mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Serological and antigenic profiles of clinical isolates of Paracoccidioides spp. from Central Western Brazil dua Queiroz Ju nior,1 Zoilo Pires de Camargo,2 Tomoko Tadano,3 Luiz de Pa Anderson Messias Rodrigues,2 Doracilde Terumi Takarara,4 Gregory Gegembauer,2 Leticia Mendes Araujo2 and Rosane Christine Hahn1 1 Mycology/Research Laboratory, Faculty of Medicine, Federal University of Mato Grosso, UFMT, Cuiaba, Mato Grosso, Brazil, 2Mycology Laboratory, Federal University of Sa~o Paulo, UNIFESP, Sa~o Paulo, Brazil, 3Julio Muller University Hospital, UFMT, Cuiaba, Mato Grosso, Brazil and 4MT Laboratory, State Department of Health, Cuiaba, Mato Grosso, Brazil
Summary
Clinical Paracoccidioides spp. isolates from patients with paracoccidioidomycosis (PCM) in Mato Grosso, Brazil exhibit different patterns of serologic reactivity. The results observed for reactions of radial immunodiffusion against the commonly used exoantigens containing a 43-kDa glycoprotein (gp43) suggest that this fungus exhibits major antigenic variability by geographic region. There is a phylogenetic gap between Paracoccidioides spp. isolates among different regions of Latin America. In particular, those from the central region of Brazil (i.e. Mato Grosso state) exhibit a lower rate of genetic similarity. We aimed at investigating the phylogenetic classification of clinical isolates of Paracoccidioides spp. in Central Brazil and the different antigenic profiles that produce. Exoantigens were obtained from five clinical isolates: two P. brasiliensis (Pb166 and Pb2880) and three P. lutzii (PL2875, PL9840, and PL2912). The protein/glycoprotein profiles of P. lutzii exoantigens were different from each other. Isolate PL9840 exhibited the most distinct bands, and isolates PL2875 and PL2912 exhibited more diffuse bands and a very intense band between 50 and 60 kDa. P. brasiliensis isolates had similar protein profiles, exhibiting a low-intensity band at 220 kDa and a diffuse band between 50 and 60 kDa. P. lutzii isolates exhibit high species-specific antigen variability, which we have already been assessed in proteomic studies.
Key words: Paracoccidioidomycosis, antigenic profile, serological diagnosis, midwest region of Brazil.
Introduction Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America, an endemic area because it possesses environmental characteristics favourable for the maintenance of the fungus in the soil. PCM is caused by a thermodimorphic fungus of Correspondence: Dr R. C. Hahn, Postgraduate Program in Health Sciences, Mycology/Research Laboratory, Faculty of Medicine, Federal University of Mato Grosso, Cuiab a, Mato Grosso CEP:78060-900, Brazil. Tel.: (+55) (65)3615 8856. Fax: (+55) (65) 3615 8809. E-mail:
[email protected] Submitted for publication 10 October 2013 Revised 11 February 2014 Accepted for publication 14 February 2014
doi:10.1111/myc.12183
the genus Paracoccidioides that currently encompasses two species: P. brasiliensis and P. lutzii.1–4 Thus, activities related to soil management are risk factors for acquiring this infection and it is estimated that about 10 million people are infected in all endemic areas, but only 2% develop the disease. About 80% of PCM cases occur in Brazil.3–9 PCM exhibits distinct clinical forms such as PCM infection, acute/subacute (i.e. juvenile) form and the chronic (i.e. adult) form10,11 and it is currently the eighth most common cause of death from chronic/recurrent infectious and parasitic diseases.12 Studies have demonstrated the mortality caused by PCM in Brazil and the results suggest that the mortality rate has decreased from 1.45 per million inhabitants in 200212 to 1.00 per million inhabitants in 2011 (Z. F. Coutinho, unpublished data). However,
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Paracoccidioides spp. from Central Brazil
the state of Mato Grosso has attracted national attention because its mortality rate has increased from 3.22 per million inhabitants in 200212 to 5.00 per million inhabitants in 2011 (Z. F. Coutinho, unpublished data). Despite findings demonstrating the importance of PCM as a public health problem, it remains a neglected disease, hindering the accurate estimation of epidemiological data related to the disease.11,12 Several studies since the late 1990s demonstrate the existence of genetic variability among cryptic species of the Paracoccidioides genus, termed ‘P. brasiliensis complex’ (S1, PS2, PS3 and ‘Pb01-like’).13–22 This enabled the identification of the high degree of genetic variation present in the cryptic species ‘Pb01-like’. In turn, this led to the suggestion of a new speciation of the genus Paracoccidioides and the proposal of a new species of this fungus, P. lutzii.2,23 Meanwhile, over the last decade, some authors demonstrated the existence of antigenic variability among Paracoccidioides spp. isolates; furthermore, geographic origin may represent an important marker in this process, which may be associated with the observed variability in the clinical presentation of PCM.24,25 Studies assessing the antigenicity of Paracoccidioides spp. isolates indicate that isolates from Central Brazil have antigenic characteristics particular to that region that directly influence the sensitivity of serological tests used in routine mycology to aid the diagnosis of PCM, such as double radial immunodiffusion (ID).25,26 Recent studies assessed the distributions of P. brasiliensis and P. lutzii in endemic areas and noted that P. lutzii is highly concentrated in the central region of Brazil, especially in the state of Mato Grosso.27 However, no studies have been carried out to explain the antigenicity of P. lutzii. Nevertheless, because of its recent classification, preliminary studies suggest that this species presents greater antigenic variability in serologic tests than P. brasiliensis.24,25 Geographical models have been developed to assess the climatic influences of El Ni~ no and La Ni~ na on the prevalence of PCM; they demonstrate that in the Botucatu region of the state of S~ ao Paulo, clay soils and higher annual precipitation are associated with more diagnosed cases of acute/subacute PCM in populations living in that hyperendemic PCM area.28 This study suggests that there is a closer relationship between climate and the saprophytic life of Paracoccidioides spp. than previously thought. Thus, this relationship should be further investigated to determine associations between climate and not only epidemiological data but also more complex
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 466–472
data related to genetic and molecular characteristics, such as phylogeny, virulence, transcriptional profile, electrophoretic profile and antigenicity presented by isolates, especially those from the central region of Brazil. We aimed at investigating the phylogenetic classification of clinical isolates of Paracoccidioides spp. in Central Brazil and the different antigenic profiles that produce.
Materials and methods Source of Paracoccidioides spp. isolates
The clinical P. lutzii isolates (PL2875, PL9840 and PL2912) were obtained from the Laboratory of Mycology, University Hospital J ulio Muller (HUJM), Federal University of Mato Grosso, from patients from the Mato Grosso region of hyperendemic PCM. Isolates were identified and preserved in the Mycology Collection Service under the direction of Dr Rosane Hahn. P. brasiliensis isolates (Pb166, Pb2880, Pb339, Pb18 and Pb2734) were obtained from patients in S~ ao Paulo and kept in the Mycology Laboratory of the Federal University of S~ ao Paulo under the direction of Professor Dr Zoilo Pires de Camargo. Paracoccidioides spp. culture and DNA extraction
Paracoccidioides spp. isolates were cultured at 25 °C in Fava Netto agar and peaked every 30 days. Genomic DNA was extracted directly from colonies and purified following the Fast DNA kit protocol (Biomedicals MP, Solon, OH, USA). Species identification
Paracoccidioides brasiliensis and P. lutzii were identified by PCR in 200-ll Eppendorf tubes. Reaction mixtures containing 12.5 ll 2 9 PCR Master Mix (Promega, Madison, WI, USA); MgCl2, dNTPs, dATP, dTTP, dCTP, dGTP and Taq polymerase), 1 ll HSPMMT1 primer (50 AACCAACCCCCTCTGTCTTG-30 ) [10 pmol ll1], 1 ll primer PLMMT1 (50 -GAAATGGGTGGCAGTATGGG-30 ) [10 pmol ll1], 9.5 ll sterile MilliQ water and 1 ll Paracoccidioides spp. DNA [100 ng ll1] in a total volume of 25 ll was incubated in a thermocycler (MyGene Series PeltierThermalCycler, MG96G; LongGeneScientific). The cycling conditions were as follows: initial denaturation at 94 °C for 5 min; 35 amplification cycles at 94 °C (denaturation) for 1 min, 60 °C (annealing) for 1 min and 68 °C (extension) for
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1 min; and final extension at 68 °C for 10 min. The primers HSPMMT1 and PLMMT1 recognise a polymorphic region of the Hsp70 gene unique to ‘Pb01-like’ isolates as described previously by Teixeira et al. [2, 23]. Isolate Pb-01 was included as a positive control for PCR. The resultant amplicons were homogenised in sample buffer (DNA) applied on 1% agarose gel containing ethidium bromide (1 mg ml1) together with 5 ll Low Ranger 100-bp DNA Step Ladder (Promega) molecular weight standard and subjected to a constant electrical current of 80 V for 60 min. After electrophoresis, amplicons were visualised by a photodocumentation system (L-PixTouch) comprising a UV transilluminator unit (Loccus Biotecnologia, S~ ao Paulo, Brazil) coupled to a digital camera (Canon, USA) using L-PixImage software (Loccus Biotecnologia). Isolates used to obtain exoantigens
The isolates were reverted to the yeast phase, peaked every 5 days in Fava Netto agar and incubated at 37 °C. Three isolates identified as P. lutzii PL2875, PL9840 and PL2912 as well as two P. brasiliensis isolates, Pb166 and Pb2880, were used in this study. Culture conditions for P. lutzii and P. brasiliensis exoantigen production
After isolates were reversed, crude exoantigens were produced as described by Camargo et al. [26]. The method used to prepare P. lutzii exoantigens was the same as that for P. brasiliensis. Protein contents were subsequently assessed by the Bradford method [37]. Analysis of exoantigens by SDS-PAGE
Vertical gel electrophoresis was prepared according to the method of Laemmli [29]. The samples (2-lg protein) were applied in a mini-gel vertical vat (Mini 8.10; GibcoBRL), diluted in sample buffer (0.068 mol l1 Tris-HCl [pH 6.8], 2.3% SDS, 10% glycerol, 0.1% bromophenol blue) and 2-mercaptoethanol (5%) and applied only at the time of use. The samples were heated at 100 °C for 5 min, centrifuged 1000 g for 5 min and applied to the stacking gel with standard molecular markers (Invitrogen, Carlsbad, CA, USA) with molecular masses ranging from 20 to 220 kDa. After electrophoresis, the gel was rinsed with double-distilled water and silver stained as described by Blum et al. [30]. After staining, the gel was vacuum dried on a hotplate (SlabDryer 443, BioRad) and saved for analysis.
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Sera of patients with PCM
Sera were obtained from patients of the HUJM, UFMT, Cuiab a, MT (anti-P. Lutzii: sera 9840, 2875 and 2912) and the Federal University of S~ ao Paulo, UNIFESP, S~ ao Paulo, SP (anti-P. brasiliensis: serum 2247) who were diagnosed with PCM. These patients were diagnosed on the basis of results of the direct examination, fungal culture and double radial ID. The three sera from patients with PCM due to P. lutzii corresponding to the P. lutzii isolates were considered the gold standard. Double radial ID
The ID reactions were performed as described by Camargo et al. [26, 38]. For the ID test, we used patient sera with PCM caused by strain 9840 for diagnosis by direct examination and culture. This serum was tested with the following antigens: (a) P. brasiliensis exoantigens (i.e. B-339 and Pb18) and (b) P. lutzii exoantigen (i.e. PL2912). Determination of the optimal antigen concentration for ELISA
An ELISA was performed as described by Camargo et al. [38]. Optical density was measured in an ELISA plate reader (MCC/340, Titertek Multiskan) at 492 nm. After reading, a chart was constructed ([optical density 9 serum dilution] [serological curve]). Western blotting
Western blotting was performed as described by Towbin et al. [39].
Results Screening of Paracoccidioides spp. isolates
The identities of the clinical isolates that were originally thought to be P. brasiliensis were reevaluated on the basis of results of the amplification of the marker gene Hsp70. These strains showed positive amplification of a 400-bp fragment using primers HSPMMT1 and PLMMT1, which was similar to that found in the amplified fragment from isolate Pb01 (P. lutzii, positive control). This indicates that these species share a region in the first intron of the Hsp70 gene limited to samples classified as ‘Pb01-like’. DNA from isolate Pb339 (S1) was used as a negative control (Fig. 1).
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 466–472
Paracoccidioides spp. from Central Brazil
(a)
(b)
Figure 1 PCR for the molecular identifi-
cation of Paracoccidioides lutzii strains. (a) PCR using Hsp70 primers that amplify only P. lutzii. (b) PCR using gp43 primers to verify that the strains are Paracoccidioides spp. M, molecular weight marker in bp. 1, PL2875; 2, PL9840; 3, PL2912; 4, Pb166; 5, Pb2880; 6, Pb339; 7, Pb18; 8, negative control of the PCR reaction.
Protein/glycoprotein profile of exoantigens
The protein/glycoprotein profiles of P. lutzii exoantigens studied and obtained in the Fava Netto medium were different from each other. The PL9840 isolate exhibited more bands overall and more distinct bands. Meanwhile, isolates PL2875 and PL2912 exhibited more diffuse bands, and a very intense band between 50 and 60 kDa was observed in isolate PL2912 (Fig. 2). ID using exoantigen preparations from Paracoccidioides spp. as antigens
The exoantigen PL9840 reacted with the serum of the respective patient as evidenced by a precipitation band. However, the exoantigens PL2912 and PL2875 did not react with sera from their respective P. lutzii PCM
patients. This indicates antigenic differences between the strains PL9840, and PL2912 and PL2875. On the other hand, as expected, the exoantigens from P. brasiliensis did not react with sera from patients with P. lutzii PCM (Fig. 3). Western blotting
Figure 4 shows the results of Western blotting using exoantigens of P. lutzii PL2875, PL9840 and PL2912 as well as those of P. brasiliensis Pb166 and Pb2880. In this experiment, we used sera from patients with P. lutzii and P. brasiliensis PCM respectively. The serum of the patient with P. lutzii PL9840 PCM recognised an antigenic fraction around 60 kDa and another at 220 kDa from exoantigen PL9840 but did not react with the other two exoantigens of P. lutzii (i.e. PL2875 and PL2912). Furthermore, there was no recognition of the antigenic fraction in the exoantigens from P. brasiliensis. On the other hand, when the same exoantigens were tested with sera from patients with P. brasiliensis PCM, there was no reactivity with the homologous system – only slight cross-reactivity with the 220-kDa antigen of P. lutzii. Optimal concentration of antigen for the ELISA
To verify the optimal concentration of antigen for the ELISA, the assay was initially performed using various concentrations of antigen and positive control serum. The optimal concentration of P. lutzii exoantigen (PL9840) to sensitise the ELISA plate was 12.5 lg ml1. Titration of P. lutzii PCM sera in ELISA P. lutzii (PL9840) antigens Figure 2 SDS-PAGE of the isolated exoantigens of Paracoccidioides lutzii produced in Fava Netto medium containing the protein/glycoprotein profile of each isolate. MW, molecular weight marker (Invitrogen). Pb, P. brasiliensis; PL, P. lutzii.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 466–472
The serological curves verified that the three sera from patients with P. lutzii PCM contained anti-P. lutzii antibodies and were titrated by a dilution of
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Figure 3 Immunodiffusion to detect anti-
bodies against Paracoccidioides lutzii. Left: The centre has the serum of a patient with paracoccidioidomycosis (PCM) due to P. lutzii (serum 9840), and the periphery (a) has P. lutzii exoantigen (PL9840). Right: The centre has serum from a patient with PCM due to P. lutzii (serum 9840), and the periphery has different exoantigens: (b) P. brasiliensis Pb339 exoantigen; (c) P. brasiliensis Pb18 exoantigen; (d) P. lutzii PL2912 exoantigen; (e) PL2875 exoantigen showing a negative reaction.
(a)
(b)
(c)
Figure 4 Western blot analysis. (a) SDS-PAGE of exoantigens of Paracoccidioides lutzii PL2875, PL9840 and PL2912 as well as exoantigens of P. brasiliensis Pb166 and Pb2880. (b) Serum from a patient with paracoccidioidomycosis (PCM) due to P. lutzii reacting with the same exoantigens in (a). (c) Serum from a patient with PCM due to P. brasiliensis reacting with the same exoantigens in (a). Western blot with serum 9840 (PL). Western blot with serum 2247 (Pb).
1 : 51 200. The reactivity of normal human serum was low. Figure 5 shows the titration of the sera from patients with P. lutzii PCM.
Discussion This study evaluated the protein and antigenic profiles of P. lutzii and P. brasiliensis strains. A new Paracoccidioides species, P. lutzii proposed by Teixeira et al. [2, 23], was recently added. Thus, it is necessary to assess the specific details of each species. To assess the protein profile, we performed molecular identification of the strains selected by amplification by a marker specific for P. brasiliensis that identifies the gene encoding gp43 kDa (as demonstrated by Cisalpino et al. [31] and Puccia et al. [32, 33]) as well as a specific marker for P. lutzii for a
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primer for the Hsp70 gene (as demonstrated by Teixeira et al. [23]). Molecular analysis of isolates PL2875, PL9840 and PL2912 from Central Brazil identified the characteristic marker of P. lutzii, while isolates Pb166, Pb2880, Pb339, and Pb18 Pb2734 from South Eastern Brazil quite clearly identified the molecular marker of P. brasiliensis. After the species were identified, the selected isolates of P. lutzii (i.e. PL2875, PL9840 and PL2912) and P. brasiliensis (i.e. Pb166 and Pb2880) were processed to obtain exoantigens. Subsequent SDS-PAGE showed divergent electrophoretic separation of bands ranging from 20 to 220 kDa when comparing the two species. Isolates Pb166 and Pb2880 (P. brasiliensis) exhibited similar protein profiles, presenting a low-intensity band at 220 kDa and a diffuse band between 50 and
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Paracoccidioides spp. from Central Brazil
Figure 5 ELISA of the sera from patients
with Paracoccidioides lutzii PCM vs. exoantigens from P. lutzii PL9840. Normal human serum (SHN) was used as a negative control.
60 kDa. Similar results are reported by Panunto-Castelo et al. [34] who also demonstrated similarities in the protein profiles of five P. brasiliensis isolates from South Eastern Brazil. In this study, the bands of electrophoretic profiles were tested by Western blotting against sera from other patients with PCM from the same region. The results showed a slight antigenic difference and high recognition of major bands such as gp43, gp70 and gp160 kDa. In contrast, this was not observed for isolates PL2875, PL9840 or PL2912 (P. lutzii), which exhibited different protein profiles compared with each other as well as P. brasiliensis isolates. It is worth noting that P. lutzii isolates exhibit protein variability amongst themselves as reported by Grossklaus [35] in 2008 as well as amongst P. brasiliensis isolates. When the fractionation of P. lutzii protein isolates was subjected to Western blotting and evaluated against sera from a patient with PCM from Mato Grosso (isolate 9840), reactivity was observed against exoantigen PL9840. However, this serum did not recognise the other two antigens of P. lutzii (i.e. PL2875 and PL2912), and no reactivity was observed when fractions were tested with serum from a patient from South Eastern Brazil (i.e. Pb2247). Again, these results differ from those of Panunto-Castelo et al. [34] who observed the recognition of P. brasiliensis antigens against sera from different patients with PCM from the same region. Batista et al. [25] and Marques-da-Silva et al. [36] report the variability in exoantigen antigenicity and their recognition in serological tests; this highlights the importance of using region-specific exoantigens in the serological diagnosis of PCM. Therefore, to increase the sensitivity and specificity of tests and antigen recognition, the serological diagnosis of PCM should be corroborated on the basis of titres that in fact express the concentration of anti-P. brasiliensis or anti-P. lutzii
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 466–472
antibodies using serum and strains obtained from the same patient. This suggests that the antigenic particularities may be much more variable than previously proposed. The present results also suggest that an exoantigen derived from a P. lutzii strain cannot be considered the gold standard, because B-339 has been considered the gold standard for a few decades; the results of this study corroborate this statement, because the exoantigen PL9840 was tested against serum 9840 by ID, demonstrating antigen recognition of the serum against its specific exoantigen. However, this same serum was unable to recognise other P. lutzii exoantigens (i.e. PL2875 and PL2912), suggesting large intraspecies antigenic variability. Thus, P. lutzii isolates do not exhibit similar patterns of electrophoretic separation or protein antigenicity as P. brasiliensis isolates.34 Research on exoantigen production from previously characterised clinical isolates highlights gaps in the current understanding of PCM; this has become the subject of interest of many researchers, particularly those trying to determine the associations between isolated species, serologic reactivity against exoantigens obtained from the corresponding strain techniques and evaluation by Western blotting and ELISA. The antigenic variation observed in different strains in this study improves our current understanding of the clinical spectrum of the disease. Finally, we note the need for further investigation of the clinical, molecular, and immunological parameters of P. brasiliensis and P. lutzii.
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