Lisa R. Bishop

Quantitation of Anti–Pneumocystis jiroveci Antibodies in Healthy Persons and Immunocompromised Patients

To facilitate studies of the epidemiology of Pneumocystis jiroveci infection in both healthy persons and immunocompromised patients, we developed a quantitative ELISA with recombinant major surface glycoprotein (MSG) fragment MSG-14, a P. jiroveci-specific protein that includes a highly conserved region of the MSG protein family. By immunoblot, all samples reacted with the carboxyl portion of MSG-14; by ELISA, immunocompromised patients with Pneumocystis pneumonia (PCP) who were immunocompromised for reasons other than AIDS had higher antibody levels than did either patients with AIDS with PCP (P=.01) or healthy persons (P=.005). Longitudinal observations of 8 patients with AIDS showed no correlation between time of diagnosis of Pneumocystis infection and change in antibody levels. Eleven percent (4/35) of healthy persons demonstrated a >4-fold change in antibody titers during 1 year of observation. This ELISA assay allows quantitation of anti-P. jiroveci antibodies in human serum samples and should be useful in better understanding the epidemiology of P. jiroveci infection in humans.

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.

Quantitative Real-Time Polymerase Chain-Reaction Assay Allows Characterization of Pneumocystis Infection in Immunocompetent Mice

Pneumocystis causes pneumonia in immunodeficient hosts but also likely causes infection in healthy hosts. To characterize infection in healthy mice, we developed and validated a real-time polymerase chain reaction assay for quantitation of Pneumocystis carinii f. sp. muris. In healthy mice exposed to Pneumocystis-infected animals, organisms were first detected at 2-3 weeks, peaked at 5-6 weeks, and were cleared by 7-9 weeks. The peak organism load in healthy animals was 2-3 logs lower than that in immunodeficient animals. This approach should facilitate studies of anti-Pneumocystis immune mechanisms in healthy hosts and provide insights into the development of Pneumocystis pneumonia in immunodeficient hosts.

Four-Antigen Mixture Containing V-Cyclin for Serological Screening of Human Herpesvirus 8 Infection

ABSTRACT Improved diagnostic reagents and testing are currently needed for the serological detection of human herpesvirus 8 (HHV-8) infections. We evaluated the luciferase immunoprecipitation systems (LIPS) for profiling antibody responses to a panel of HHV-8 proteins for diagnosis of Kaposi sarcoma (KS)-infected individuals. Using a pilot serum set, LIPS detected robust antibody responses to several known antigens, and a screen of 14 additional HHV-8 proteins identified v-cyclin as a potentially new diagnostic antigen. In evaluating a training-serum set, a four-antigen panel (K8.1, v-cyclin, ORF65, and a LANA fragment) was found to provide sufficient information for diagnosis. Analysis of a validation serum set using the combined results from these four separate antigen tests showed 100% sensitivity and 100% specificity. Furthermore, a LIPS format using a mixture of the four antigens, which simplifies data collection and analysis, closely matched the diagnostic performance of the combined separate tests (R = 0.95). This four-antigen mixture format analyzed with the validation serum set also showed 100% sensitivity and 100% specificity but was not statistically different from two separate enzyme-linked immunosorbent assays (94% sensitivity and 100% specificity) using baculovirus-produced LANA and bacterially produced K8.1. Heat map analysis of KS patient antibody titers revealed marked heterogeneity in humoral responses to this four-antigen panel. Overall, the LIPS assay showed 97% sensitivity, and positive anti-v-cyclin antibodies were detected in approximately 75% of the KS sera. These results suggest that LIPS screening using an antigen mixture is a sensitive and high-throughput method for serological screening of HHV-8 infection in individuals with KS.

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

Immune responses to Pneumocystis murina are robust in healthy mice but largely absent in CD40 ligand-deficient mice

Pneumocystis is a pathogen of immunocompromised hosts but can also infect healthy hosts, in whom infection is rapidly controlled and cleared. Microarray methods were used to examine differential gene expression in the lungs of C57BL/6 and CD40 ligand knockout (CD40L‐KO) mice over time following exposure to Pneumocystis murina. Immunocompetent C57BL/6 mice, which control and clear infection efficiently, showed a robust response to infection characterized by the up‐regulation of 349 primarily immune response‐associated genes. Temporal changes in the expression of these genes identified an early (Week 2), primarily innate response, which waned before the infection was controlled; this was followed by primarily adaptive immune responses that peaked at Week 5, which coincided with clearance of the infection. In conjunction with the latter, there was an increased expression of B cell‐associated (Ig) genes at Week 6 that persisted through 11 weeks. In contrast, CD40L‐KO mice, which are highly susceptible to developing severe Pneumocystis pneumonia, showed essentially no up‐regulation of immune response‐associated genes at Days 35–75. Immunohistochemical staining supported these observations by demonstrating an increase in CD4+, CD68+, and CD19+ cells in C57BL/6 but not CD40L‐KO mice. Thus, the healthy host demonstrates a robust, biphasic response to infection by Pneumocystis; CD40L is an essential upstream regulator of the adaptive immune responses that efficiently control infection and prevent development of progressive pneumonia.

Discordant antibody and cellular responses to Pneumocystis major surface glycoprotein variants in mice

BackgroundThe major surface glycoprotein (Msg) of Pneumocystis is encoded by approximately 50 to 80 unique but related genes. Msg diversity may represent a mechanism for immune escape from host T cell responses. We examined splenic T cell proliferative and cytokine as well as serum antibody responses to recombinant and native Pneumocystis antigens in immunized or Pneumocystis-infected mice. In addition, immune responses were examined in 5 healthy humans.ResultsProliferative responses to each of two recombinant Msg variant proteins were seen in mice immunized with either recombinant protein, but no proliferation to these antigens was seen in mice immunized with crude Pneumocystis antigens or in mice that had cleared infection, although the latter animals demonstrated proliferative responses to crude Pneumocystis antigens and native Msg. IL-17 and MCP-3 were produced in previously infected animals in response to the same antigens, but not to recombinant antigens. Antibody responses to the recombinant P. murina Msg variant proteins were seen in all groups of animals, demonstrating that all groups were exposed to and mounted immune responses to Msg. No human PBMC samples proliferated following stimulation with P. jirovecii Msg, while antibody responses were detected in sera from 4 of 5 samples.ConclusionsCross-reactive antibody responses to Msg variants are common, while cross-reactive T cell responses are uncommon; these results support the hypothesis that Pneumocystis utilizes switching of Msg variant expression to avoid host T cell responses.

β-glucans are Masked but Contribute to Pulmonary Inflammation During Pneumocystis Pneumonia

β-glucans, which can activate innate immune responses, are a major component in the cell wall of the cyst form of Pneumocystis In the current study, we examined whether β-1,3-glucans are masked by surface proteins in Pneumocystis and what role β-glucans play in Pneumocystis-associated inflammation. For 3 species, including Pneumocystis jirovecii, which causes Pneumocystis pneumonia in humans, Pneumocystis carinii, and Pneumocystis murina, β-1,3-glucans were masked in most organisms, as demonstrated by increased exposure following trypsin treatment. Using quantitative polymerase chain reaction and microarray techniques, we demonstrated in a mouse model of Pneumocystis pneumonia that treatment with caspofungin, an inhibitor of β-1,3-glucan synthesis, for 21 days decreased expression of a broad panel of inflammatory markers, including interferon γ, tumor necrosis factor α, interleukin 1β, interleukin 6, and multiple chemokines/chemokine ligands. Thus, β-glucans in Pneumocystis cysts are largely masked, which likely decreases innate immune activation; this mechanism presumably was developed for interactions with immunocompetent hosts, in whom organism loads are substantially lower. In immunosuppressed hosts with a high organism burden, organism death and release of glucans appears to be an important contributor to deleterious host inflammatory responses.

Outcomes of four patients with homocysteine remethylation disorders detected by newborn screening

Purpose:We evaluated the clinical outcome in homocysteine remethylation disorders following newborn screening (NBS) and initiation of early specific treatment.Methods:Five patients with remethylation disorders were included in this study.Results:Two asymptomatic patients (one with cblG and one with cblE) were identified by NBS using an approach that combines a postanalytical interpretive tool (available on the Region 4 Stork (R4S) collaborative project website, http://www.clir-r4s.org) and a second-tier test for total homocysteine determination. Both the initial screening and the second-tier test are performed on the same blood spot, with no additional patient contact, resulting in no false-positive outcomes. Two additional patients with methylenetetrahydrofolate reductase deficiency were detected by NBS using low methionine as a marker. Although already symptomatic despite the early diagnosis, the latter two patients greatly improved with treatment and their outcomes are compared with that of another patient with methylenetetrahydrofolate reductase deficiency and significant morbidity who was diagnosed clinically at 3 months of age.Conclusion:Early detection by NBS and timely and specific treatment considerably improve at least short-term outcomes of homocysteine remethylation disorders. When a remethylation disorder is suspected, group-specific treatment could be started prior to the completion of in vitro confirmatory testing because all disorders from this group require similar intervention.Genet Med 18 2, 162–167.

Galleria mellonella are Resistant to Pneumocystis murina Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleriamellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.