DOI 10.1007/s10517-015-2936-3
258
Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 IMMUNOLOGY AND MICROBIOLOGY
Cell-Mediated Hemolytic Activity of Nosocomial Pseudomonas Aeruginosa Strains M. V. Kuznetsova*,** and T. I. Karpunina** Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 159, No. 2, pp. 219-223, February, 2015 Original article submitted December 22, 2013 Cell-mediated hemolysis and adhesion index of nosocomial P. aeruginosa strains were experimentally studied. The highest hemoglobin release was recorded after centrifugation of erythrocyte and bacterial cell suspension preincubated at 37oC. All cultures were referred to highly adherent variants. The relationship between P. aeruginosa adhesion activity and erythrocyte lysis was found only in “passive” cell–cell contact. No correlation between cellassociated hemolysis and hemolysis caused by secreted factors was detected. It seems that the cytotoxicity of the studied P. aeruginosa strains was determined by ExoU and ExoS third type secretion effectors. Key Words: P. aeruginosa; cell-mediated hemolysis; adhesion; ExoU; ExoS
Pseudomonas aeruginosa is an opportunistic pathogen inducing a pyoseptic disease in immunodeficiency caused by stress factors associated with severe somatic diseases, injury, burn, and surgical interventions. The mechanisms of pathogenic effects of ubiquitous bacteria, one of which is P. aeruginosa, can be different and remain not quite clear. Obviously, the principle of the only dominant pathogenic factor cannot be applied to these bacteria, because the unfolding infectious process and its course including cell damage are determined by a group of effectors. Hemolytic activity of P. aeruginosa is most often determined by extracellular substances: thermolabile phospholipases C (PlcH, PlcN, and PlcB) and a thermostable component, based on glycolipids: monorhamnolipid (Rha-C10-C10) and dirhamnolipid (Rha-Rha-C10-C10) [2,3]. Cell-associated hemolysis is described for P. aeruginosa manifesting only under conditions of a direct contact between the bacteria and erythrocytes and mediated, similarly as in other gram-negative bacteria, by type III secretion system (TTSS) proteins. P. aeruginosa uses TTSS for translocation of four ef*Institute of Ecology and Genetics of Microorganisms, Ural Division of the Russian Academy of Sciences; **E. A. Wagner Perm State Medical Academy, Ministry of Health of the Russian Federation, Perm, Russia. Address for correspondence: [email protected]. M. V. Kuznetsova
fector proteins: ExoT, ExoS, ExoU, and ExoY [7]. Two of them, ExoS and ExoT, have two functional domains: the carboxyterminal fragment of the enzymes has ADP-ribosyltransferase activity, while the aminoterminal fragment activates GTPases [9]. Exoenzyme ExoY exhibits adenylate cyclase activity, ExoU is the most potent cytotoxin functioning as a phospholipase and can be classified with this group of enzymes [14]. We studied the probable mechanisms of cell-mediated hemolysis in nosocomial P. aeruginosa strains.
MATERIALS AND METHODS P. aeruginosa reference strain ATCC 27853 (F-53) from L. A. Tarasevich State Institute of Viral Preparations Standardization and Control, Ministry of Health of Russian Federation was used in the study. Clinical strains (n=12) were isolated from patients with pyoseptic diseases in surgical hospitals of Perm. Erythrocytes were collected from a donor with blood group 0(I), Rh(+). Hemolysis was evaluated on 5% blood agar after 24-h incubation of the cultures at 37oC. A coefficient was derived (hemolysis zone diameter/colony diameter, mm) for more correct interpretation of the results. Cell-associated hemolysis was studied as described previously [5]. Erythrocytes were washed
0007-4888/15/15920258 © 2015 Springer Science+Business Media New York
M. V. Kuznetsova and T. I. Karpunina
259
twice in PBS (10 mM potassium-sodium-phosphate buffer on 0.9% NaCl, pH 7.2) and resuspended in the same medium to the final concentration of 2% erythrocytes at 4oC. The bacteria were cultured in Luria-Bertani broth (LB-broth) for 24 h, centrifuged at 8000 rpm, and resuspended in PBS to the opacity equivalent to 2.0 according to McFarland’s standard. Washed erythrocytes and bacterial culture suspension were mixed (1 ml each) in Eppendorf tubes, which were then centrifuged at 1000 rpm for 10 min or not centrifuged (parallel experiments) and incubated on a vortex at 100 rpm, 37oC or 25oC for 1 h. After incubation the tubes were centrifuged at 1000 rpm for 10 min. The hemolytic activity of P. aeruginosa supernatants was studied as follows. Overnight cultures, standardized to 2.0 after McFarland, were inoculated in tubes with LB-broth (1 ml standard culture and 5 ml LB-broth). Supernatants of 24-h cultures, filtered through Millex-MP (0.22 μ; Nihon Millipore) were mixed in equal volumes with erythrocytes washed twice. The erythrocytes with native supernatants were incubated at 37oC for 1 h. After exposure the tubes were centrifuged for 10 min at 1000 rpm and the hemolytic activity was evaluated. Hemoglobin release was measured in 200 μl of supernatant in the wells of a 96-well polystyrene plate by optical density (OD) at =540 nm on a Benchmark
Plus spectrophotometer (Bio-Rad). The percentage of lysis was evaluated by the formula [5]: X-B ×100%, T-B where B was negative control (basal level, solution OD 540 after incubation of erythrocytes with 1 ml broth), T was positive control (OD540 of solution after erythrocyte lysis under the effect of 1 ml 0.1% SDS inducing maximum hemoglobin release), and X was OD540 of analyzed specimen. The level of bacterial adhesion to erythrocytes was measured as described previously [1]. Formalintreated human erythrocytes (blood group 0(I), Rh(+)) washed twice in PBS and brought to 100×10 6/ml served as the cell substrate. The bacterial cells adhering to one erythrocyte were counted (at least 25 erythrocytes). The cell adhesion characteristics were evaluated by the microorganism adhesion index (MAI) – the mean number of bacteria adhering to one erythrocyte, participating in the adhesion process [1]. The microorganisms were considered nonadherent at MAI≤1.75, poorly adherent at MAI of 1.76-2.5, medium adherent at 2.51 to 4.0 MAI, and highly adherent at MAI≥4.0. The genes coding for TTSS effectors were detected by PCR with primers for genes exoS (5’-GGCCCAGGATCGGCTTGCAA and 5’-GATCCGCTGCCGAGCCAAGA; fragment 1800 b. p.)
TABLE 1. Hemolysis of P. aeruginosa Strains Hematolytic activity, % of lysis after 1-h incupation at 37оC P. aeruginosa stain
АТСС 27853
KA
cells without centrifugation
cells after centrifugation
native supernatants
1.8±0.4
10.53±2.17
67.60±7.48*
58.07±13.40
801
1.4±0.3
12.72±3.78
74.81±23.24*
68.60±18.91
5
1.5±0.4
21.60±5.23
66.34±21.23*
56.56±23.12
3-7
2.5±0.5
25.31±4.08
72.62±19.98*
32.01±8.34
2459
1.5±0.2
20.04±3.16
54.07±18.51*
51.11±15.67
63
2.4±0.3
32.34±7.34
93.65±25.92*
22.51±7.32
BALF
2.8±0.3
23.62±2.72
68.99±17.39*
91.90±23.34
Sputum
3.0±0.5
13.67±3.74
68.03±14.28*
58.88±13.45
9-3
1.1±0.2
11.18±1.95
97.02±26.08*
72.69±34.30
Drainage
2.1±0.3
6.25±1.23
46.02±6.51*
68.88±12.35
29а
1.0±0.2
5.74±2.03
46.62±7.72*
28.87±6.35
ITT
1.8±0.4
13.48±4.20
62.22±12.54*
67.30±14.65
565
1.2±0.2
22.66±5.04
74.80±26.07*
41.39±12.02
Note. KA: coefficient hemolysis zone diameter/colony diameter in blood agar, arb. units. *p
258
Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 IMMUNOLOGY AND MICROBIOLOGY
Cell-Mediated Hemolytic Activity of Nosocomial Pseudomonas Aeruginosa Strains M. V. Kuznetsova*,** and T. I. Karpunina** Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 159, No. 2, pp. 219-223, February, 2015 Original article submitted December 22, 2013 Cell-mediated hemolysis and adhesion index of nosocomial P. aeruginosa strains were experimentally studied. The highest hemoglobin release was recorded after centrifugation of erythrocyte and bacterial cell suspension preincubated at 37oC. All cultures were referred to highly adherent variants. The relationship between P. aeruginosa adhesion activity and erythrocyte lysis was found only in “passive” cell–cell contact. No correlation between cellassociated hemolysis and hemolysis caused by secreted factors was detected. It seems that the cytotoxicity of the studied P. aeruginosa strains was determined by ExoU and ExoS third type secretion effectors. Key Words: P. aeruginosa; cell-mediated hemolysis; adhesion; ExoU; ExoS
Pseudomonas aeruginosa is an opportunistic pathogen inducing a pyoseptic disease in immunodeficiency caused by stress factors associated with severe somatic diseases, injury, burn, and surgical interventions. The mechanisms of pathogenic effects of ubiquitous bacteria, one of which is P. aeruginosa, can be different and remain not quite clear. Obviously, the principle of the only dominant pathogenic factor cannot be applied to these bacteria, because the unfolding infectious process and its course including cell damage are determined by a group of effectors. Hemolytic activity of P. aeruginosa is most often determined by extracellular substances: thermolabile phospholipases C (PlcH, PlcN, and PlcB) and a thermostable component, based on glycolipids: monorhamnolipid (Rha-C10-C10) and dirhamnolipid (Rha-Rha-C10-C10) [2,3]. Cell-associated hemolysis is described for P. aeruginosa manifesting only under conditions of a direct contact between the bacteria and erythrocytes and mediated, similarly as in other gram-negative bacteria, by type III secretion system (TTSS) proteins. P. aeruginosa uses TTSS for translocation of four ef*Institute of Ecology and Genetics of Microorganisms, Ural Division of the Russian Academy of Sciences; **E. A. Wagner Perm State Medical Academy, Ministry of Health of the Russian Federation, Perm, Russia. Address for correspondence: [email protected]. M. V. Kuznetsova
fector proteins: ExoT, ExoS, ExoU, and ExoY [7]. Two of them, ExoS and ExoT, have two functional domains: the carboxyterminal fragment of the enzymes has ADP-ribosyltransferase activity, while the aminoterminal fragment activates GTPases [9]. Exoenzyme ExoY exhibits adenylate cyclase activity, ExoU is the most potent cytotoxin functioning as a phospholipase and can be classified with this group of enzymes [14]. We studied the probable mechanisms of cell-mediated hemolysis in nosocomial P. aeruginosa strains.
MATERIALS AND METHODS P. aeruginosa reference strain ATCC 27853 (F-53) from L. A. Tarasevich State Institute of Viral Preparations Standardization and Control, Ministry of Health of Russian Federation was used in the study. Clinical strains (n=12) were isolated from patients with pyoseptic diseases in surgical hospitals of Perm. Erythrocytes were collected from a donor with blood group 0(I), Rh(+). Hemolysis was evaluated on 5% blood agar after 24-h incubation of the cultures at 37oC. A coefficient was derived (hemolysis zone diameter/colony diameter, mm) for more correct interpretation of the results. Cell-associated hemolysis was studied as described previously [5]. Erythrocytes were washed
0007-4888/15/15920258 © 2015 Springer Science+Business Media New York
M. V. Kuznetsova and T. I. Karpunina
259
twice in PBS (10 mM potassium-sodium-phosphate buffer on 0.9% NaCl, pH 7.2) and resuspended in the same medium to the final concentration of 2% erythrocytes at 4oC. The bacteria were cultured in Luria-Bertani broth (LB-broth) for 24 h, centrifuged at 8000 rpm, and resuspended in PBS to the opacity equivalent to 2.0 according to McFarland’s standard. Washed erythrocytes and bacterial culture suspension were mixed (1 ml each) in Eppendorf tubes, which were then centrifuged at 1000 rpm for 10 min or not centrifuged (parallel experiments) and incubated on a vortex at 100 rpm, 37oC or 25oC for 1 h. After incubation the tubes were centrifuged at 1000 rpm for 10 min. The hemolytic activity of P. aeruginosa supernatants was studied as follows. Overnight cultures, standardized to 2.0 after McFarland, were inoculated in tubes with LB-broth (1 ml standard culture and 5 ml LB-broth). Supernatants of 24-h cultures, filtered through Millex-MP (0.22 μ; Nihon Millipore) were mixed in equal volumes with erythrocytes washed twice. The erythrocytes with native supernatants were incubated at 37oC for 1 h. After exposure the tubes were centrifuged for 10 min at 1000 rpm and the hemolytic activity was evaluated. Hemoglobin release was measured in 200 μl of supernatant in the wells of a 96-well polystyrene plate by optical density (OD) at =540 nm on a Benchmark
Plus spectrophotometer (Bio-Rad). The percentage of lysis was evaluated by the formula [5]: X-B ×100%, T-B where B was negative control (basal level, solution OD 540 after incubation of erythrocytes with 1 ml broth), T was positive control (OD540 of solution after erythrocyte lysis under the effect of 1 ml 0.1% SDS inducing maximum hemoglobin release), and X was OD540 of analyzed specimen. The level of bacterial adhesion to erythrocytes was measured as described previously [1]. Formalintreated human erythrocytes (blood group 0(I), Rh(+)) washed twice in PBS and brought to 100×10 6/ml served as the cell substrate. The bacterial cells adhering to one erythrocyte were counted (at least 25 erythrocytes). The cell adhesion characteristics were evaluated by the microorganism adhesion index (MAI) – the mean number of bacteria adhering to one erythrocyte, participating in the adhesion process [1]. The microorganisms were considered nonadherent at MAI≤1.75, poorly adherent at MAI of 1.76-2.5, medium adherent at 2.51 to 4.0 MAI, and highly adherent at MAI≥4.0. The genes coding for TTSS effectors were detected by PCR with primers for genes exoS (5’-GGCCCAGGATCGGCTTGCAA and 5’-GATCCGCTGCCGAGCCAAGA; fragment 1800 b. p.)
TABLE 1. Hemolysis of P. aeruginosa Strains Hematolytic activity, % of lysis after 1-h incupation at 37оC P. aeruginosa stain
АТСС 27853
KA
cells without centrifugation
cells after centrifugation
native supernatants
1.8±0.4
10.53±2.17
67.60±7.48*
58.07±13.40
801
1.4±0.3
12.72±3.78
74.81±23.24*
68.60±18.91
5
1.5±0.4
21.60±5.23
66.34±21.23*
56.56±23.12
3-7
2.5±0.5
25.31±4.08
72.62±19.98*
32.01±8.34
2459
1.5±0.2
20.04±3.16
54.07±18.51*
51.11±15.67
63
2.4±0.3
32.34±7.34
93.65±25.92*
22.51±7.32
BALF
2.8±0.3
23.62±2.72
68.99±17.39*
91.90±23.34
Sputum
3.0±0.5
13.67±3.74
68.03±14.28*
58.88±13.45
9-3
1.1±0.2
11.18±1.95
97.02±26.08*
72.69±34.30
Drainage
2.1±0.3
6.25±1.23
46.02±6.51*
68.88±12.35
29а
1.0±0.2
5.74±2.03
46.62±7.72*
28.87±6.35
ITT
1.8±0.4
13.48±4.20
62.22±12.54*
67.30±14.65
565
1.2±0.2
22.66±5.04
74.80±26.07*
41.39±12.02
Note. KA: coefficient hemolysis zone diameter/colony diameter in blood agar, arb. units. *p
