Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites

BMC Microbiology, Dec 2009

Background Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes paracoccidiodomycosis (PCM). Glycoprotein gp43 is the fungal main diagnostic antigen, which can also protect against murine PCM and interact with extracellular matrix proteins. It is structurally related to glucanases, however not active, and whose expression varies considerably. We have presently studied polymorphisms in the PbGP43 flanking regions to help understand such variations. Results we tested the protein-binding capacity of oligonucleotides covering the PbGP43 proximal 5' flanking region, including overlap and mutated probes. We used electrophoretic mobility shift assays and found DNA binding regions between positions -134 to -103 and -255 to -215. Only mutation at -230, characteristic of P. brasiliensis phylogenetic species PS2, altered binding affinity. Next, we cloned and sequenced the 5' intergenic region up to position -2,047 from P. brasiliensis Pb339 and observed that it is composed of three tandem repetitive regions of about 500 bp preceded upstream by 442 bp. Correspondent PCR fragments of about 2,000 bp were found in eight out of fourteen isolates; in PS2 samples they were 1,500-bp long due to the absence of one repetitive region, as detected in Pb3. We also compared fifty-six PbGP43 3' UTR sequences from ten isolates and have not observed polymorphisms; however we detected two main poly(A) clusters (1,420 to 1,441 and 1,451 to 1,457) of multiple cleavage sites. In a single isolate we found one to seven sites. Conclusions We observed that the amount of PbGP43 transcripts accumulated in P. brasiliensis Pb339 grown in defined medium was about 1,000-fold higher than in Pb18 and 120-fold higher than in Pb3. We have described a series of features in the gene flanking regions and differences among isolates, including DNA-binding sequences, which might impact gene regulation. Little is known about regulatory sequences in thermo-dimorphic fungi. The peculiar structure of tandem repetitive fragments in the 5' intergenic region of PbGP43, their characteristic sequences, besides the presence of multiple poly(A) cleavage sites in the 3' UTR will certainly guide future studies.

Article PDF cannot be displayed. You can download it here:

http://www.biomedcentral.com/content/pdf/1471-2180-9-277.pdf

Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites

BMC Microbiology BioMed Central Research article Open Access Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites Antonio A Rocha1, Flávia V Morais2 and Rosana Puccia*1 Address: 1Department of Microbiology, Immunology and Parasitology, Federal University of São Paulo (UNIFESP), 04023-062 São Paulo, SP, Brazil and 2Laboratory of Molecular Genetics and Genomics, University of Vale do Paraíba (UNIVAP), SP, Brazil Email: Antonio A Rocha - ; Flávia V Morais - ; Rosana Puccia* - * Corresponding author Published: 30 December 2009 BMC Microbiology 2009, 9:277 doi:10.1186/1471-2180-9-277 Received: 23 June 2009 Accepted: 30 December 2009 This article is available from: http://www.biomedcentral.com/1471-2180/9/277 © 2009 Rocha et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Paracoccidioides brasiliensis is a thermo-dimorphic fungus that causes paracoccidiodomycosis (PCM). Glycoprotein gp43 is the fungal main diagnostic antigen, which can also protect against murine PCM and interact with extracellular matrix proteins. It is structurally related to glucanases, however not active, and whose expression varies considerably. We have presently studied polymorphisms in the PbGP43 flanking regions to help understand such variations. Results: we tested the protein-binding capacity of oligonucleotides covering the PbGP43 proximal 5' flanking region, including overlap and mutated probes. We used electrophoretic mobility shift assays and found DNA binding regions between positions -134 to -103 and -255 to -215. Only mutation at -230, characteristic of P. brasiliensis phylogenetic species PS2, altered binding affinity. Next, we cloned and sequenced the 5' intergenic region up to position -2,047 from P. brasiliensis Pb339 and observed that it is composed of three tandem repetitive regions of about 500 bp preceded upstream by 442 bp. Correspondent PCR fragments of about 2,000 bp were found in eight out of fourteen isolates; in PS2 samples they were 1,500-bp long due to the absence of one repetitive region, as detected in Pb3. We also compared fifty-six PbGP43 3' UTR sequences from ten isolates and have not observed polymorphisms; however we detected two main poly(A) clusters (1,420 to 1,441 and 1,451 to 1,457) of multiple cleavage sites. In a single isolate we found one to seven sites. Conclusions: We observed that the amount of PbGP43 transcripts accumulated in P. brasiliensis Pb339 grown in defined medium was about 1,000-fold higher than in Pb18 and 120-fold higher than in Pb3. We have described a series of features in the gene flanking regions and differences among isolates, including DNA-binding sequences, which might impact gene regulation. Little is known about regulatory sequences in thermo-dimorphic fungi. The peculiar structure of tandem repetitive fragments in the 5' intergenic region of PbGP43, their characteristic sequences, besides the presence of multiple poly(A) cleavage sites in the 3' UTR will certainly guide future studies. Page 1 of 13 (page number not for citation purposes) BMC Microbiology 2009, 9:277 Background Paracoccidioides brasiliensis is a thermo-dimorphic pathogenic fungus. It causes paracoccidiodomycosis (PCM) in man, which is an endemic mycosis in Latin America that affects mostly the lungs, but can disseminate to other organs [1]. P. brasiliensis is multinucleated in both pathogenic yeast and infectious mycelial phases. Genetic transformation in the species has recently been optimized [2], however genetic manipulation is still in its infancy. It is now recognized that most P. brasiliensis isolates diversified into an S1 main species, which is genetically close to the PS3 group of Colombian isolates, while PS2 is composed of a few isolates that constitute a phylogenetically cryptic species [3]. Gp43 is the main diagnostic and prognostic antigen so far characterized in P. brasiliensis [4,5]. It is a secretory glycoprotein whose peptide structure bears antigenic properties that are peculiar to the species [6]. Therefore, it confers high levels of sensitivity and specificity for PCM patients' sera when used as antigen in diagnostic tests such as immunodiffusion and capture ELISA, as well as by antigen detection in biological fluids [7]. Antibody titers are directly proportional to the severity of active PCM; they are probably not protective in advanced stages of the disease, but experimental protocols in mice point to the immunotherapeutic potential of anti-gp43 monoclonal antibodies [8]. On the other hand, gp43 contains T cell epitopes that are protective to vaccinated mice [5]. The best studied T-cell epitope is 15 aminoacid-long P-10, which showed additive effect in the treatment of murine PCM when administered with anti-fungal agents [9]. In addition, gp43 has adhesive properties to extracellular matrix proteins that may help fungal dissemination [10,11]. The complete PbGP43 ORF has originally been found in a cloned 3,800-bp EcoRI genomic region from the Pb339 (B-339) isolate. It comprises 1,329 bp that contain a unique 78-bp intron [12]. The EcoRI genomic fragment includes 326 bp from the PbGP43 5' intergenic proximal region and about 500 bp of the 3' intergenic sequence, which is shared by a neighboring RanBP homologue. This gene encodes a nuclear Ran-binding protein in Schizosaccharomyces pombe, or importin 11 in Aspergillus fumigatus, that transports ribosomal proteins to the nucleus [13]. PbGP43 and PbRanBP are linked in twelve P. brasiliensis isolates, as observed by Feitosa et al. [14]. Our group has carried out original and detailed studies on sequence polymorphism in the PbGP43 ORF [15] and 5' intergenic proximal region [16], which defined at least five genotypes [17]. When compared to a consensus sequence, the most polymorphic A genotype carries three substitutions in the 5' intergenic proximal region and up to fifteen informative sites in the ORF, mostly concen- http://www.biomedcentral.com/1471-2180/9/277 trated in exon 2. So far, the A genotype has been detected in all six PS2 isolates [3]. It is of note that PbGP43 was the most polymorphic gene in the multilocus analysis performed by Matute et al. [3] in P. brasiliensis. Isolates Pb2, Pb3 and Pb4, which belong in PS2 group [3], evoked milder experimental PCM in B10. A mice than representative isolates from the main species S1, including Pb18 [16]. This isolate has been long used in experimental PCM due to its high virulence. P. brasiliensis Pb339 has traditionally been employed in antigen preparation [18]. It secretes high amounts of gp43, however that is not a rule among isolates [19]. (...truncated)


This is a preview of a remote PDF: http://www.biomedcentral.com/content/pdf/1471-2180-9-277.pdf
Article home page: http://www.biomedcentral.com/1471-2180/9/277

Antonio A Rocha, Flávia V Morais, Rosana Puccia. Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites, BMC Microbiology, 2009, pp. 277, 9, DOI: 10.1186/1471-2180-9-277