Upstream Conserved Sequence Research Articles

Genotyping of Pneumocystis jirovecii isolates from human immunodeficiency virus-negative patients in China

Pneumocystis jirovecii is a fungus that causes Pneumocystis pneumonia in immuno-compromised patients. To analyze the genetic diversity of P. jirovecii in HIV-negative patients in China, respiratory specimens were obtained from 105 patients who tested PCR-positive for the presence of the P. jirovecii mitochondrial large subunit ribosomal RNA (mtLSU rRNA) between 2011 and 2013. P. jirovecii isolates were genotyped based on the upstream conserved sequence (UCS) of the major surface glycoprotein (MSG) gene and the internal transcribed spacer (ITS) of nuclear rRNA operon. Eighty-one of the 105 isolates showed a positive PCR for the UCS region. We identified six different patterns comprising two, three, four, or five UCS repeats, including 1, 2, 3 (69.14%), 1, 2, 3, 3 (22.22%), 1, 2 (3.7%), 1, 1, (2.47%), 2, 2, 3, 3 (1.23%), and 1, 1, 2, 3, 3 (1.23%). In regard to the ITS region, 58 of the 105 isolates were cloned and sequenced successfully. Six known ITS1 alleles (A, B, DEL1, E, N, and SYD1), two new alleles (designated as BTM3 and BTM4), six known ITS2 alleles (a, b, i, g, h and O) and one new allele (designated as btm6) were observed. A total of 19 P. jirovecii ITS haplotypes were identified. The most frequent type was Bi (25.9%), followed by Ai (13.8%), Eb (10.3%), and SYD1g (6.9%). Among the 58 specimens examined, 49 (84.5%) were found to contain a single type of P. jirovecii, while 9 (15.5%) contained multiple genotypes. A total of 34 allelic profiles were observed in 58 isolates when the two loci were combined with each other. A Fisher’s exact test revealed that there was no statistically significant (P=0.330) association between the most frequent UCS and ITS genotypes. An analysis of the phylogenetic relationship between different patient groups identified two major groups based on the sequence variations of concatenated UCS and ITS sequences in 49 isolates. Our results demonstrated the high genetic variability of P. jirovecii in HIV-negative patients in China.

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

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.

Genetic diversity ofPneumocystis jiroveciistrains based on sequence variation of different DNA region

Pneumocystis jirovecii is an important opportunistic pathogen that causes severe pneumonia in immunocompromised patients. The aim of the present study was to investigate the genetic diversity of P. jirovecii strains by direct sequencing and analysis of the Upstream Conserved Sequence (UCS) region, mitochondrial large-subunit (mtLSU) rRNA and dihydrofolate reductase (DHFR) genes. We identified the polymorphisms in P. jirovecii strains of 15 immunocompromised patients, as well as detecting a new tandem repeat of 5 nucleotides in UCS region. The following three different types of repeat unit were found: type a GCCCA; type b GCCCT; and type c GCCTT. In addition, we identified the repeat unit which consisted of 10 nucleotides and three different patterns of UCS repeats with 3 and 4 repeats, i.e., 1, 2, 3 (86.7%), 1, 2, 3, 3 (6.6%) and a new genotype 2, 2, 3, 3 (6.6%). The polymorphism in the mtLSUrRNA gene was seen primarily at position 85 where we detected three different genotypes. Genotype 3 and genotype 2 were the most abundant with frequencies of 53.3% and 40%, respectively. With regard to the DHFR gene, only two (20%) patients had nucleotide substitution in position 312. In conclusion, the multilocus analysis facilitated the typing of P. jirovecii strains and proved the important genetic biodiversity of this fungus.

Genetic characterization of UCS region of Pneumocystis jirovecii and construction of allelic profiles of Indian isolates based on sequence typing at three regions

Pneumocystis jirovecii is an opportunistic pathogen that causes severe pneumonia in immunocompromised patients. To study the genetic diversity of P. jirovecii in India the upstream conserved sequence (UCS) region of Pneumocystis genome was amplified, sequenced and genotyped from a set of respiratory specimens obtained from 50 patients with a positive result for nested mitochondrial large subunit ribosomal RNA (mtLSU rRNA) PCR during the years 2005–2008. Of these 50 cases, 45 showed a positive PCR for UCS region. Variations in the tandem repeats in UCS region were characterized by sequencing all the positive cases. Of the 45 cases, one case showed five repeats, 11 cases showed four repeats, 29 cases showed three repeats and four cases showed two repeats. By running amplified DNA from all these cases on a high-resolution gel, mixed infection was observed in 12 cases (26.7%, 12/45). Forty three of 45 cases included in this study had previously been typed at mtLSU rRNA and internal transcribed spacer (ITS) region by our group. In the present study, the genotypes at those two regions were combined with UCS repeat patterns to construct allelic profiles of 43 cases. A total of 36 allelic profiles were observed in 43 isolates indicating high genetic variability. A statistically significant association was observed between mtLSU rRNA genotype 1, ITS type Ea and UCS repeat pattern 4.

Genetic characterization of the UCS and Kex1 loci of Pneumocystis jirovecii

Nucleotide variation in the Pneumocystis jirovecii upstream conserved sequence (UCS) and kexin-like serine protease (Kex1) loci was studied in pulmonary specimens from Portuguese HIV-positive patients. DNA was extracted and used for specific molecular sequence analysis. The number of UCS tandem repeats detected in 13 successfully sequenced isolates ranged from three (9 isolates, 69%) to four (4 isolates, 31%). A novel tandem repeat pattern and two novel polymorphisms were detected in the UCS region. For the Kex1 gene, the wild-type (24 isolates, 86%) was the most frequent sequence detected among the 28 sequenced isolates. Nevertheless, a nonsynonymous (1 isolate, 3%) and three synonymous (3 isolates, 11%) polymorphisms were detected and are described here for the first time.

Autonomous cytoplasmic linear plasmid pPac1‐1 of Pichia acaciae: molecular structure and expression studies

The genome organization of the linear DNA-element pPac1-1 from Pichia acaciae was determined. It turned out to be the smallest autonomous cytoplasmic yeast plasmid known so far, consisting of only 12 646 bp, carrying the shortest terminal inverted repeats yet found (138 bp). As for other cytoplasmic linear yeast plasmids, it is characterized by a strikingly high A + T content (75.35%). Ten putative genes (open reading frames, ORFs) reside on the element, leaving only 2.9% of the sequence outside a coding region. Highest similarities of the predicted proteins were obtained for proteins encoded by the three hitherto known autonomous cytoplasmic linear yeast plasmids. Amino acid sequences correspond to predicted polypeptides encoded by ORFs 2-11 of the linear plasmids pGKL2 of Kluyveromyces lactis, pSKL of Saccharomyces kluyveri and pPE1B of Pichia etchellsii. As for the latter, ORF1 existing on the two other plasmids is lacking on pPac1-1. Consistent with cytoplasmic localization, a cytoplasmic promoter termed upstream conserved sequence (UCS) is located in front of each reading frame. RT-PCR transcript analyses for ORFs 8, 9 and 11 proved expression of such genes but functions could not be attributed. The genome organization of pPac1-1 and other autonomous linear elements was found to be almost congruent, irrespective of the accompanying smaller elements, which may or may not encode their own element-specific DNA polymerases.

Expression of the Pneumocystis carinii major surface glycoprotein epitope is correlated with linkage of the cognate gene to the upstream conserved sequence locus.

The major surface glycoprotein (MSG) is a variable surface antigen of the pathogenic fungus Pneumocystis carinii. Many forms of MSG are encoded by a gene family. Expression of the MSG gene family is believed to be controlled in a cis-dependent fashion. Transcription of a given MSG gene is correlated with linkage of that gene to a unique locus called the upstream conserved sequence (UCS). These data predict that the MSG protein on a given organism will match that encoded by the MSG gene at the UCS locus in that organism. To test this hypothesis, a monoclonal antibody (mAb) that recognizes a small number of MSG isoforms was identified, and the DNA sequence encoding the mAb epitope (epitope-encoding sequence, EES) was determined. Western blotting, immunofluorescence and DNA hybridization showed that expression of the mAb epitope was associated with the presence of the EES at the UCS locus. Correlation of epitope expression and UCS linkage supports the hypothesis that expression of a particular MSG on the surface requires UCS linkage of the gene encoding it.

Sequence diversity of transcripts from Pneumocystis carinii gene families MSR and PRT1.

P. carinii has three gene families, MSG, PRT1, and MSR, the members of which are interspersed at the ends of chromosomes. Only one of these families, MSG, has been characterized with respect to transcription. It appears that only one MSG gene is transcribed per organism at any given time because MSG transcripts come from a unique locus called the UCS. This locus encodes the Upstream Conserved Sequence (UCS) present at the beginning of messenger RNA molecules that encode different MSG proteins [4–6]. The UCS in messenger RNA molecules encodes the signal peptide motif needed to direct nascent MSG proteins into the endoplasmic reticulum, the first step in their journey to the cell surface. MSG on the surface lacks the UCS-encoded peptide, suggesting that it is removed by proteolytic cleavage. A possible site for such processing is marked by a conserved lysine-arginine motif, which would presumably be a target for socalled dibasic processing proteases (DPP), which are also known as subtilisin-like proteases. Interestingly, the PRT1 family encodes DPPs. The number of PRT1 genes has not been determined, but most if not all of the members of this gene family are linked to MSG genes, and 80% of characterized telomeric gene clusters contain at least one PRT1 gene. These clusters average 1.5 PRT1 genes each. There are approximately 34 telomeres in the P. carinii genome. Therefore, there would appear to be between 34 and 51 PRT1 genes. Based on antibody studies, at least one PRT1 protein appears to be located on the surface of the microbe [2]. P. carinii telomeric gene clusters also contain members of a third gene family, MSR, the members of which encode peptides that are related to MSG. MSR and MSG genes are distinguished by dependence, or lack thereof, on the UCS locus for expression [3]. MSG genes that are not linked to the UCS locus appear to be silent, but MSR genes have not been found at the UCS, and MSR messenger RNA molecules lack the UCS. Instead of the UCS, MSR messenger RNA molecules begin with sequences that map to MSR genes. These regions of MSR messenger RNA molecules encode a motif that would be expected to direct MSR proteins into the secretory pathway. Therefore, it seems probable that MSR proteins either are secreted or are on the surface of P. carinii. There are probably between 34 and 51 copies of the MSR gene family. Previous studies of RNA made from P. carinii organisms from the lungs of immunosuppressed rats have established that transcripts from multiple MSG, PRT1, and MSR genes are often present in a single animal. While these observations show that multiple genes can be expressed in a population of P. carinii, they do not show that multiple genes are expressed in an individual organism. In the case of MSG, the involvement of the UCS provided a molecular tag that could be used to discern transcriptional regulation even though all of the organism populations analyzed were expressing more than one MSG. In these studies, populations of P. carinii were found to differ with respect to the MSG genes that were present at the UCS locus. Comparisons of UCS-linked genes and MSG messenger RNA molecules in different populations indicated that genes at the UCS are represented by mRNA, while genes not at the UCS are not. This correlation can be explained with a simple model whereby linkage to the UCS locus is necessary and sufficient for MSG gene transcription. Recently, we discovered that the number of MSG genes at the UCS locus could be drastically reduced by restricting the number of organisms that contribute to the original infection [1]. We found that immunosuppression of a P. carinii-colonized rat that has been kept in isolation since weaning and remains in isolation during immunosuppression produced P. carinii populations expressing one MSG in at least 90% of organisms. The discovery of these populations provided an opportunity to detect co-regulation, if it occurs. In the present study, we used a P. carinii population dominated by organisms with one UCS-MSG sequence to address two questions. (1) Do transcripts encoding MSG have the same sequence as the UCS locus? (2) Is transcription of the other two gene families, MSR and PRT, coregulated with MSG?

Characterization of the expression site of the major surface glycoprotein of human-derived Pneumocystis carinii.

The major surface glycoprotein (MSG) of Pneumocystis carinii, a pathogen responsible for pulmonary infection in AIDS and other immunocompromised patients, is an abundant surface protein that potentially allows the organism to evade host defences by antigenic variation. MSG is encoded by a multicopy gene family; in two specific forms of rat-derived P. carinii, regulation of MSG expression uses a single expression site, termed the upstream conserved sequence (UCS), through two related but distinct mechanisms. In the current study, the UCS of the MSG from human-derived P. carinii was obtained using an RNA ligase-mediated rapid amplification of cDNA ends technique. Southern blot analysis demonstrated that the UCS was present in a single copy per genome, whereas multiple copies of the downstream MSG gene were present. Sequencing and restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from pulmonary samples of patients with P. carinii pneumonia demonstrated that multiple MSG genes were expressed in a given host, and that different patterns of MSG expression were seen among different patients. Tandem repeats present in the single intron occurred with varying frequency in different patient isolates, potentially providing a new method for typing human isolates. Thus, human-derived P. carinii regulates MSG expression in a manner similar to P. carinii f. sp. carinii and, in immunosuppressed patients, in whom immune pressures that probably drive antigenic variation are functioning inadequately, P. carinii can express a broad repertoire of MSG variants.

A new family of Pneumocystis carinii genes related to those encoding the major surface glycoprotein.

A new family of Pneumocystis carinii genes (called MSR for MSG-related) that encodes peptides related to the major surface glycoprotein (MSG) is described. Members of the MSR sequence family are linked to MSG genes and are located near the ends of at least 13 chromosomes. Transcripts encoding different MSR isoforms were present in a single population of P. carinii f. sp. carinii, showing that multiple MSR genes were expressed. Two size classes of MSR mRNA, 2.4 and 3.5 kb, were detected. Both sizes of MSR mRNA lacked the upstream conserved sequence (UCS), which is found on the 5' end of MSG mRNAs because MSG genes must be linked to the UCS to be transcribed. The absence of the UCS from MSR mRNAs suggests that expression of MSR genes does not require linkage to the UCS locus.

Identification of a Putative Precursor to the Major Surface Glycoprotein ofPneumocystis carinii

The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii is a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSGs each begin with the same 365-bp sequence, called the Upstream Conserved Sequence (UCS), which is in frame with the contiguous MSG sequence. The UCS contains several potential start sites for translation. To determine if translation of MSG mRNAs begins in the UCS, polyclonal antiserum was raised against the 123-amino-acid peptide encoded by the UCS. The anti-UCS serum reacted with a P. carinii protein that migrated at 170 kDa; however, it did not react with the mature MSG protein, which migrates at 116 kDa. A 170-kDa protein was immunoprecipitated with anti-UCS serum and shown to react with a monoclonal antibody against a conserved MSG epitope. To explore the functional role of the UCS in the trafficking of MSG, the nucleotide sequence encoding the UCS peptide was ligated to the 5' end of an MSG gene and incorporated into a recombinant baculovirus. Insect cells infected with the UCS-MSG hybrid gene expressed a 160-kDa protein which was N-glycosylated. By contrast, insect cells infected with a baculovirus carrying an MSG gene lacking the UCS expressed a nonglycosylated 130-kDa protein. These data suggest that in P. carinii, translation begins in the UCS to produce a pre-MSG protein, which is subsequently directed to the endoplasmic reticulum and processed to the mature form by proteolytic cleavage.

Molecular characterization of mouse Pneumocystis carinii surface glycoprotein A.

Since the mouse offers an easily manipulated experimental animal model for the study of the immunopathogenesis of pneumonia caused by the opportunist Pneumocystis carinii, we cloned and characterized cDNAs encoding an abundant, immunogenic surface antigen termed glycoprotein A (gpA) from mouse P. carinii. A cDNA library was constructed in bacteriophage lambda gt11 from P. carinii-infected mouse lung poly(A+) RNA. Using a nucleic acid probe derived from a conserved region of the mouse P. carinii gpA structural gene, cDNAs encoding gpA were identified. A composite full-length gpA coding sequence was assembled from two overlapping cDNA clones. A DNA element homologous to the rat P. carinii upstream conserved sequence (UCS) was identified at the 5' end of several of the mouse P. carinii gpA cDNA clones, just upstream of the sequences encoding gpA structural gene isoforms. Using primer extension analysis, two neighboring putative transcriptional start sites were located on UCS-gpA mRNAs approximately 25 and 30 nt, respectively, upstream of the most 5' gpA cDNA clone isolated, suggesting a 5' UCS of 489 or 494 nucleotides in mouse P. carinii gpA. A comparative alignment of the composite mouse P. carinii gpA deduced amino acid sequence with gpA homologs from rat, human and ferret P. carinii demonstrated 156 identical residues, including 46 cysteines, further supporting the hypothesis for conserved secondary structure, as well as function, for gpA from all P. carinii.

Residence at the expression site is necessary and sufficient for the transcription of surface antigen genes of Pneumocystis carinii.

The major surface glycoprotein (MSG) of P. carinii f. sp. carinii is a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSG isoforms start with the same sequence, called the upstream conserved sequence (UCS), which is encoded by a single locus. The mechanism by which the UCS becomes part of different MSG mRNAs is not obvious because at least 15 loci, which are distributed throughout the genome, encode MSGs. One possibility is that attachment to the UCS locus is required for the transcription of an MSG gene. The alternative to this expression site model is that mRNAs acquire the UCS by RNA splicing. To distinguish between these two models, UCS/MSG junctions in the genome were compared with UCS/MSG junctions in mRNA. The UCS/MSG junctions in the mRNA matched those in the genome, as would be expected if splicing did not contribute to the attachment of the UCS to the 5' ends of MSG mRNAs. Given that few if any MSG mRNAs lack the UCS, the correspondence between the UCS/MSG junctions in transcripts and those in the genome indicates that attachment to the UCS is both necessary and sufficient for transcription of an MSG gene.