Giovanna Fantoni

Mutations in the Dihydropteroate Synthase Gene of Human-Derived Pneumocystis carinii Isolates from Italy Are Infrequent but Correlate with Prior Sulfa Prophylaxis

Mutations in the human-derived Pneumocystis carinii dihydropteroate synthase (DHPS) gene have been reported with increasing frequency and have been linked to prior sulfa prophylaxis and possible emergence of sulfa resistance. This study was done to examine the prevalence and clinical significance of P. carinii DHPS mutations in Italian patients. A previously described single-strand conformation polymorphism technique was used to identify P. carinii DHPS mutations in 107 patients with acquired immunodeficiency syndrome. Overall prevalence (8%) was low compared with that in other reports. Mutations were observed in 19% (6/31) of patients exposed to sulfa prophylaxis, compared with 4% (3/76) of patients not exposed to sulfa prophylaxis (P=.017). No significant association was observed between the presence of DHPS mutations and mortality, CD4 cell count, or demographic factors. The study confirms the association between DHPS mutations and prior sulfa prophylaxis and shows that the prevalence of DHPS mutations in an Italian patient population is lower than that in other populations.

Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses.

Genotyping of Pneumocystis jiroveci pneumonia in Italian AIDS patients. Clinical outcome is influenced by dihydropteroate synthase and not by internal transcribed spacer genotype.

Background:Two Pneumocystis jiroveci independent genomic regions, internal transcribed spacer (ITS) 1 and ITS2, and dihydropteroate synthase (DHPS) gene have been used for typing a cohort of HIV-infected Italian patients with P. jiroveci pneumonia (PcP). Methods:Bronchoalveolar lavage samples isolated from 207 HIV-infected adults were ITS and DHPS genotyped by DNA sequencing and by restriction fragment length polymorphism analysis, respectively. Mutant DHPS samples were cloned and ITS typed. Data on severity, treatment, and outcome of PcP were obtained by chart review. Results:High diversity with 46 different ITS genotypes was observed. At the DHPS locus, 9.1% of samples analyzed were found to be mutated. A correlation was observed between DHPS mutants and greater severity of PcP, as defined by higher lactate dehydrogenase (P = 0.015) and need for intubation (P = 0.002), and worse outcomes, as defined by failure of sulfa treatment (P = 0.04), death, and/or relapse of PcP (P = 0.008). There was a significant difference in ITS genotype patterns between DHPS wild-type and mutants (P = 0.028). Conclusions:The present data suggest the absence of a correlation between P. jiroveci ITS types and specific clinical characteristics. DHPS mutations correlate with possible failure of anti-P. jiroveci sulfa therapy, and a trend of association is shown between DHPS mutations and some clinical PcP features.

Sequencing and characterization of the complete mitochondrial genomes of three Pneumocystis species provide new insights into divergence between human and rodent Pneumocystis

Pneumocystis jirovecii is an important opportunistic pathogen associated with AIDS and other immunodeficient conditions. Currently, very little is known about its nuclear and mitochondrial genomes. In this study, we sequenced the complete mitochondrial genome (mtDNA) of this organism and its closely related species Pneumocystis carinii and Pneumocystis murina by a combination of sequencing technologies. Our study shows that P. carinii and P. murina mtDNA share a nearly identical number and order of genes in a linear configuration, whereas P. jirovecii has a circular mtDNA containing nearly the same set of genes but in a different order. Detailed studies of the mtDNA terminal structures of P. murina and P. carinii suggest a unique replication mechanism for linear mtDNA. Phylogenetic analysis supports a close association of Pneumocystis species with Taphrina, Saitoella, and Schizosaccharomyces, and divergence within Pneumocystis species, with P. murina and P. carinii being more closely related to each other than either is to P. jirovecii. Comparative analysis of four complete P. jirovecii mtDNA sequences in this study and previously reported mtDNA sequences for diagnosing and genotyping suggests that the current diagnostic and typing methods can be improved using the complete mtDNA data. The availability of the complete P. jirovecii mtDNA also opens the possibility of identifying new therapeutic targets.—Ma, L., Huang, D. W., Cuomo, C. A., Sykes, S., Fantoni, G., Das, B., Sherman, B. T., Yang, J., Huber, C., Xia, Y., Davey, E., Kutty, G., Bishop, L., Sassi, M., Lempicki, R. A., Kovacs, J. A. Sequencing and characterization of the complete mitochondrial genomes of three Pneumocystis species provide new insights into divergence between human and rodent Pneumocystis. FASEB J. 27, 1962–1972 (2013). www.fasebj.org

Restriction fragment length polymorphism typing demonstrates substantial diversity among Pneumocystis jirovecii isolates.

Better understanding of the epidemiology and transmission patterns of human Pneumocystis should lead to improved strategies for preventing Pneumocystis pneumonia (PCP). We have developed a typing method for Pneumocystis jirovecii that is based on restriction fragment length polymorphism (RFLP) analysis after polymerase chain reaction amplification of an approximately 1300 base-pair region of the msg gene family, which comprises an estimated 50-100 genes/genome. The RFLP pattern was reproducible in samples containing >1000 msg copies/reaction and was stable over time, based on analysis of serial samples from the same patient. In our initial analysis of 48 samples, we found that samples obtained from different individuals showed distinct banding patterns; only samples obtained from the same patient showed an identical RFLP pattern. Despite this substantial diversity, samples tended to cluster on the basis of country of origin. In an evaluation of samples obtained from an outbreak of PCP in kidney transplant recipients in Germany, RFLP analysis demonstrated identical patterns in samples that were from 12 patients previously linked to this outbreak, as well as from 2 additional patients. Our results highlight the presence of a remarkable diversity in human Pneumocystis strains. RFLP may be very useful for studying clusters of PCP in immunosuppressed patients, to determine whether there is a common source of infection.

Assessment of Variability in the SOMAscan Assay

SOMAscan is an aptamer-based proteomics assay capable of measuring 1,305 human protein analytes in serum, plasma, and other biological matrices with high sensitivity and specificity. In this work, we present a comprehensive meta-analysis of performance based on multiple serum and plasma runs using the current 1.3 k assay, as well as the previous 1.1 k version. We discuss normalization procedures and examine different strategies to minimize intra- and interplate nuisance effects. We implement a meta-analysis based on calibrator samples to characterize the coefficient of variation and signal-over-background intensity of each protein analyte. By incorporating coefficient of variation estimates into a theoretical model of statistical variability, we also provide a framework to enable rigorous statistical tests of significance in intervention studies and clinical trials, as well as quality control within and across laboratories. Furthermore, we investigate the stability of healthy subject baselines and determine the set of analytes that exhibit biologically stable baselines after technical variability is factored in. This work is accompanied by an interactive web-based tool, an initiative with the potential to become the cornerstone of a regularly updated, high quality repository with data sharing, reproducibility, and reusability as ultimate goals.

Characterization of the meiosis-specific recombinase Dmc1 of pneumocystis.

The life cycle of Pneumocystis, which causes life-threatening pneumonia in immunosuppressed patients, remains poorly defined. In the present study, we have identified and characterized an orthologue of dmc1, a gene specific for meiotic recombination in yeast, in 3 species of Pneumocystis. dmc1 is a single-copy gene that is transcribed as ∼1.2-kb messenger RNA, which encodes a protein of 336-337 amino acids. Pneumocystis Dmc1 was 61%-70% identical to those from yeast. Confocal microscopy results indicated that the expression of Dmc1 is primarily confined to the cyst form of Pneumocystis. By sequence analysis of 2 single-copy regions of the human Pneumocystis jirovecii genome, we can infer multiple recombination events, which are consistent with meiotic recombination in this primarily haploid organism. Taken together, these studies support the occurrence of a sexual phase in the life cycle of Pneumocystis.

DHPS‐Mutated Isolates of Pneumocystis jirovecii from HIV‐Infected Individuals: Analysis of Related ITS Genotypes

PNEUMONIA caused by Pneumocystis jirovecii remains the most common opportunistic pneumonia in AIDS and other immunocompromised patients (Morris et al. 2004). Most drugs used for prevention and treatment of Pneumocystis pneumonia (PcP) target enzymes involved in the biosynthesis of folic acid; sulfa drugs, in particular sulfamethoxazole (SMX), inhibit the dihydropteroate synthase (DHPS). Emergence of sulfa drug-resistant P. jirovecii has been suggested to be due to exposure to sulfa drugs and leading to DHPS mutations. Two point mutations corresponding to amino acid positions 55 and 57 of the P. jirovecii DHPS protein are the genetic markers for possible sulphonamide resistance that have been reported with increasing frequency (Kazanjian et al. 2004, 2000; Lane et al. 1997; Ma et al. 1999). In this study, the DHPS and the internal transcribed spacers (ITS) loci of P. jirovecii isolates from HIV-infected patients were typed to investigate the possible association between specific ITS and DHPS types and what that might mean in the epidemiology of PcP. MATERIALS AND METHODS

Pneumocystis carinii ITS Typing: Doubtful Evidence of Genotype-Related Virulence

Pnewnocystis carinii is an oppurtunistic pulmonary pathogen particularly associated with HIV-infected patients. Controversial results have been reparted on the possible occurrence of genotyperelated virulence [l-21 based on Internal Transcribed Spacers (ITS) typing. Wide ITS variability has been identified. and the locus is described as a single copy gene, thus allowing accurate description of genotypes during P. carinii pneumonia (PCP) episodes [3, 41. Based on paired cliical and molecular analysis of lTS sequences, we examined if there is a correlation between these two parameters among P. carinii isolated fkom patients in Italy.

Web Tool for Navigating and Plotting SomaLogic ADAT Files

SOMAscan™ is a complex proteomic platform created by SomaLogic. Experimental data resulting from the assay is provided by SomaLogic in a proprietary text-based format called ADAT. This manuscript describes a user-friendly point and click open source, platform-independent software tool designed to be used for navigating and plotting data from an ADAT file. This tool was used either alone or in conjunction with other tools as a first pass analysis of the data on several different on-going research projects. We have seen a need from our experience for a web interface to the ADAT file so that users can navigate, generate plots, perform QC and conduct statistical analysis on their own data in a point and click manner. After several rounds of interacting with biologists and their requirements with respect to data analysis, we present an online interactive Shiny Web Tool for Navigating and Plotting data contained within the ADAT file. Extensive video tutorials, example data, the tool and the source code are available online.