1 Central
European Institute for Technology (CEITEC), Masaryk University, Brno, Czech
Republic.
2 National
Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno,
Czech Republic.
3 Department
of Animal Physiology and Immunology, Institute of Experimental Biology, Masaryk
University, Brno, Czech Republic.
4 Department
of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic.
Photorhabdus
asymbiotica is gram-negative
bioluminescent bacteria living in a symbiotic relationship with nematodes from
the genus Heterorhabditis. Together with
nematode it forms a complex that is highly pathogenic for insects. However,
while other three recognized species of the Photorhabdus
genus are strictly entomopathogenic, P.
asymbiotica is unique in its ability to act as an emerging human pathogen
as well.
Analysis of the P.
asymbiotica genome identified a novel lectin designated PHL with a sequence
similarity to the recently described lectin PLL from P. luminescens. Recombinant PHL protein was purified and
characterized. 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. PHL was shown to interact with all
types of red blood cells and insect haemocytes. It inhibits the production of
reactive oxygen species in human blood and antimicrobial activity both in human
blood, serum and insect haemolymph.
In order to further examine its binding abilities, we
crystallized the protein and soaked it with methyl-α,L-fucoside, D-galactose
and BGH trisaccharide – the human blood cell epitope. The X-ray diffraction
data were collected at BESSY synchrotron in Berlin, with resolution of 1.9 –
2.2 Å. The structure analysis of these complexes revealed an unusual
organization of binding sites that was not observed in any other lectin so far.
Surprisingly, up to twelve binding sites per monomer can be capable of
saccharide ligand binding. Additionally, PHL forms a dimer, which is further
stabilized by intramonomer disulfide bridge. The presence of high number of
binding sites per monomer together with protein dimerization enables high
affinity of the lectin towards potential interacting surfaces, e.g. bacteria,
immune cells or host epithelia. These results suggest that PHL might play a
crucial role in the interaction of P.
asymbiotica with both human and insect hosts.
This
work was supported by the Czech Science Foundation (project 13-25401S) and by
the Ministry of Education, Youth and Sports of the Czech Republic under the
project CEITEC 2020 (LQ1601). 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 electron storage ring
(Berlin-Adlershof, Germany) for access to their synchrotron data collection
facilities and allocation of synchrotron radiation beam time.