A comparison of capillary electrophoresis and direct sequencing in upstream conserved sequence region analysis of Pneumocystis jirovecii strains

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1 Journal of Medical Microbiology (2013), 62, DOI /jmm A comparison of capillary electrophoresis and direct sequencing in upstream conserved sequence region analysis of Pneumocystis jirovecii strains M. A. Jarboui, 1 F. Mseddi, 1 H. Sellami, 1 A. Sellami, 1 N. Mahfoudh, 2 F. Makni, 1 H. Makni 2 and A. Ayadi 1 Correspondence M. A. Jarboui mohamedalijarboui@gmail.com 1 Fungal and Parasitic Molecular Biology Laboratory, School of Medicine, University of Sfax, Magida Boulila Street, 3029 Sfax, Tunisia 2 Laboratory of Immunology, Hedi Chaker hospital, Sfax, Tunisia Received 4 April 2012 Accepted 15 January 2013 The major surface glycoprotein (MSG) of Pneumocystis jirovecii is the most abundant surface protein and appears to play a critical role in the pathogenesis of pneumocystosis. The expressed MSG gene is located immediately downstream of a region called the upstream conserved sequence (UCS). The UCS contains a region of tandem repeats that vary in number and sequence. In the present study, we have used capillary electrophoresis and direct sequencing to detect the variability in the repeat units of UCS. By direct sequencing the PCR products from samples of 13 patients, we have identified three types of repeat units which consisted of 10 nt and three different patterns in the UCS region with three and four repeats: 1, 2, 3 (84.6 %); 1, 2, 3, 3 (8.2 %); and a new genotype 2, 2, 3, 3 (8.2 %). The same samples were analysed by capillary electrophoresis. Three samples (23 %) contained a mixture of two or three different patterns of UCS repeats. In conclusion, quantifying the number of repeat units in the UCS by capillary electrophoresis provides a potential new method for the rapid typing of P. jirovecii and the detection of mixed infection. INTRODUCTION The major surface glycoprotein (MSG) of Pneumocystis jirovecii, a pathogen responsible for pulmonary infection in immunocompromised patients, is the most abundant surface protein and appears to play a critical role in the pathogenesis of pneumocystosis by acting as an attachment ligand to alveolar epithelial cells. MSG is also a target of both humoral and cellular immune responses (Kutty et al., 2008; Theus et al., 1997). The MSG is encoded by a multi-copy gene family, with an estimated 80 copies per genome, which are clustered in tandem arrays near the telomeres of each chromosome. Pneumocystis can vary its expressed MSG, presumably as a mechanism to avoid the host immune system (Keely & Stringer, 2009; Cornillot et al., 2002; Keely et al., 2005). The MSG gene is located immediately downstream of a region called the upstream conserved sequence (UCS). The UCS is considered to be essential for the expression of MSG and contains a region of 10 nt tandem repeats with variation in the sequence and in the number of repeats (Kutty et al., 2001; Ma et al., 2002; Esteves et al., 2009). Abbreviations: BAL, bronchoalveolar lavage; MSG, major surface glycoprotein; UCS, upstream conserved sequence. The aim of the present study was to analyse the tandem repeats in the UCS intron of MSG by direct sequencing and to develop a simple and rapid capillary electrophoresis method for typing P. jirovecii and for the detection of mixed infection. METHODS The study was performed on samples from 13 immunocompromised patients, including 10 HIV-positive patients, two kidney transplant patients and one leukaemia patient. These patients were identified as positive after the amplification of the P. jirovecii mtlsurrna gene by PCR (Jarboui et al., 2010). Clinical specimens [bronchoalveolar lavage (BAL) and sputum] isolated from these patients were centrifuged at 1500 g for 5 min and the pellet was digested overnight by proteinase K at 56 uc. DNA was extracted using a commercial kit from Qiagen (QIAamp DNA Minikit; Qiagen). PCR amplification. The PCR assay for the mtlsurrna gene was carried out in 50 ml, containing 2.5 mm MgCl 2 ; 0.25 mm each of datp, dttp, dctp and dgtp; 0.25 mm of each primer; and 2.5 U of Taq DNA polymerase (Promega). For the amplification of the UCS genomic DNA sequence of the P. jirovecii MSG gene, we used four primers (Fig. 1): ML40 (59- TTCAGCGCAGGTTGGTTG-39; corresponding to nt ), ML664 (59-CGAGGCTCCCCCAAATG-39; complementary to nt G 2013 SGM Printed in Great Britain

2 Analysis of UCS region in P. jirovecii strains ML40 JQ8 CRJE Exon 1 Intron Exon 2 MSG JQ14 ML664 UCS MSG-coding sequence (multigene family) Fig. 1. Illustration of the UCS region and MSG sequence of a Pneumocystis strain. The DNA encoding the UCS extends from exon 1, through the intron into a second block, the beginning of exon 2 and a last block (black rectangle), corresponding to the conserved recombination junction element (CRJE) ), JQ8 (59-CTGTGGATTGAGCTATTTCTTGTATCTAT- GCGCT-39; corresponding to nt ) (VIC fluorescin primer) and JQ14 (59-GCATGCAAGCTGACATTCCGCGCAAAAATAAGC- ACT-39; complementary to nt ) (Ma et al., 2002). Two rounds of PCR were used to amplify two fragments of the UCS region. The first round is a touch-down thermal cycling protocol using primers ML40 and ML664, and consisted of 10 cycles of 45 s at 95 uc and 1 min at 65 uc, with a decrease by 1 uc for every cycle to reach 55 uc in the last cycle of the period, and 1 min at 72 uc, followed by 25 cycles of 45 s at 95 uc, 1 min at 55 uc and 1 min at 72 uc. The DNA extracted was used to amplify a second fragment by primers JQ8 and JQ14. The thermal cycling protocol consisted of 35 cycles of 95 uc for 45 s, 55 uc for 45 s and 72 uc for 45 s. To avoid contamination, each step (DNA extraction, master mix preparation and amplification) was performed in separate rooms with different sets of micropipettes and using barrier tips. PCR mixtures were prepared in a laminar-flow cabinet. Several controls were included in the PCR experiments. All amplifications were performed in parallel with a negative control (ultrapure distilled water) and a positive control (BAL samples of a patient with definite Pneumocystis pneumonia and P. jirovecii positive detection by toluidine blue O dye, Giemsa stain and immunofluoresence assay). DNA sequencing and sequence analysis. The PCR products of the first amplification were purified by using the MiniElute PCR purification kit (Qiagen) and were sequenced with the same primers used to amplify the fragment of the UCS region (ML40 and ML664). Sequencing reactions were performed using the BigDye Terminator technology according to the manufacturer s protocol (Applied Biosystems), and products were analysed in an ABI 3100 automated DNA sequencer (Applied Biosystems). Data obtained with forward and reverse sequencing primers were combined, and the alignments were obtained using BioEdit. Fragment analysis by automated fluorescent capillary electrophoresis. One microlitre of fluorescent PCR products was combined with 24 ml formamide and 0.5 ml LIZ 500 marker (Applied Biosystems); these samples were then denatured for 1 min at 95 uc, with rapid cooling to 4 uc (and then to 280 uc). The PCR products were resolved by capillary electrophoresis with polymer POP-7 in an ABI Prism 310 genetic analyser. The reading of the signal and data were collected by ABI Prism 310 collection software and analysed with Gene Mapper software (Applied Biosystems). Gene Mapper software automatically calculates the PCR fragment length. The numbers of repeats in each sample could be easily determined on the basis of the size of DNA fragments. PCR products of tandem repeats were shown as green fluorescent peaks. RESULTS Analysis of UCS repeats by direct sequencing The UCS region was amplified and sequenced successfully for the samples from 13 patients using the ML40 and ML664 primers. We have identified the repeat unit which consisted of 10 nt, with sequence variability occurring in the first and fourth positions among adjacent repeat units. There are three different sequences for the repeat unit: ACAGGCGCAT (type 1), ACATGCGCAT (type 2) and G- CAGGCGCAT (type 3). From the 13 patient samples, we identified three different patterns of UCS repeats with three and four repeats: 1, 2, 3 (84.6 %); 1, 2, 3, 3 (8.2 %); and a new genotype 2, 2, 3, 3 (8.2 %). We attempted to analyse the genetic relationship among our different P. jirovecii strains. Numbers and combinations of three repeat units generate the allelic profile of a strain (Table 1). Analysis of UCS repeats by capillary electrophoresis We have analysed the UCS repeats in the same samples by capillary electrophoresis after PCR amplification with JQ8 and JQ14 primers. As shown in Fig. 2, this method permitted the resolution of all the patterns of UCS repeats. The presence of a single strain infection, as demonstrated by sequencing, has been confirmed in 10 cases by capillary electrophoresis. Only three samples (23 %), corresponding to patients 5, 11 and 13, were found to contain a mixture of two or three different patterns of UCS repeats. Patient no. 13 (genotype 2, 2, 3, 3) had an association of two strains with three and four repeats. Patients nos 5 and 11 (genotype 1, 2, 3) had an association of strains with two, three and four repeats. In addition, the isolates from all patients included in our study have previously been typed at the DHPS gene by PCR RFLP (Jarboui et al., 2011). Table 1 shows a comparison of genotypes and infection type (single or mixed) determined 561

3 M. A. Jarboui and others Table 1. Characteristics of P. jirovecii strains analysed by direct sequencing and capillary electrophoresis in 13 immunocompromised patients CE, capillary electrophoresis. Patient Clinical status No. of repeats by direct sequencing Genotype No. of repeats by CE DHPS 1 HIV 3 1, 2, 3 3 Wild-type 2 HIV 3 1, 2, 3 3 Wild-type 3 HIV 3 1, 2, 3 3 Wild-type 4 HIV 4 1, 2, 3, 3 4 Wild-type 5 HIV 3 1, 2, 3 2, 3 and 4 Wild-type and mutant 6 Kidney transplant 3 1, 2, 3 3 Wild-type 7 HIV 3 1, 2, 3 3 Wild-type 8 Leukaemia 3 1, 2, 3 3 Wild-type 9 Kidney transplant 3 1, 2, 3 3 Wild-type 10 HIV 3 1, 2, 3 3 Wild-type 11 HIV 3 1, 2, 3 2, 3 and 4 Wild-type 12 HIV 3 1, 2, 3 3 Wild-type 13 HIV 4 2, 2, 3, 3 3 and 4 Wild-type and mutant in the present study and the DHPS genotypes identified previously. DISCUSSION The intron of the UCS contains a region of tandem repeats that vary in number and sequence in different isolates. The biological role of these tandem repeats in the UCS region is unknown but the UCS is considered to be essential for the expression of MSG. A similar MSG gene organization has also been identified in rat, mouse and ferret Pneumocystis (Kutty et al., 2001; Haidaris et al., 1998; Wright et al., 1995). Since Pneumocystis is haploid and the UCS is present as a single copy per genome, quantifying the number of repeats provides a potential easy method for rapidly typing P. jirovecii. Every organism can express only a single MSG variant, but recombination may play a role in generating multiple MSG variants in different P. jirovecii strains. This process facilitates the evasion of immune responses in hosts (Hauser et al., 1998; Daly et al., 2009; Edman et al., 1996; Wada & Nakamura, 1996; Underwood et al., 1996; Stringer & Keely, 2001; Stringer, 2007). In the present study, we have used direct sequencing and capillary electrophoresis to detect the variability in the repeat units of UCS. By direct sequencing, we have identified three different types of repeat unit. The presence of these units and the variable numbers of tandem repeats with varying frequency in different strain isolates was first described by Kutty et al. (2001). The types 2 and 3 each differ from type 1 by a single nucleotide substitution, which suggests that they arose independently from type 1, perhaps during the a b c d Fig. 2. Representative electropherograms of four different patients: (a) a patient with a strain with three repeats, (b) a patient with an association of three strains (strain with two repeats, strain with three repeats and strain with four repeats), (c) a patient with a strain with four repeats, and (d) a patient with an association of two strains (strain with three repeats and strain with four repeats). 562 Journal of Medical Microbiology 62

4 Analysis of UCS region in P. jirovecii strains duplication process. The repeat type (1, 2, 3) is the major genotype in our population (84.6 %). The strains with four repeats (1, 2, 3, 3) were identified in 8.2 %. The detection of the novel tandem repeat pattern (2, 2, 3, 3) was an interesting result. As reported in another study, most P. jirovecii strains contain three or four repeats (Ma et al., 2002; Esteves et al., 2009). A disadvantage of the direct sequencing method is that it is not always able to detect mixed infection by different strains of P. jirovecii from the same patient. For this purpose, we have developed a simple and rapid capillary electrophoresis for typing P. jirovecii. This tandem repeat typing method is based primarily on the detection of DNA length polymorphisms resulting from variation numbers of tandem repeats. By examining P. jirovecii isolates, we have demonstrated that the number of UCS tandem repeats varies from two to four, with three repeats being the most common. The capillary electrophoresis method offers highresolution of DNA fragments by size, allowing an easy interpretation of the results and detection of a minor population in mixed populations. The presence of multiple repeat patterns in a single clinical sample was also confirmed by denaturing gel electrophoresis analysis (Ma et al., 2002). The capillary electrophoresis technique was able to identify coinfection with more than two UCS repeat patterns. In our study, coinfection with two or three strains of P. jirovecii was found in 23 % of cases. We have detected two cases of coinfection by three strains and one case of coinfection by two strains. These cases were detected in BAL specimens of three HIV patients with typical symptoms of Pneumocystis pneumonia (dyspnoea, dry cough and fever) and typical chest X-ray with interstitial infiltration. The rate of coinfection was reported elsewhere in studies of other genetic loci using the single strand conformation polymorphism or type-specific oligonucleotides techniques and varies between 20 and 69 % (Hauser et al., 1998; Lee et al., 1998; Ma & Kovacs, 2001). A disadvantage of capillary electrophoresis is that it is unable to differentiate the type of repeat units in the UCS region. To identify variant repeat units, DNA sequencing is needed. The isolates from all patients included in our study had been characterized previously at the DHPS gene by the PCR RFLP technique (Jarboui et al., 2011), and we have detected two cases of coinfection by wild-type and mutant P. jirovecii strains. These same patients had a coinfection with two and three UCS repeat patterns detected by capillary electrophoresis. This result proves the efficacy of PCR RFLP and capillary electrophoresis in the study of mixed infection by P. jirovecii. In conclusion, quantifying the number of repeat units of the UCS by capillary electrophoresis proves a potential new method for the rapid typing of P. jirovecii and the detection of mixed infection. The combination of this technique and direct sequencing facilitates the molecular and epidemiological study of P. jirovecii. ACKNOWLEDGEMENTS This study was done with the financial support of the Ministry of Higher Education and Scientific Research of Tunisia. REFERENCES Cornillot, E., Keller, B., Cushion, M. T., Méténier, G. & Vivarès, C. P. (2002). Fine analysis of the Pneumocystis carinii f. sp. carinii genome by two-dimensional pulsed-field gel electrophoresis. Gene 293, Daly, K., Koch, J., Respaldiza, N., de la Horra, C., Montes-Cano, M. 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