Colourimetric Determination of Phospholipase Activities in Balamuthia mandrillaris

Balamuthia mandrillaris is a recently identified protozoan pathogen that can cause fatal granulomatous encephalitis however the pathogenesis and pathophysiology associated with Balamuthia encephalitis remain unclear. We have recently isolated B. mandrillaris from a 33-years old male who died of encephalitis. Using this isolate, we demonstrated for the first time that B. mandrillaris exhibited phospholipase activities. More specifically, B. mandrillaris exhibited phospholipase A2 and phospholipase D activities. For the first time we used colourimetric technique based on spectrophotometer and designed phospholipases assays to determine these phospholipase activities. The functional role of phospholipases was determined in in vitro assays using human brain microvascular endothelial cells (HBMEC). We observed that PLA2-specific inhibitor i.e., cytidine 5’diphosphocholine significantly inhibited B. mandrillaris binding to HBMEC. Similarly PLD inhibitor i.e., compound 48/80 inhibited B. mandrillaris binding to HBMEC. Moreover, both inhibitors inhibited B. mandrillaris-mediated HBMEC cytotoxicity. Overall these results clearly demonstrate that phospholipases are important virulence determinants in B. mandrillaris. Further studies will identify the precise role of phospholipases in the pathogenesis of B. mandrillaris, which may help develop therapeutic interventions. Using a novel spectrophotometric-based assay we demonstrated for the first time that B. mandrillaris exhibit phospholipase activities.


INTRODUCTION
Balamuthia mandrillaris is a recently discovered protozoan pathogen that can cause fatal granulomatous encephalitis. It is believed that this organism is widely distributed in fresh water, soil and dust throughout the world. The first known isolate of this amoeba was from a mandrill Baboon who died of B. mandrillaris granulomatous encephalitis (BGE) [1]. The first time B. mandrillaris was recognised as causing encephalitis in humans in 1990 when first case was reported at Santa Cruz hospital California U.S.A [2]. Since then more than 105 fatalities attributed to B. mandrillaris [3] [4]. There have been only two survivors of BGE [5]. Thus the mortality is more than 98%. Phospholipases are the enzymes, which catalyse the hydrolysis of specific ester bonds in phospholipids. Individuals enzymes are grouped on the basis of the bond they hydrolyse and are further categorized. The phospholipases are thus called phospholipase A, B, C and D. For the phospholipase A, a subscript 1 or 2 is added depending on whether the cleaved bond involved is at the sn-1 or sn-2, position of the phospholipid substrate. The term phospholipase A is used for those enzymes, which catalyse the hydrolysis of the terminal or central acyl group from a membrane phospholipid. Phospholipase A is further classified into phospholipase A 1 (PLA 1 ) and phospholipase A 2 (PL A 2 ). Phospholipase A 1 catalyzes the hydrolysis of the terminal acyl group from a phospholipid, generating a free fatty acid and a lysophospholipid and referred to as PLA 1 because of its 1-acyl specificity. Phospholipase A 2 represents a class of heat-stable calcium-dependent enzymes catalyzing the hydrolysis of the 2-acyl bond of 3-n-phosphoglycerides, generating a free fatty acid and a lysophospholipid. This enzyme is named phospholipase A 2 to denote its 2-acyl specificity. Phospholipase A 2 enzyme plays a significant role in the liberation of arachidonic acid from the sn-2 position of cellular phospholipids in most mammalian cells. The released arachidonic acid is used in many cases of the biosynthesis. Phospholipase B (PLB) (synonyms: lysophospholipase, lysophospholipase-transacylase) refers to an enzyme that can remove both sn-1 and sn-2 fatty acids, its nomenclature is confusing. This confusion arises because PLB has both hydrolase (fatty acid release) and lysophospholipase-transacylase (LPTA) activities. The hydrolase activity allows the enzyme to cleave fatty acids from both phospholipids (PLB activity) and lysophospholipids [lysophospholipase (Lyso-PL] activity)], while the transacylase activity allows enzyme to produce phospholipid by transferring a free fatty acid to lysophospholipid. Phospholipase C enzymes catalyze the hydrolysis of the phosphoric ester bond of a membrane phospholipid and generate a phosphorylated alcohol and diacylglycerol. They are important in the digestion of dietary phospholipids and in various processes dependent on hormonally induced calcium mobilization or arachidonic acid production, they occur in all mammalian tissues and as toxic secretion products of pathogens. Phospholipase D enzymes catalyze the hydrolysis of the alcohol group from a phospholipid, and generate the corresponding phosphatidate. They occur in various forms, predominantly in plants (e.g. cabbage), but in humans they may be a part of a mechanism to generate diacylglycerol for the mobilization of calcium in response to hormones. These enzymes are nonhaemolytic but only in the presence of the cholesterol oxidase these PLDs show haemolytic activity. These enzymes have generally not been well studied but some of them are haemolysin, which are active preferentially towards sphingomyelin and degrades phospholipids [6]. The aim of this study was to determine Balamuthia mandrillaris special emphasis on their possible role in the virulence of this pathogen.

MATERIALS AND METHODS
Human brain microvascular endothelial cells (HBMEC): Primary brain microvascular endothelial cells from human origin were obtained from our collaborator (Prof. K. S. Kim, John Hopkins University Baltimore, MA, USA). HBMEC were routinely grown in tissue culture flasks in RPMI containing 10% heat inactivated fetal bovine serum, 10% Nu-Serum, 2 mM glutamine, 1 mM pyruvate, penicillin (100 U/ml), streptomycin (100 µg/ml), 1% non-essential amino acids and1% vitamins as previously described [7]. Media reagents were filtered using 0.2 µM pore size filter and stored at 4°C and used within two weeks. Briefly, HBMEC were collected using trypsin and transferred into a 50 ml centrifuge tube. The cells were centrifuged at 750 x g for 5 min. The supernatants were aspirated and the pellet resuspended into fresh HBMEC media and inoculated into flasks. Flasks were incubated at 37°C in 5% CO 2 incubator. For cytotoxicity and adhesion assays, HBMEC were grown into 24-well plates by inoculating 5 x 10 5 cells/well/ml. At this cell density, HBMEC formed confluent monolayers within 24 h and used for assays.
Balamuthia mandrillaris cultures: Two isolates of B. mandrillaris were used. First isolate was from the brain of a mandrill baboon (ATCC VO39) and the second isolate from the brain of a 33-year old patient who died of encephalitis [8]. Both isolates were cultured using HBMEC monolayers as food source. Briefly, 5 x 10 5 B. mandrillaris were incubated with HBMEC in serum free media (RPMI containing 2 mM glutamine, 1 mM pyruvate and 1% non-essential amino acids). Flasks were incubated at 37°C in a 5% CO2 incubator and observed daily. HBMEC monolayer degradation was observed within 3-4 days and resulted in approximately 5 x 10 6 amoebae (more than 95% amoebae in trophozoite forms). Balamuthia mandrillariswere subsequently used in phospholipases, adhesion and cytotoxicity assays.
x Vs = Units per ml activity. OD is optical density, Vt is total volume of the cuvette, Vs is sample volume 13.3 is millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm 2 /micromole), 0.5 is a factor based on the fact that one mole of H 2 O 2 produces half a mole of quinoneimine dye, 1 is light path length (cm).

Adhesion assays:
The purpose of the experiment was to determine if B. mandrillaris bind to in vitro to HBMEC. A 75 cm 2 tissue culture flask containing the amoebae were examined under a light microscope. The amoebae could be assumed to be healthy if present in the trophozoite stage and appeared to be actively attached feeding trophozoites on the flask base. The amoebae cultures were collected by flask agitation. The flask was then examined under the microscope to check that amoebae were free in the RPMI 1640 medium and collected by centrifugation as described above. Next, amoebae were counted (amoebae/ml) using a haemocytometer. Balamuthia mandrillaris (5 x 10 5 cells/well/500 µl) were incubated with HBMEC grown to monolayers in 24-well plates as previously described [9]. Plates were incubated at 37°C in 5% CO2 incubator for 2 h. HBMEC incubated alone in RPMI 1640 were used as negative controls. Each condition was performed in duplicate for each experiment. After this time the plates were removed and the wells gently mixed. Six µl was pipetted out from each well for a haemocytometer count. The counts were recorded. The numbers of B. mandrillaris recorded can be related to the numbers bound to the HBMEC since we know how many were inoculated into each well initially.

Adhesion assays in the presence of phospholipase inhibitors:
To determine the involvement of phospholipases in B. mandrillaris binding to HBMEC, adhesion assays were performed in the presence of phospholipase inhibitors, i.e., cytidine 5'diphosphocholine, a PLA 2 inhibitor; and compound 48/80, a PLD inhibitor. These inhibitors were used at various concentrations. Briefly, various concentrations of inhibitors were incubated with B. mandrillaris for 30 min prior to adhesion assays. Adhesion assays were carried out as described above in the presence of inhibitors.
Cytotoxicity assays: To examine the pathogenic potential of each isolate used in this study, cytotoxicity assays were performed as previously described [10]. Briefly, B. mandrillaris isolates (5 x 10 5 parasites/well/500 µl) were incubated with HBMEC monolayers in serum free media (RPMI 1640 containing 2 mM glutamine, 1 mM pyruvate and nonessential amino acids) at 37°C in 5% CO 2 incubator. HBMEC monolayers were observed periodically for cytopathic effects for up to 24 h. At the end of this incubation period, cytopathic effects were assessed visually after hematoxylin staining. In addition, supernatants were collected and cytotoxicity was determined by measuring lactate dehydrogenase (LDH) activity release (Cytotoxicity detection kit; Roche Applied Science, Lewes, UK) as previously described [10]. This assay is based on the measurement of LDH activity released from damaged cells using the 96-well plates. Briefly, cell supernatant containing LDH catalyzes the conversion of lactate (solution from kit) to pyruvate, generating NADH and H + . In the second step, the catalyst (diaphorase, solution from kit) transfers H and H + from NADH and H + to the tetrazolium salt piodo-nitrotetrazolium (INT) yellow, which is reduced to formazan red, and absorbance is read at 492 nm. Percentage LDH release was detected as follows: (sample value -control value / total LDH releasecontrol value x 100 = % cytotoxicity). Control values were obtained from HBMEC incubated alone. Total LDH release was determined from HBMEC treated with 1% Triton-X-100 (w/v).

Cytotoxicity assays in the presence of phospholipase inhibitors:
To determine the involvement of PLA 2 and PLD in B. mandrillaris cytotoxicity to HBMEC, cytotoxicity assays were performed in the presence of phospholipase inhibitors. These inhibitors were used at various concentrations.
Briefly, various concentrations of inhibitors were incubated with B. mandrillaris for over night prior to cytotoxicity assays. Cytotoxicity assays were carried out as described above in the presence of inhibitors

Balamuthia mandrillaris exhibit phospholipase A 2 and phospholipase D activities:
To determine whether B. mandrillaris exhibit phospholipase activities, assays were performed as described in materials and methods. Our results demonstrated that both B. mandrillaris isolates possess phospholipase A 2 and phospholipase D activities. As observed in Fig. 1, 10 6 B. mandrillaris exhibited more than 0.1 U/ml PLA 2 activities, but showed up to 0.48 U/ml PLD activities. Interestingly, B. mandrillaris exhibited higher PLD than PLA 2 activities. Maximal PLA 2 and PLD activities were observed at 37°C: To determine the effects of temperature on phospholipase activities, various temperatures were tested. We observed optimal PLA 2 and PLD activities at 37 °C, i.e., 0.144 U/ml and 0.255 U/ml respectively (Fig. 3). Interestingly, both PLA 2 and PLD activities were inhibited at 60 °C. Overall, these findings suggest that B. mandrillaris exhibit optimal PLA 2 and PLD activities at 37 °C indicating their physiological relevance. Maximal PLA 2 and PLD activities were observed at pH 8: To determine the effects of pH on phospholipase activities, various pHs were tested. As shown in Fig. 4, that optimal PLA 2 and PLD activities were observed at pH 8 i.e., 0.178 U/ml and 0.53 U/ml respectively.

PLA 2 and PLD inhibitors partially inhibited B. mandrillaris binding to the host cells:
In order to understand the B. mandrillaris adherence with the HBMEC, adhesion assays were performed in the presence and absence of phospholipase inhibitors. As shown in Fig. 6, these inhibitors play important roles to inhibit the binding of B. mandrillaris to HBMEC. It was observed that in the presence of cytidine (PLA 2 inhibitor) B. mandrillaris binding to HBMEC is 17% while in the presence of compound 48/80 (PLD inhibitor) this binding is more than 20%.

PLA 2 and PLD inhibitors partially inhibited B. mandrillaris-mediated host cells cytotoxicity:
The major tasks to perform these assays were to confirm PLA 2 and PLD activities in B. mandrillaris, to understand B. mandrillaris roles in the blood brain barrier changes, to know how could we inhibit these activities and what is the effect of inhibitors on HBMEC. These assays were done by using cytidine (PLA 2 inhibitors) and compound 48/80 (PLD inhibitor). As shown in Fig. 7 [11]. However, it is not clear how circulating amoebae cross the blood brain barrier. Thus understanding the molecular mechanism associated with B. mandrillaris-interactions with HBMEC and crossing the blood brain barrier may provide opportunities to develop novel strategies for treatment. Due to the ability of B. mandrillaris to penetrate host tissues, we hypothesize that this amoeba exhibits hydrolytic activities. In this study, for the first time we have shown the presence of phospholipase A 2 (PLA 2 ) and phospholipase D (PLD) activities in B. mandrillaris. This is an important finding as both PLA 2 and PLD have been shown to be important in the pathogenesis of other microbes including bacteria such as Vibrio parahaemolyticus [12] and Vibrio damsella [6]; fungal pathogens including Aspergillus fumigatus [13], and protozoans such as Acanthamoeba [14]. For example, Langton and Cesareo [15] have shown that PLA 2 produced by Helicobacter pylori can degrade phosphatidylcholine-rich gut lining which can directly lead to gastric ulcer disease. Using PLA 2 -specific inhibitors, it was further shown that H. pylori were significantly less able to produce gastric ulcer in human [16] clearly indicating that PLA 2 plays important roles in pathogenesis of disease. Similarly, Muckle and Gyles [17] have shown that PLD produced by Cornybacterium pseudotuberclosis causes increased vascular permeability and leads to the lymphadenitis or lymphangitis disease in ruminants or horses. This was further supported with the finding that PLD mutant of C. pseudotuberclosis might itself be used as a vaccine [18]. Similar roles may be attributed to B. mandrillaris phospholipases, however it remains to be determined. Among variable temperatures, the optimal B. mandrillaris phospholipase activities were observed at 37ºC. Similarly a range of pH was tested to determine the optimal activities and alkaline pH exhibited optimal activities indicating their physiological relevance. Next we determined PLA 2 and PLD activities in B. mandrillaris conditioned medium, which could suggest that both enzymes are extracellular. Our results showed that B. mandrillaris exhibit extracellular PLA 2 and PLD activities. These findings are novel as they suggest that PLA 2 and PLD may be used as contactindependent virulence factors of B. mandrillaris. Next we tested the role of phospholipases on B. mandrillaris binding to the host cells as well as their cytotoxicity on the host cells. We made use of HBMEC as host cells and performed adhesion assays. Our findings revealed that in the presence of cytidine (PLA 2 inhibitors) 17% B. mandrillaris bind to the HBMEC and in the presence of compound 48/80 (PLD inhibitor) this binding increased up to 25%. It is important to observe that in the absence of these inhibitors Balamuthia mandrillaris-mediated HBMEC binding increased up to 90% indicating that phospholipases of B. mandrillaris may play important roles in the HBMEC disruption. It is interesting to note that, of the two major phospholipases observed in this study, lectate dehydrogenase (LDH) assays in the presence of cytidine (PLA 2 inhibitor) showed only 17% B. mandrillaris-mediated HBMEC cytotoxicity while compound 48/80 (PLD inhibitor) showed only 42% cytotoxicity which is an another evidence to prove that B. mandrillaris PLA 2 and PLD are extracellular and play major roles in Balamuthia mandrillaris-mediated HBMEC cytotoxicity. These findings suggest that B. mandrillaris phospholipases play important roles in the blood brain-barrier changes. However, the precise targets and underlying mechanism of phospholipasesmediated HBMEC cytotoxicity remain unknown. It was also observed in adhesion and LDH assays that cytidine showed significant inhibition, which suggests that cytidine is a good inhibitor against B. mandrillaris PLA 2 . Moreover cytidine showed very less cytotoxic effects on HBMEC even at higher concentration which indicating that these inhibitors could be important for therapeutic interventions

CONCLUSION
In conclusion, we have shown for the first time that B. mandrillaris exhibit phospholipase activities i.e., phospholipase A 2 and phospholipase D activities. Our results show that these phospholipases are involved in B. mandrillaris binding to HBMEC and play vital roles in B. mandrillaris-mediated HBMEC cytotoxicity, which may lead to amoebae traversal of the blood-brain barrier. Thus understanding the molecular mechanism associated with B. mandrillaris phospholipases may provide opportunities to develop novel strategies for treatment. In this study we have shown the roles of phospholipases in B. mandrillaris virulence, the future work will be to use these enzymes in drug discovery efforts to identify and design inhibitors and/or their use as a vaccine. The utility of phospholipases as diagnostic markers of B. mandrillaris infections is yet another area of study that may prove to be fruitful.