Page 75 - Zbornik_Konf_2019

https://doi.org/10.15414/2019.9788055220703

4 International Scientific Conference Abstracts Book
th
CHARACTERIZATION OF THE PSEUDOMONAS PUTIDA LIPOPOLYSACCHARIDES,
CAUSATIVE AGENTS OF CHESTNUT DISEASES
Оksana Brovarska, Liudmyla Varbanets, Аrtur Likhanov

Zabolotny Institute of Microbiology and Virology, NAS of Ukraine, Kyiv, Ukraine;
E-mail.: oksanabro2016@gmail.com
At present, much attention is being paid to the search for new natural
immunomodulators, including promising lipopolysaccharides (LPS), which are the main
components of the outer membrane of gram-negative bacteria. Despite the fact that the LPS of
phytopathogenic species of Pseudomonas is well-studied today, representatives of
Pseudomonas putida, isolated from chestnut leaves, causing large-scale destruction of trees,
studied very little.
In this regard, the purpose of this work was to investigate the LPS of Pseudomonas
putida, isolated from the leaves of chestnut in Kyiv. LPS was extracted from dried cells with 45
% aqueous solution of phenol. The amount of carbohydrates was determined by the Dubois
method, the content of proteins was analyzed by Lowry method with Folin reagent.
Identification of neutral monosaccharides and fatty acids was carried out on an Agilent
6890N/5973 inert chromatography-mass spectrometry system. The antigenic activity of LPS
was studied by the method of double immunodiffusion in agar according to Ouchterlony. The
study of toxicity (LD50) was performed on healthy white mice, introducing them the various
LPS concentrations. The pyrogenic effect of LPS was determined on rabbits weighing 2.5–3.0
kg by thermometry after an introduction on the minimum pyrogenic dose of LPS. It was
shown that yielding of Pseudomonas putida LPS was 7.5 % of the dry cell mass. When studying
the biopolymer composition it is shown that the content of carbohydrates in preparations of
LPS was 63.2 %, protein 1.36 %, and 2-keto-3-deoxyoctonic acid, which is always presented in
LPS and is a kind of marker of the molecule, found in small amounts to 0.1 %. In PAAG
electrophoresis with the coloration of gels by silver salts, it was shown that P. putida
synthesizes S-type LPS, presented in the form of heterogeneous bands with the formation of
"stairs". By the chromato-mass-spectrometric method, it was established that LPS contains
saturated and hydroxy acids with a length of the carbon chain from C11 to C20. Hydroxy acids
were predominant, the total percentage of which was 45.61%. The length of the chain and the
amount of fatty acids in lipid A play an essential role in the manifestation of toxic effects of
LPS. It was found that LD50 of P. putida LPS for white mice sensitized with D-galactosamine
was 29.73 μg/mouse. Compared to LPS of other Enterobacteriaceae: Rahnella aquatilis (5–6
μg/mouse) E. coli 126 (11.25 μg/mouse), the investigated P. putida LPS was less toxic. It was
also non-pyrogenic: an hour after its introduction there was a decrease of rectal temperature
at 0.2 °С. Hypothermia was observed for three hours. When studying the immunochemical
properties of LPS, polyclonal O-antiserum was obtained by immunizing rabbits with a boiled
P. putida cells. The titre in the reaction of ring precipitation was 1:320 000. In the reaction of
immunodiffusion in Ouchterlony O-antiserum against to P. putida cells gave with homologous
LPS only one clear line of precipitation in a concentration of 1 mg/ml, indicating its
serological activity.
Since LPS is a major factor in the pathogenicity of gram-negative bacteria, the obtained
results can be used in further work to combat chestnut diseases caused by P. putida.

Keywords: Pseudomonas putida, lipopolysaccharide, lipid A fatty acid composition, toxicity,
pyrogenicity, serological activity.

4 International Scientific Conference Agrobiodiversity Nutrition, Health and Quality of Human and Bees Life |75
th
September 11–13, 2019