Bulletin of Experimental Biology and Medicine, Vol. 154, No. 1, November, 2012
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IMMUNOLOGY AND MICROBIOLOGY Differential Analysis of Bactericidal Systems of Blood Serum with Recombinant Luminescent Escherichia coli and Bacillus subtilis Strains D. G. Deryabin, I. F. Karimov, I. V. Manukhov*, N. A. Tolmacheva, and V. P. Balabanov*
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 154, No. 7, pp. 68-73, July, 2012 Original article submitted April 13, 2011 Luminescence intensity of recombinant Escherichia coli and Bacillus subtilis strains with cloned luxCD(AB)E genes of the natural luminescent microorganism Photobacterium leiognathi was studied under the influence of 30 individual samples of human blood serum of different component composition. A relationship was found between the level of residual bioluminescence and degree of the bactericidal effect. Moreover, the inhibition of E. coli lux+ luminescence was shown to be related to activity of the complement-lysozyme system. The reaction of B. subtilis lux+ primarily depended on the presence of β-lysin in the blood serum. These data provide an experimental substantiation of a new method of differential analysis of humoral factors of nonspecific innate immunity with recombinant luminescent bacteria. Key Words: bioluminescence; E. coli; B. subtilis; complement; β-lysin Studies of bactericidal systems in human and animals, which form the system of nonspecific innate immunity, are the urgent problems of modern biology and medicine [8,9]. These studies include analysis of basic bactericidal systems in the human serum. Emil von Behring first designated them as thermolabile α-lysin and thermostable β-lysin. α-Lysin was then identified as the complement system [7]. Activation of this system is followed by an effective lysis of gram-negative microorganisms (MO), while β-lysin exhibiting maximum affinity for gram-positive MO is often associated with platelet-derived cationic protein [1]. Experimental, clinical, and diagnostic studies for activity of the complement-β-lysin system involve a Orenburg State University; *State Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia. Address for correspondence:
[email protected]. D. G. Deryabin
routine bacteriological test with special laboratory cultures that are highly sensitive to an adverse effect of these factors [6]. Differences in the composition of surface structures in MO allow us to perform a relatively specific analysis of each bactericidal system. Further improvement of these tests (i.e. development of rapid analyses and highly technological methods) is closely related to the use of recombinant luminescent MO as a target object. A change (inhibition) in luminescence of these MO is in direct proportion to the bactericidal effect recorded by bacteriological methods or cytofluorometry [3,11]. The reliability of this approach was confirmed by published data on the dependence of bioluminescence of the recombinant strain E. coli K12 TG1 with cloned luxCD(AB)E genes from Photobacterium leiognathi [4] and other model gram-negative bacteria [10] on the appropriate function of the classical and alternative pathways of
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Bulletin of Experimental Biology and Medicine, Vol. 154, No. 1, November, 2012 IMMUNOLOGY AND MICROBIOLOGY
complement activation. It should be emphasized that luminescence of the original strain B. subtilis carrying Vibrio harveyi luxAB genes is highly sensitive to platelet-derived cationic protein [5]. Here we compared the changes in luminescence of E. coli and B. subtilis strains with P. leiognathi lux genes in response to treatment with blood sera of different component composition. A special attention was paid to the evaluation of dependence of luminescence inhibition on the quantitative presence of individual bactericidal factors.
MATERIALS AND METHODS Experiments were performed with the following target objects: recombinant strain E. coli K12 TG1 with a complete cassette of luxCDABE genes from the marine luminescent bacterium Photobacterium leiognathi 54D10 [2] (designated as E. coli lux+); and original strain B. subtilis lux+ (developed in the present work) from the All-Russian Collection of Industrial Microorganisms (State Institute of Genetics and Selection of Industrial Microorganisms; strain No. B-10548). The ribosome-binding site typical of gram-positive bacteria was introduced before each luxAB gene in P. leiognathi. Hence, B. subtilis lux+ is characterized by high level of translation of constitutively transcribed genes of bacterial luciferase after addition of the luminescent reaction substrate (long-chain aldehyde decanal). Luminescence of this strain was at least 10 times greater than that of B. subtilis with unmodified ribosomebinding sites of luxAB genes from Vibrio harveyi [5]. The bacteria were grown on LB agar in the presence of a selective factor (100 g/ml ampicillin for E. coli; or 40 g/ml kanamycin for B. subtilis) at 37oC for 24 h. The biomass was washed out with sterile LB broth immediately before the study. Then the biomass was standardized to an optical density of 1.2 rel. units at 540 nm. The measurements were performed in cuvettes (optical path length 1 cm) on a Flyuorat-02 Panorama spectrofluorometer (Lyumeks). Thirty individual samples of the human serum from healthy donors were used as a source of bactericidal factors. The component composition of each sample was evaluated by immunochemical methods. Total complement activity (CH50) was estimated from the lysis of 50% sheep erythrocyte suspension after sensitization with specific rabbit antibodies (Mikrogen). The quantitative presence of C3, C4, and C5 complement components and factor H was estimated by means of EIA with test systems (Tsitokin). The total concentrations of IgA, IgM, and IgG and amount of surface-specific antibodies (for E. coli and B. subtilis) were measured by the same method with test systems (Vektor-Best). The activities of lysozyme and β-lysin
were estimated by nephelometric methods [1,6]. To study the effect of blood sera on luminescence of E. coli lux+ and B. subtilis lux+, they were mixed in the 1:1 ratio and incubated at 37oC. Aliquots of the reaction system (250 l) were sampled on the 0th and 30th minutes. In experiments with B. subtilis lux+, 2 μl decanal (7×10–6 M) was added to these aliquots. The same luminescent bacteria were mixed with 0.85% NaCl (v/v ratio 1:1) and served as the control. Luminescence was recorded on a BLM-8802M luminometer (Nauka; for E. coli lux+) or Biotoks-7 luminometer (Nera; for B. subtilis lux+). The effect of blood sera on bacterial luminescence was determined as follows: lk0×lt30 , lk30×lt0 where lk and lt are the intensities of luminescence in the control and treated samples, respectively, on the 0th and 30th minutes of the measurement. The bactericidal effect of these sera on E. coli lux+ and B. subtilis lux+ on the 30th minute was studied simultaneously by the bacteriological method [3,5]. The measurements were performed in at least 3 repetitions. The effect of the component composition of blood sera on luminescence of E. coli lux+ and B. subtilis lux+ was analyzed by the multiple correlation test and factor analysis.
RESULTS Quantitative study of E. coli lux+ and B. subtilis lux+ bioluminescence after treatment with individual sera from 30 healthy donors revealed general and specific reactions of each sensory MO that are potentially determined by differences in the composition of surface structures in target cells. The influence of blood sera on E. coli lux+ was followed by a significant decrease in bioluminescence (15-85% of the control; Fig. 1, a). A positive correlation was found between these changes and number of viable E. coli lux+ cells (estimated by the bacteriological method; r=0.530, p
