Differential sensitivity of New World Leishmania spp. promastigotes to complement-mediated lysis:...

Acta Tropica 69 (1998) 17–29

Differential sensitivity of New World Leishmaniaspp. promastigotes to complement-mediated lysis:Correlation with the expression of three parasite

polypeptides

Fatima S.M. Noronha a, Alvaro C. Nunes b, Katia T. Souza b,M. Norma Melo c, F. Juarez Ramalho-Pinto d,*

a Departamento de Microbiologia, Uni6ersidade Federal de Minas Gerais, Belo Horizonte MG, Brazilb Departmento de Bioquımica-Imunologia, Uni6ersidade Federal de Minas Gerais,

Belo Horizonte MG, Brazilc Departmento de Parasitologia, ICB, Uni6ersidade Federal de Minas Gerais,

Belo Horizonte MG, Brazild Departamento de Parasitologia, Microbiologia e Imunologia,

Faculdade de Medicina de Ribeirao Preto, Uni6ersidade de Sao Paulo, Ribeirao Preto, Brazil

Received 1 April 1997; received in revised form 30 June 1997; accepted 1 August 1997

Abstract

American tegumentary leishmaniasis is caused by Leishmania of the subgenera Leishmaniaand Viannia. In this paper, we demonstrate that promastigotes of these two subgeneradisplay distinct characteristic patterns of complement sensitivity during growth in vitro.Using fresh normal human serum in lytic assays, we show that while promastigotes of twospecies of the subgenus Leishmania differentiate into forms that are more resistant to the lyticaction of complement, promastigotes of three species of the subgenus Viannia remainsensitive to complement mediated lysis during all stages of their growth in vitro. Complementresistance of the subgenus Leishmania is temporary, reaching its peak at the beginning of thestationary phase of growth, and decreasing thereafter. By sodium dodecyl sulfate-polyacry-lamide gel electrophoresis (SDS-PAGE) we detected in L. amazonensis (subgenus Leishma-nia), but not in L. guyanensis (subgenus Viannia), three polypeptides whose expression

* Corresponding author. Tel.: +55 16 6023230; fax: +55 16 6336631; e-mail: [email protected]

0001-706X/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S0001 -706X(97 )00108 -3

F.S.M. Noronha et al. / Acta Tropica 69 (1998) 17–2918

parallels the resistance of promastigotes to complement-mediated lysis. © 1998 ElsevierScience B.V. All rights reserved.

Keywords: Promastigotes; Complement resistance; L. amazonensis ; L. mexicana ; L. guyanen-sis ; L. panamensis ; L. braziliensis

1. Introduction

Leishmania spp. is the causative agent of leishmaniasis, one of the majorproblems of public health in several tropical countries. In the New World, leishma-niasis is grouped into two classes: American visceral leishmaniasis caused by someparasites of subgenus Leishmania and American cutaneous leishmaniasis involvingparasites of the two subgenera Leishmania and Viannia (Lainson and Shaw, 1987;Grimaldi and Tesh, 1993).

During its life cycle, Leishmania spp. presents basically three forms: promastig-otes, paramastigotes and amastigotes. Promastigotes and paramastigotes are flagel-lated, motile forms that are found in the alimentary tract of phlebotomine sandflies.Amastigotes are non-motile forms, found inside mononuclear phagocytes of themammalian host. Metacyclic infective promastigotes are transmitted by the femalesandflies to mammals, where they invade and multiply as amastigotes withinmacrophages (Molyneux and Killick-Kendrick, 1987; Killick-Kendrick, 1990).

One of the earliest events after promastigotes have entered the mammalian hostis their contact with plasma proteins. It has been shown that fresh normal humanserum (f-NHS) can cause the lysis of Leishmania spp. in vitro (Pearson andSteigbigel, 1980; Franke et al., 1985) through the alternative pathway of comple-ment (Mosser and Edelson, 1984; Mosser et al., 1986; Brittingham et al., 1995).Therefore, they must escape the lytic effect of serum before they can invademacrophages (Jokiranta et al., 1995). In fact, it has been shown that non-infectivepromastigotes from logarithmic stage of growth, which are susceptible to comple-ment (Franke et al., 1985; Bandyopadhyay et al., 1991), differentiate into stationaryphase promastigotes (Sacks and Perkins, 1984, 1985) with an increased resistance tocomplement and higher infectivity (Franke et al., 1985; Barral-Netto et al., 1987;Howard et al., 1987; Grimm et al., 1991).

Differences in the pattern of promastigotes sensitivity to complement, lead to thehypothesis that complement resistance of Leishmania spp. is species-specific (Frankeet al., 1985), and that their resistance could be related to the severity of the diseasecaused by each species (Barral-Netto et al., 1987).

In this report we have compared the sensitivity of promastigotes of five NewWorld Leishmania species, two of subgenus Leishmania and three of subgenusViannia, to lysis mediated by human complement. We show that promastigotes ofthe subgenera Leishmania and Viannia present marked differences in their patternsof sensitivity to complement during their growth in vitro. Complement resistance inthe subgenus Leishmania was associated to an increase in the expression of threeparasite polypeptides.

F.S.M. Noronha et al. / Acta Tropica 69 (1998) 17–29 19

2. Material and methods

2.1. Parasite culture

The following species and strains of Leishmania spp. were used in thepresent work: Leishmania (Leishmania) amazonensis (IFLA/BR/67/PH8), Leish-mania (L.) mexicana (MAKO/BR/78/BH77R), Leishmania (Viannia) braziliensis(MHOM/BR/75/M2903), Leishmania (V.) panamensis (ITRA/CO/81/M9116),Leishmania (V.) guyanensis (MHOM/BR/70/M1176).

Promastigotes were cultured at 23°C in RPMI-1640 (Sigma, St. Louis, MO)supplemented with 20 mM Hepes (Sigma), 15% heat-inactivated Fetal BovineSerum (Cultilab, Campinas, Brazil) and gentamicin (50 mg/ml). Cultures wereseeded by inoculation with early stationary phase promastigotes in the startingdensity of 1×106/ml. Parasites were counted daily using a Neubauer bright-line hemocytometer.

2.2. Serum

A pool of fresh normal human serum (f-NHS) was used as source of com-plement. Blood was collected from individuals with no history of leishmaniasis,with negative immunofluorescence to promastigotes. The blood was allowed toclot for 30 min at room temperature, then for 3 h at 4°C. After centrifuga-tion, the serum was collected, pooled, aliquoted and stored at −80°C untiluse.

2.3. Lytic assay

Promastigotes were collected daily, washed and 1×106 parasites in 100 m l ofculture medium were incubated with f-NHS serially diluted in 100 m l of cul-ture medium. Initially, all the Leishmania promastigotes were tested for theirserum sensitivity using f-NHS ranging from 0.5 to 32%. For the standardtests, the final dilution of f-NHS ranged from 1 to 32% for species belongingto subgenus Leishmania, and from 0.5 to 8% for species of the subgenus Vian-nia. As controls, promastigotes were incubated without f-NHS. After incuba-tion at 37°C for 45 min, the reaction was stopped by diluting the suspensionin 1 ml of ice-cold phosphate buffered saline (PBS). Live and dead parasiteswere counted in a hemocytometer at 400× magnification after the addition of200 m l of 0.4% Erythrosin B in PBS (Riedel-de-Haen). Non-motile parasitesstained in red were considered damaged. Those which excluded the dye andremained refringent were counted as alive. LD50, defined as the concentrationof f-NHS that causes lysis of 50% of promastigotes, was calculated by linearregression of the curves corresponding to percentage of lysis versus serum con-centration.


2.4. Characterization of NHS lytic action

To ascertain that the lytic action of f-NHS was due to complement activation,lytic assays were performed with: (1) NHS heat-inactivated at 56°C for 30 min(hi-NHS) to inactivate factor B and C2, blocking both classical and alternativepathways; (2) f-NHS in the presence of 10 mM EDTA, that chelates Ca2+ andMg2+, blocking both classical and alternative pathways; or (3) with sera devoid ofthe complement components, C1q or factor B.

2.5. SDS-polyacrylamide gel electrophoresis and gel densitometry

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), wasperformed by the discontinuous buffer method according to Laemmli (1970), using10% separating and 4% stacking gels. Each day of culture, promastigotes werewashed with PBS and maintained at −80°C until used in the SDS-PAGE. For thisassay, aliquots of the thawed out parasites containing 50 mg of proteins in 10–20m l (determined by a protein microassay, Bio-Rad, Hercules, CA,), were mixed withan equal volume of sample buffer (2.3% SDS, 10% glycerol, 5% 2-mercaptoethanol,0.01% bromophenol blue in 0.125 M Tris–HCl pH 6.8), boiled at 100°C for 5 minand electrophoresed. Gels were then soaked in 50% methanol (Merck) and silverstained according to the method described by Wray et al. (1981). Densitometry ofproteins bands was performed at l=470 nm in the CS-9301PC Dual WavelengthFlying Spot Scanning Densitometer (Shimadzu, Japan).

3. Results

3.1. Lytic effect of normal human serum is due to complement alternati6e pathwayacti6ation

All five species of Leishmania studied in this paper were sensitive to 32% off-NHS in a dose-dependent manner, either at the logarithmic or at the stationaryphase (not shown). Fig. 1 shows a typical result of L. amazonensis promastigotelysis using 32% f-NHS. Heat inactivation or the presence of EDTA completelyabolished the lytic effect of normal serum against the promastigotes, indicating thatparasite killing is dependent on complement activation.

In order to determine which pathway of the complement system was active inkilling the promastigotes by f-NHS, lytic assays were performed with sera devoid ofthe complement components C1q or factor B. In the presence of C1q-deficientserum, in which only the alternative pathway is functional, lysis was comparable tothat observed with f-NHS (Fig. 1). Conversely, when the factor B-deficient serum,that is defective on the alternative pathway, was used, no lytic activity forpromastigotes was detected (Fig. 1). Similar results were consistently obtained forthe five Leishmania species studied (not shown).


3.2. Promastigotes of subgenera Leishmania and Viannia ha6e different patterns ofcomplement sensiti6ity during growth in 6itro

The New World species of Leishmania studied in the present work showed typicalgrowth curves when cultivated in RPMI/FBS. All of the species studied presentedwell defined logarithmic and stationary phases and reached similar populationlevels at day 4 or 5, the beginning of their stationary phase (data not shown).

The use of lytic assays at various serum concentrations and the daily determina-tion of serum LD50 values allowed us to evaluate complement sensitivity of the fiveNew World Leishmania species during their growth in axenic culture. Promastigotesfrom the species belonging to subgenus Leishmania, L. amazonensis and L. mexi-cana, show a variable susceptibility to complement during their growth in vitro(Fig. 2). These species are very sensitive to complement at the beginning of theculture with 50% of promastigotes being damaged by less than 6% of f-NHS.However, parasites gradually acquire a resistance to complement-mediated lysisthat reaches a maximum at day 4, when they enter the stationary phase. At thisstage, 50% of L. (L.) amazonensis promastigotes are resistant to 12% f-NHS and L.

Fig. 1. Activation of the complement system by L. (L.) amazonensis promastigotes. 1×106 promastig-otes of L. (L.) amazonensis were incubated with or without 32% f-NHS, f-NHS plus 10 mM EDTA,hi-NHS, Factor B- or C1q-deficient sera in culture medium at 37°C for 45 min. Damage to parasites wasassessed using the vital dye Erythrosin B. Values represent the mean of duplicates9S.D.


Fig. 2. Sensitivity of promastigotes of the subgenera Leishmania and Viannia to complement duringgrowth in axenic culture. Every 24 h, parasites were exposed to serial dilutions of 1–32% f-NHS, for thesubgenus Leishmania, and 0.5–8% for the subgenus Viannia at 37°C for 45 min. Damage to parasiteswas assessed using the vital dye Erythrosin B and the sensitivity of promastigotes to complement isrepresented by LD50, the concentration of f-NHS that causes lysis of 50% of promastigotes. Valuesrepresent the mean of duplicates9S.D. The results are representative of three experiments performedfor each species.

(L.) mexicana promastigotes to 18% of f-NHS. During the stationary phase,parasites from these species decreased in their complement resistance, presentingLD50 values similar to those observed at the early logarithmic phase of growth. Ourresults with L. (L.) amazonensis and L. (L.) mexicana promastigotes clearlydemonstrate that complement resistance varies during growth of parasites in vitroand show that promastigotes with the highest resistance to complement are foundat the early stationary phase.

Unlike the subgenus Leishmania, parasites of subgenus Viannia, are markedlysensitive to complement during the whole period of growth in vitro, not presentinga stage of heightened resistance (Fig. 2). Doses as low as 2% of f-NHS were

Fig. 3. SDS-PAGE analysis of L. (L.) amazonensis promastigotes. Aliquots containing 1×106 parasites(approximately 50 mg of proteins) collected at each day of culture (day 1–7) were resuspended in samplebuffer and analysed by 10% SDS-PAGE. Gels were silver stained (A) and scanned in a densitometer at470 nm (B). Straight lines correspond to days 1 and 7 and dashed line correspond to day 4.


Fig. 3.


sufficient to lyse 50% of the promastigotes, either in logarithmic or stationaryphase.

3.3. Complement resistance correlates with the expression of three parasitepolypeptides

Using SDS-PAGE and silver staining, we examined the protein profile of L. (L.)amazonensis (Fig. 3) and L. (V.) guyanensis (Fig. 4) collected each day of theirgrowth in vitro. Albeit the complexity of the parasite protein pattern in silverstained gels, we were able to identify in L. (L.) amazonensis promastigotes,polypeptides whose expression parallels the pattern of resistance to complement

Fig. 4. SDS-PAGE analysis of L. (V.) guyanensis promastigotes. Aliquots containing 1×106 parasites(approximately 50 mg of proteins) collected at each day of culture (day 2–6) were resuspended in samplebuffer and analysed by 10% SDS-PAGE. Gels were silver stained (A) and scanned in a densitometer at470 nm (B). Straight lines correspond to days 2 and 6 and dashed line corresponds to day 4.


Fig. 4.

observed in these parasites. The densitometry of the gel confirmed this observa-tion and three bands corresponding to the molecular masses of 46, 32 and 27 kDaincrease in intensity three, two and 13 times, respectively. These polypeptidesreach this maximum expression at day 4 (early stationary phase) of promastigotesgrowth, coincident with maximum complement resistance (Fig. 3A,B), and thenreturn to values similar to those observed at the beginning of the culture. Expres-sion of other proteins, such as the PSP, promastigote surface protease gp63, doesnot change during the whole period of in vitro growth. This is in agreement withour previous results using monoclonal antibodies anti-gp63, that showed that theexpression of gp63 is constant during the growth of L. (L.) amazonensis in vitro(Nunes and Ramalho-Pinto, 1996). The analysis of L. (V.) guyanensis proteinprofile does not show any major alteration of the level of polypeptide expressioncorroborating the correlation between complement resistance and expression ofthese particular proteins (Fig. 4A,B). Interestingly, at least one of the abovementioned bands, 46 kDa, is not found in the protein profile of L. (V.) guyanensispromastigotes.


4. Discussion

The five species studied here, three from subgenus Viannia and two fromsubgenus Leishmania, comprise the most common agents causing human leishma-niasis in the New World. They are responsible for a variety of clinical featuresranging from the self-healing cutaneous leishmaniasis, caused by L. (V.) guyanensis,to the disforming diffuse cutaneous form or even the visceral leishmaniasis, causedby L. (L.) amazonensis (Grimaldi et al., 1989; Grimaldi and Tesh, 1993).

Studying the complement sensitivity of L. (L.) amazonensis, L. (L.) mexicana, L.(V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis, we observed thatconcentrations as high as 32% f-NHS are lytic to either logarithmic or stationarypromastigotes of all these species. These findings confirm other reports, showingthat, at high serum concentration and at 37°C, no resistant parasite is found for thedifferent Leishmania species (Mosser and Edelson, 1984; Mosser et al., 1986;Barral-Netto et al., 1987; Grimm et al., 1991). Lytic assays performed in thepresence of hi-NHS, chelating agents and sera deficient in complement components(Fig. 1), clearly demonstrate that the lytic effect of f-NHS is due to activation of thealternative pathway of complement. Our results are in agreement with thosereported by others, that f-NHS is lytic for promastigotes from subgenera Leishma-nia and Viannia (Franke et al., 1985; Barral-Netto et al., 1987; Howard et al., 1987;Grimm et al., 1991) due to the activation of the alternative pathway of complement(Mosser and Edelson, 1984; Mosser et al., 1986).

The use of lytic assays at various serum concentrations, and the daily determina-tion of LD50 values allowed the comparison of complement sensitivity of the fiveNew World Leishmania species during their growth in axenic culture. Our resultsshow that promastigotes of the species from subgenus Leishmania, L. amazonensisand L. mexicana, differentiate into stationary forms with increased resistance tocomplement during growth in vitro (Fig. 2). On the other hand, no degree ofresistance was detected in promastigotes of species, of subgenus Viannia, L.braziliensis, L. panamensis and L. guyanensis (Fig. 2).

Studies using promastigotes from different growth phases have shown thatstationary promastigotes display an increased resistance to complement (Franke etal., 1985; Howard et al., 1987; Grimm and Jenni, 1993). In relation to promastig-otes from subgenus Viannia, our results do not corroborate these findings. In ourassay system, promastigotes from this subgenus were not able to develop anyresistance to complement, even at the stationary phase of their growth.

Parasites from subgenus Viannia are usually associated with self-healing forms ofdisease, whereas parasites of the subgenus Leishmania often cause more hazardousforms of disease, some species causing the visceral form. It is well known thatpromastigotes from subgenera Viannia and Leishmania differ in many aspects, suchas type of lesion, size, motility (Lainson and Shaw, 1987) and expression of proteins(McMahon-Pratt et al., 1992). It is also shown in this paper, that in relation tocomplement resistance, they seem to behave differently. Although it is becomingincreasingly apparent that the clinical expression of leishmanial infection in thehuman host is dependent on a number of different factors, of which the parasite


species is only one (Grimaldi and Tesh, 1993), it is tempting to hypothesize arelationship between complement resistance of Leishmania promastigotes and theseverity of the disease they cause. We propose that the differential sensitivity tocomplement could be used to distinguish parasites from the subgenera Viannia andLeishmania, at least the species that are most commonly involved in causing humanleishmaniasis in the New World. This study needs, however, to be extended to alarger number of species and stocks of the two subgenera.

A very interesting observation was that resistance to complement by subgenusLeishmania promastigotes varies during the stationary phase of growth. In thispaper, L. (L.) amazonensis and L. (L.) mexicana promastigotes have a definedpattern of complement resistance, showing a peak at the beginning of the stationaryphase (Fig. 2). This variation in complement resistance suggests a remarkablealteration on the parasite’s surface. Although shedding of previously synthesizedmolecules involved in protection against complement lytic action (Sacks and DaSilva, 1987) could happen, it is also possible that newly synthesized molecules couldplay a role in the parasite protection. Our results indicate that this could be thecase. By gel densitometry after SDS-PAGE of extracts, of promastigotes collectedduring their growth in vitro, we detected three polypeptides in parasites of L. (L.)amazonensis, whose pattern of expression parallels complement resistance duringgrowth in vitro. These molecules of 46, 32 and 27 kDa, reach their maximumexpression at the beginning of the stationary phase, when promastigotes reach theirmaximum resistance to complement. An interesting finding is the fact that one ofthese polypeptides, 46 kDa, is absent in promastigotes of the subgenus Viannia, thatin our culture conditions, do not express any resistance to complement. A veryabundant surface glycoprotein of promastigotes of L. (L.) amazonensis is gp46(McMahon-Pratt and David, 1982), a molecule not found in species of thesubgenus Viannia (McMahon-Pratt et al., 1992). Interestingly, gp46 presents twowell-defined structural domains. One of them is a 27 kDa-polypeptide which isgenerated by proteolytic cleavage of the intact protein and is very resistant tofurther proteolysis (Khal and McMahon-Pratt, 1987). Coincidently, 46 kDa and 27kDa are the molecular masses of two out of the three polypeptides that varyaccording to the parasite complement-resistance pattern. Since promastigotes fromsubgenus Viannia, which do not differentiate into complement-resistant forms anddo not express gp46 (McMahon-Pratt et al., 1992), it is plausible to speculate thatmembers of the gp46 family could be somehow involved in complement evasion bypromastigotes from subgenus Leishmania. We are now investigating how pro-mastigotes of L. amazonensis evade complement lysis and the role these proteinsplay in the protection of the parasite against complement mediated lysis.

Acknowledgements

This work was supported by Financiadora de Estudos e Projetos (FINEP),Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico (CNPq), Fun-dacao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), Brazil.


F.S.M.N. was supported by Coordenadoria de Aperfeicoamento de Professores doEnsino Superior (CAPES), Brazil. We thank Elimar de Faria and Soraia Oliveirafor the excellent technical assistance. We are grateful to Drs Maria de FatimaHorta and Jorge Gonzales for their critical review of the manuscript.

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