Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 104-112

ISSN: 2319-7706 Volume 3 Number 4 (2014) pp. 104-112 http://www.ijcmas.com

Original Research Article Flow cytometry assay of adenylate cyclase (CyaA) preparations of B.pertussis on

S.A.Khosavani1, S.M.A.Mansorian1, Majid Amouei2 and A.Sharifi1*

1Yasuj University of Medical Sciences,Yasuj, Iran 2The Ministry of Health and Medical Education, Iran *Corresponding author

A B S T R A C T

Bordetella pertussis is the etiological agent of , a highly K e y w o r d s contagious childhood respiratory disease, characterized by bronchopneumonia and paroxysmal coughing interrupted by inspiratory whoops. Two purified forms of CyaA with different enzymic and invasive properties were produced. These were: pertussis; the native enzymatically-active, invasive toxin (CyaA), an invasive derivative childhood lacking AC enzymic activity (CyaA*). Different concentrations of CyaA and respiratory CyaA* were used to investigate dose-dependent effects of the on phagocytosis in U937 human monoblastic cells, J774.2 mouse -like disease; cells and fresh human granulocyte cells (whole blood used). Important effects were invasive seen with 0.2 mg /ml of CyaA. In instance, there was almost complete toxin (80%) inhibition of phagocytosis by J774.2 cells and 70% inhibition of phagocotosis by human granulocyte cells, but CyaA* did not have a significant effect on either. The results of this study showed that both enzymatic and invasive functions are required for the cytotoxic effects of adenylate cyclase toxin.

Introduction

Adenylate cyclase toxin (CyaA) is one of intoxicating and the major virulence factors produced by causing impotence and , the whooping cough inducing macrophage (Confer, agent. Among the variety of toxins and Eaton, 1982; Gueirard, et al., 1998). produced by B.pertussis, the adenylate The role of CyaA in the pathogenesis of cyclase (CyaA) is a crucial factor in the B.pertussis was clearly demonstrated in virulence strategy of the during the mouse respiratory model. Indeed, the early phases of respiratory tract genetically modified B. pertussis strains colonization (Mohammed El-Azami-El- deficient in the expression of CyaA were Idrissi et al., 2003; Smith et al., 2001). The impaired in their ability to induce toxin allows the pathogen to escape host pulmonary lesions and to cause lethal immune surveillance, mainly by (Khelef, et al., 1994; Weiss, and

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Goodwin, 1989). Inaddition, CyaA was al., 1998). At high concentrations, CyaA shown to induce protective forms pores or channels which makes the against B. pertussis colonization in a toxin cytolytic (El Azami El Idrissi et al., mouse model (Betsou, et al., 1993; Betsou 2002). Anti-CyaA have been et al., 1995). CyaA is a polypeptide chain shown to enhance phagocytosis of B. of 1,706 aa residues belonging to the RTX pertussis through, neutralisation of CyaA (repeat in toxin) family of bacterial toxins. which normally inhibits phagocytosis by CyaA is synthesized as an inactive polymorphonuclear leukocytes protoxin that is converted to an active (Mobberley-Schuman et al., 2003). toxin by posttranslational fatty acylation. The N-terminal part of the protein contains An immune response to this toxin might the catalytic domain, whereas the C- therefore be useful in preventing terminal part mediates its binding to the colonisation of the host by B. pertussis. target and delivery of the Immunisation with CyaA, purified from B. catalytic moiety into the cytosol. After pertussis or in recombinant form from membrane translocation, the catalytic , protected mice against domain is activated by Ca2+/, intranasal challenge with virulent B. thereby acquiring the ability to effectively pertussis (Betsou et al., 1993; Hormozi et convert cellular ATP into cAMP (Ladant al., 1999). In addition, coadministration of and Ullmann . 1999; Hewlett, et al., 2006). CyaA or CyaA*, a derivative lacking AC CyaA can also form cation-selective pores enzymic activity, with an ACV was shown in cell membranes independently of to enhance the protective effects of an translocation, thereby perturbing ion ACV in mice (Cheung et al., 2006). homeostasis (Benz et al., 1994). CyaA triggers the sustained elevation of CyaA requires to acquire a intracellular Ca2+ through cAMP- translocation-specific conformation that dependent, L-type Ca2+ channels. allows the delivery of the catalytic domain into the cell cytosol (Rogel, A., and The suppressive activities of CyaA on Hanski, E. (1992; Rose, et al., 1995) CyaA immune cells have been largely ascribed can penetrate at least to some extent a to its capacity to increase intracellular wide range of cell types, including the cAMP (Paccani, et al., 2008; Martín, et al., mammalian erythrocytes lacking 2010), which acts as a potent membrane trafficking (Basar, et al., 2001; immunosuppressant by interfering with the Bellalou, et al., 1990). However, CyaA signaling pathways initiated by toxicity effects such as the abrogation of immunoreceptors (Taskén, and Stokka . phagocytic capacity and the induction of 2006). Upon entry into the cell, the N- apoptosis were mainly elucidated on terminal AC enzymic moiety is activated immune cells, namely neutrophils and by host calmodulin to produce macrophages (Khelef, N., and Guiso, N. supraphysiological levels of cyclic AMP (1995; Khelef, et al., 1993). In this study, (cAMP). In immune effector cells, this we investigated the role of different impairs their phagocytic and bactericidal concentrations of CyaA and CyaA* on capabilities and induces apoptosis, features phagocytosis in U937 human monoblastic that are assumed to assist survival of the cells, J774.2 mouse macrophage-like cells bacterium in the initial stages of and fresh human granulocyte cells (whole respiratory tract colonisation (Gueirard et blood used).

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Materials and Methods Oxidative Burst Procedure

Preparation of Recombinant CyaA The phagotest Kit (OPREGEN Pharma; From E. coli BD Biosciences, Oxford, UK) allows the quantitative determination of leukocyte Twenty mL of an overnight culture of E. phagocytosis. It contains fluorescently coli BL21/DE3 containing the relevant (FITC- fluorescein isothiocyanate) plasmids were diluted into 500 mL of LB labelled, opsonized bacteria (E.coli-FITC) containing appropriate , and measures the overall percentage of incubated at 37C with shaking at 200 rpm macrophages and granulocytes showing until an OD600nm of between 0.4-0.45 phagocytosis in general (ingestion of one was obtained (~3 h). Isopropyl-1-thio--D- or more bacteria per cell) and the galactoside (IPTG) was added to a final individual cellular phagocytic activity concentration of 1 mM and shaking (number of bacteria per cell). continued at 37C for 3 h. Finally, cells The investigation of phagocytosis can be were harvested at 10,000 xg for 25 min performed either by flow cytometry or by and the supernatant discarded. Cells fluorescence microscopy. Because of the pellets could be stored at -20C if quantitative analysis, very accurate work necessary. is important, especially when day to day Expression and Purification of CyaA comparisons are required. The detailed instructions result from specific E. coli BL21/DE3 (F ompT rB mB ) experience and precise validation assays. was used as the host strain for production The ingestion activity of peripheral human of CyaA. The source of plasmids used in granulocyte cells in whole blood, J774.2 this study (pGW44 and pGW54) was de- and U937 cell lines was evaluated in the scribed previously (Khosravani et al., presence and absence of recombinant 2007; Westrop et al., 1996; Paccani, et al., CyaA protein using the flow-cytometry 2008; Martín, et al., 2010). Co-expression based Phagotest kit according to the of pGW44 with pGW54 generates fully manufacturer's instructions for conjugated active acylated, invasive CyaA, pGW44- E. coli. Briefly, 100 l of whole blood or a 188, pGW54 generates Non-active AC/ volume of 0.4 x 106 of J774.2 mouse invasive (CyaA*). The recombinant macrophages or human monoblastic U937 were purified as described cells were added at the bottom of a 5 ml previously with the following Falcon tube. Samples were incubated (120 modifications; the CyaA inclusion bodies minutes, 37ºC, 5% CO2) with either CyaA were washed twice with 1% (w/v) N-octyl or CyaA* diluted in 8 M urea, 20mM -d glucopyranoside (Sigma, Sweden in histidine (0.05, 0.1 or 0.2 g/ml) or PBS, 20mM histidine buffer (pH 6.0), twice then incubated for another 20 minutes at with 2M urea in 20mM histidine buffer 37ºC in 5% CO2 with opsonized E. coli- (pH 6.0) and once with pyrogen-free water FITC cells (6 cells per leukocyte). A before solubilisation in 8M urea, 20mM sample with PBS and no Bioparticle and histidine buffer (pH 6.0). The solubilised another sample with PBS (120 min) and crude CyaA was purified by Q-Sepharose, opsonized FITC-labeled E. coli cells (20 Germany Amersham) and phenyl- min) remained on ice for the whole period Sepharose chromatography (Figures 1 and of experiment, these acted as negative 2). controls.

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At the end of the incubation time all monoblastic cells was determined after samples were placed on ice in order to exposure to different concentrations of stop phagocytosis. A volume of 100 l of CyaA and CyaA*. This technique was ice-cold quenching solution was added and developed for the evaluation of mixed gently. Then 2ml of washing phagocytosis activity in human peripheral solution was added to each tube after blood (neutrophils and ) and which the tubes were centrifuged at 4 C other cells. Ingestion activity was for 5 min at 250 x g. The cell samples measured as the mean fluorescence were incubated in washing solution intensity (MFI) produced after 20 minutes containing 1% paraformaldehyde for 10 of incubation with FITC-conjugated E. min, washed again and centrifuged as coli. All tubes were read by flow described. Finally, 200 l of DNA staining cytometry at a wavelength of 488 nm after solution were added to each tube, mixed quenching and DNA staining. and incubated for 10 min on ice. Cells were analysed by flow cytometry (FACS Exposure of granulocytes or J774.2 cells Calibur, BD Biosciences) and 5000 to different concentration of CyaA and granulocyte events acquired to obtain the CyaA* (0.05, 0.1, 0.2 g/ml final percentage and number of cells ingesting concentrations) resulted in significantly bioparticles, as well as their mean lower mean neutrophil ingestion in the fluorescence intensity (MFI) using presence of CyaA compared to CyaA* at Cellquest Pro software (BD Biosciences). the same concentrations, and the The nucleated cells were discriminated by unexposed controls cells (Table 1 and a setting in the red fluorescence channel Figure 1). The results also showed that (DNA staining FL2-H,), detecting those phagocytosis was significantly impaired events which have the DNA content of a by increases in CyaA concentrations for human diploid cell (to exclude bacterial granulocytes and J774.2 cells compared to aggregates). The nucleated events were cells treated with PBS or CyaA*. With 0.2 then discriminated; into lymphocytes, g protein/ml of CyaA there was almost monocytes and polymorphonuclear cells complete (92%) inhibition of phagocytosis by combined measurements of the forward by J774.2 cells and 63% inhibition of angle light scatter (FSC) and side angle phagocyotosis by human granulocyte cells. light scatter (SSC). The granulocyte CyaA* had no obvious affect on cluster was then gated in the analysis phagocytosis except at the highest program in the scatter diagram (FSC vs concentration of toxin used (0.2 g/ml) SSC), and its green fluorescence where 10 % inhibition with granulocytes histogram was analysed). The SSC vs FL- was seen but this was not significant. The 1 diagram also was also set to make sure flow cytometry histograms that represents that the appropriate cell population data the amount of ingested E. coli, also was collected. indicated the dose-dependent inhibitory effect of CyaA on J774.2 and granulocytes Results and Discussion (Fig 1, images 2 and 4), while there was lack of inhibitory effect of CyaA* on Using flow cytometry, the phagocytic J774.2 and granulocytes (Fig 2, images 1 ingestion of E. coli by human peripheral and 3). In similar experiments, blood cells from healthy donors, J774.2 undifferentiated and differentiated U937 mouse macrophages and U937 human human monocytes were treated with CyaA

107 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 104-112 only at 0.1 g/ml. As Table 3.7.1 shows, fluorochromes. These authors used FITC- only differentiated cells were affected by E. coli for phagocytosis experiments and CyaA, showing 42% inhibition of the extracellular fluorescence was phagocytosis. Overall the results of this quenched by trypan blue to differentiate it assay would imply that inhibition of from that due to intracellular bacteria. phagocytosis by CyaA was due mainly to the AC enzymic activity of the protein. Host defence against B. pertussis relies to some extent upon phagocytosis and killing The statistical analysis of data by t test of the bacterial cells. The adenylate was as follows: CyaA (0.0 5 g/ml) vs cyclase toxin appears to be an important PBS, P < 0.044,, ; CyaA ( 0.1, g/ml) vs virulence factor against phagocytosis, as PBS, P < 0.004, CyaA (0.2 g/ml) vs evidenced by the efficient phagocytosis of PBS, p < 0.005, CyaA* (0.2 g/ml) vs mutants that fail to express this toxin PBS, p < 0.19, CyaA* (0.2 g/ml) vs (Weingart, and Weiss, 2000). These CyaA, p < 0.001 Phagocytosis provides a authors also showed that CyaA, added specialised mechanism for regulated separately, blocks phagocytosis even when ingestion and intracellular destruction of bacteria are not expressing the toxin. microbial pathogens as well as of apoptotic host cells and debris. In general, In this study, different concentrations of professional , including CyaA and CyaA* were used to evaluate neutrophils (which migrate from the blood their modulatory effects on phagocytosis. to a site of infection) and macrophages The results of the ingestion activity (which constitutively reside in tissues and experiment suggested that CyaA inhibits are less motile than neutrophils), the phagocytosis of E. coli by J774.2 accomplish most phagocytosis. By macrophage-like cells and by human confining the mechanisms of microbial granulocytes as it caused a decrease in the killing and digestion to distinct mean fluorescent intensity (MFI) intracellular compartments (lysozomes) of measured by flow cytometry. these cells, damage to host cells and tissues is minimised during the process of Although the concentration of CyaA used killing offending microbes. In addition to in the assay caused appreciable cell killing disposing of microbial pathogens, of J774.2 cells as determined by the MTT phagocytosis (especially by macrophages assay, it should be emphasised that the and dendritic cells) initiates the process of effect of CyaA on phagocytosis was antigen processing and presentation for measured only in viable cells, by the flow development of adaptive immune cytometric method here. In contrast, no responses (Ramachandra et al., 1999). significant decrease of phagocytosis activity was noted in response to treatment In order to better understand the of J774.2 macrophage-like cells or human importance of this defence mechanism, a granulocytes with various concentrations flow cytometry assay was developed to of CyaA*. The poor AC enzymic activity study oxidative burst of neutrophils of CyaA* is presumably responsible for stimulated by bacteria (Khosravani et al., the lack of effect on phagocytosis activity 2013). A flow cytometry investigation was in these cells, when compared with CyaA. made in order to choose appropriate target cells, opsonisation conditions, and

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Table.1 Suppression of phagocytosis activity of granulocytes, J774.2 and U937 cells by CyaA preparations

Toxin conc % Inhibition of % Inhibition of % Inhibition of % Inhibition ( g/ml) granulocytes U937 cells U937 cells of J774.2 cells (Human (human (human (mouse peripheral monocytes) monocytes) macrophages) blood) (without PMA) (with PMA) CyaA (0.05) 26 28 0 18 CyaA (0.1) 79 58 0 42 CyaA (0.2) 92 63 0 59 CyaA* (0.05) 0 0 CyaA* (0.1) 0 0 CyaA* (0.2) 0 10 PBS 0 0

Figure 1 ffffFi1The effect of CyaA and CyaA* on phacytosis of E. coli by J774.2 cells and fffffffffffffhumanImage granulocyte 1 cells Image 2

Image 4

Image 3

Image 3 Image 4

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An additional experiment was performed However, the results of this study show under the same conditions on the U937 that the full activity of CyaA is necessary human monoblastic cell line in the absence for phagocytic inhibitory activity as or in the presence of PMA. PMA is used CyaA* had little effect on phagocytosis. as a stimulator to differentiate this cell-line These results are keeping with the results into -like cells. The effect of reported by Galgiani et al. (1988). They PMA shown in this study was that it showed that an adenylate cyclase toxin- potentiated the phagocytosis activity of containing extract blocked neutrophil- U937 cells. However, the cytotoxic effect mediated inhibition of N- of CyaA was greater on PMA- acetylglucosamine incorporation by differentiated U937 cells than on arthroconidia of Coccioides immitis in a undifferentiated cells. Thus, it can be dose-dependent manner. The authors suggested that PMA at 0.020 g at final indicated that CyaA, rather than other concentration could stimulate the virulence factors of B. pertussis, was immature cells to differentiate, which responsible for the inhibitory effects on CyaA could, then affect. Further PMN. In the present study, the effects of investigation is recommended to show CyaA preparations were examined and the how differentiation of U937 cells might interactions of human granulocytes and influence their susceptibility to CyaA, for J774.2 mouse phagocyte-like cells with E. example by increasing the number of coli in vitro were compared. The results receptors for the toxin. suggest that CyaA impairs the functions of human granulocytes and J774.2 cells It is very clear that phagocytic functions, phagocytosis in a dose dependent manner such as migration, ingestion and release of and that this activity is dependent on the reactive oxygen intermediates (ROI), AC activity of the toxin. which appear to be responsible for the killing activity, are important host defence References mechanisms against Bordetella. Evasion of these functions may be the principal Basar, T., Havlicek, V., Bezouskova, S., tools by which the bacteria can persist in Hackett, M., and Sebo, P. 2001. the sites of colonization and proliferate Acylation of 983 is sufficient further. One obvious strategy in defence for toxin activity of Bordetella against phagocytosis is direct attack by the pertussis adenylate cyclase. bacteria upon the professional phagocytes Substitutions of alanine 140 modulate using extracellular or toxins that acylation site selectivity of the toxin kill or at least block the activity of acyltransferase CyaC. J. Biol. Chem. phagocytes. Extracellular proteins that 276, 348 354. inhibit phagocytosis include the exotoxin Bellalou, J., Sakamoto, H., Ladant, D., A of , which Geoffroy, C., and Ullmann, A. 1990. kills macrophages, and the bacterial Deletions affecting hemolytic and exotoxins that are adenylate cyclases e.g. toxin activities of Bordetella pertussis EF and B. pertussis CyaA adenylate cyclise. Infect. Immun. 58, which decrease phagocytic activity. Thus, 3242 3247. the reduction of ingestion and oxygen- Benz, R., E. Maier, D. Ladant, A. dependent killing functions is a Ullmann, P. Sebo . 1994. Adenylate predominant activity of B. pertussis CyaA. cyclase toxin CyaA of Bordetella

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