Geetha Kutty

Molecular Characterization and Developmental Expression of a Retinoid- and Fatty Acid-binding Glycoprotein from Drosophila A PUTATIVE LIPOPHORIN

A detailed understanding of the mechanism of lipid transport in insects has been hampered by the inability to identify the proapolipophorin gene that encodes apolipophorins I and II, the principal protein components of lipophorin, the lipid transport vehicle. Here we provide the first molecular description of the Drosophila gene encoding a retinoid- and fatty acid-binding glycoprotein (RFABG) and present evidence that it is a member of the proapolipophorin gene family. The gene, localized to the chromosome 4 (102 F region), encodes a 3351-amino acid protein that could serve as the precursor for the ∼70-kDa and >200-kDa polypeptides associated with RFABG. The N-terminal sequence of the ∼70-kDa polypeptide and that predicted for the >200-kDa polypeptide showed high sequence similarity to blowfly apolipophorin II and apolipophorin I, respectively. The RFABG precursor contains a signal peptide and exhibits a multidomain mosaic protein structure, which is typical of extracellular proteins. It has structural domains similar to lipid-binding proteins, namely vitellogenins and apolipoprotein B. The protein also contains a domain similar to the D domain of von Willebrand factor and mucin. The gene is expressed in the Drosophila embryo during development in cells that make up the amnioserosa and fat bodies. Immunolocalizations using specific antibodies against RFABG reveal that the protein is initially dispersed through the embryonic amnioserosa sac and latter concentrated at skeletal muscle-epidermis apodemeal contact junctions during larval development. This novel gene may play an important role in the transport of lipids, including retinoids and fatty acids, in insects.

Outbreaks of Pneumocystis Pneumonia in 2 Renal Transplant Centers Linked to a Single Strain of Pneumocystis: Implications for Transmission and Virulence

BACKGROUND There have been numerous reports of clustered outbreaks of Pneumocystis pneumonia (PCP) at renal transplant centers over the past 2 decades. It has been unclear whether these outbreaks were linked epidemiologically to 1 or several unique strains, which could have implications for transmission patterns or strain virulence. METHODS Restriction fragment length polymorphism (RFLP) analysis was used to compare Pneumocystis isolates from 3 outbreaks of PCP in renal transplant patients in Germany, Switzerland, and Japan, as well as nontransplant isolates from both human immunodeficiency virus (HIV)-infected and uninfected patients. RESULTS Based on RFLP analysis, a single Pneumocystis strain caused pneumonia in transplant patients in Switzerland (7 patients) and Germany (14 patients). This strain was different from the strain that caused an outbreak in transplant patients in Japan, as well as strains causing sporadic cases of PCP in nontransplant patients with or without HIV infection. CONCLUSIONS Two geographically distinct clusters of PCP in Europe were due to a single strain of Pneumocystis. This suggests either enhanced virulence of this strain in transplant patients or a common, but unidentified, source of transmission. Outbreaks of PCP can be better understood by enhanced knowledge of transmission patterns and strain variation.

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.

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 Single-Copy Gene Encodes Kex1, a Serine Endoprotease of Pneumocystis jiroveci

ABSTRACT We have cloned and characterized the kex1 gene of Pneumocystis jiroveci. Unlike the case for Pneumocystis carinii, in which the homologous PRT-1 genes are multicopy, kex1 is a single-copy gene encoding a protein homologous to fungal serine endoproteases, which localize to the Golgi apparatus. Thus, substantial biological differences can be seen among Pneumocystis species.

Analysis of Variation in Tandem Repeats in the Intron of the Major Surface Glycoprotein Expression Site of the Human Form of Pneumocystis carinii

Variation in the tandem repeats in the expression site of the human-derived Pneumocystis carinii major surface glycoprotein gene was characterized by denaturing gel electrophoresis. The number of repeats in 147 isolates ranged from 2 to 6, with 2, 3, and 4 repeats being the most common. Sequence analysis identified 3 types of repeat units that differed by 1 nucleotide, which suggests a hierarchy of evolution of human-derived P. carinii. Examination of sequential samples obtained from 6 patients at an interval of 10-90 days showed an identical repeat pattern in each patient. However, in 2 of 4 patients with 2-3 different samples obtained within 4 days, different repeat patterns were observed among the samples. Quantifying the number of repeats by denaturing gel electrophoresis is a simple and rapid-typing method that can be used alone or in combination with other methods to study the epidemiology of P. carinii.

Outbreak of Pneumocystis Pneumonia in Renal and Liver Transplant Patients Caused by Genotypically Distinct Strains of Pneumocystis jirovecii

Background An outbreak of 29 cases of Pneumocystis jirovecii pneumonia (PCP) occurred among renal and liver transplant recipients (RTR and LTR) in the largest Danish transplantation centre between 2007 and 2010, when routine PCP prophylaxis was not used. Methods P. jirovecii isolates from 22 transplant cases, 2 colonized RTRs, and 19 Pneumocystis control samples were genotyped by restriction fragment length polymorphism and multilocus sequence typing analysis. Contact tracing was used to investigate transmission. Potential risk factors were compared between PCP cases and matched non-PCP transplant patients. Results Three unique Pneumocystis genotypes were shared among 19 of the RTRs, LTRs, and a colonized RTR in three distinct clusters, two of which overlapped temporally. In contrast, Pneumocystis control samples harbored a wide range of genotypes. Evidence of possible nosocomial transmission was observed. Among several potential risk factors, only cytomegalovirus viremia was consistently associated with PCP (P=0.03; P=0.009). Mycophenolate mofetil was associated with PCP risk only in the RTR population (P=0.04). Conclusion We identified three large groups infected with unique strains of Pneumocystis and provide evidence of an outbreak profile and nosocomial transmission. LTRs may be infected in PCP outbreaks simultaneously with RTRs and by the same strains, most likely by interhuman transmission. Patients are at risk several years after transplantation, but the risk is highest during the first 6 months after transplantation. Because patients at risk cannot be identified clinically and outbreaks cannot be predicted, 6 months of PCP chemoprophylaxis should be considered for all RTRs and LTRs.

Variation in the Major Surface Glycoprotein Genes in Pneumocystis jirovecii

The genome of Pneumocystis, which causes life-threatening pneumonia in immunosuppressed patients, contains a multicopy gene family that encodes the major surface glycoprotein (Msg). Pneumocystis can vary the expressed Msg, presumably as a mechanism to avoid host immune responses. Analysis of 24 msg-gene sequences obtained from a single human isolate of Pneumocystis demonstrated that the sequences segregate into 2 branches. Results of a number of analyses suggest that recombination between msg genes is an important mechanism for generating msg diversity. Intrabranch recombination occurred more frequently than interbranch recombination. Restriction-fragment length polymorphism analysis of human isolates of Pneumocystis demonstrated substantial variation in the repertoire of the msg-gene family, variation that was not observed in laboratory isolates of Pneumocystis in rats or mice; this may be the result of examining outbred versus captive populations. Increased diversity in the Msg repertoire, generated in part by recombination, increases the potential for antigenic variation in this abundant surface protein.

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.