
forming a new type of lectin called bangle lectin [1].
As being promising target for treatment of P. asymbiotica related infections, we tested a wide range of various mono-, di- and oligovalent carbohydrate-based molecules. Their ability to bind PHL and subsequently inhibit its interactions with natural ligands was studied using heamagglutination, isothermal titration calorimetry and surface plasmon resonance [2]. For several of them, we also succeeded in preparing crystals of corresponding PHL complexes and analyzing their structures (Fig. 1). Data gained so far show the way for design and synthesis of potential therapeutics.
This work was supported by the Czech Science Foundation (project 18-18964S) and from European Regional Development Fund-Project „CIISB4HEALTH“ (No. CZ.02.1.01/0.0/0.0/16_013/0001776). CIISB research infrastructure project LM2015043 funded by MEYS CR is also gratefully acknowledged for the financial support of the measurements at the CF Biomolecular Interactions and Crystallization, CF X-ray Diffraction and Bio-SAXS and CF Proteomics at CEITEC (Brno, Czech Republic). We wish to thank the BESSY II (Berlin-Adlershof, Germany) and PETRA III (Hamburg, Germany) for access to their synchrotron data collection facilities and allocation of synchrotron radiation beam time.
Figure 1: PHL dimer in complex
with one of the studied inhibitors (shown as ball and sticks). Individual
monomers shown in white and black, respectively, and recognized binding sites
highlighted in orange.