Expression Pattern of the Pneumocystis jirovecii Major Surface Glycoprotein Superfamily in Patients with Pneumonia.

BACKGROUND: The human pathogen Pneumocystis jirovecii harbors 6 families of major surface glycoproteins (MSGs) encoded by a single gene superfamily. MSGs are presumably responsible for antigenic variation and adhesion to host cells. The genomic organization suggests that a single member of family I is expressed at a given time per cell, whereas members of the other families are simultaneously expressed. METHODS: We analyzed RNA sequences expressed in several clinical samples, using specific weighted profiles for sorting of reads and calling of single-nucleotide variants to estimate the diversity of the expressed genes. RESULTS: A number of different isoforms of at least 4 MSG families were expressed simultaneously, including isoforms of family I, for which confirmation was obtained in the wet laboratory. CONCLUSION: These observations suggest that every single P. jirovecii population is made of individual cells with distinct surface properties. Our results enhance our understanding of the unique antigenic variation system and cell surface structure of P. jirovecii.

A critical role for CARD9 in pneumocystis pneumonia host defence.

Caspase recruitment domains-containing protein 9 (CARD9) is an adaptor molecule critical for key signalling pathways initiated through C-type lectin receptors (CLRs). Previous studies demonstrated that Pneumocystis organisms are recognised through a variety of CLRs. However, the role of the downstream CARD9 adaptor signalling protein in host defence against Pneumocystis infection remains to be elucidated. Herein, we analysed the role of CARD9 in host defence against Pneumocystis both in CD4-depleted CARD9-/- and immunocompetent hosts. Card9 gene-disrupted (CARD9-/- ) mice were more susceptible to Pneumocystis, as evidenced by reduced fungal clearance in infected lungs compared to wild-type (WT) infected mice. Our data suggests that this defect was due to impaired proinflammatory responses. Furthermore, CARD9-/- macrophages were severely compromised in their ability to differentiate and express M1 and M2 macrophage polarisation markers, to enhanced mRNA expression for Dectin-1 and Mincle, and most importantly, to kill Pneumocystis in vitro. Remarkably, compared to WT mice, and despite markedly increased organism burdens, CARD9-/- animals did not exhibit worsened survival during pneumocystis pneumonia (PCP), perhaps related to decreased lung injury due to altered influx of inflammatory cells and decreased levels of proinflammatory cytokines in response to the organism. Finally, although innate phase cytokines were impaired in the CARD9-/- animals during PCP, T-helper cell cytokines were normal in immunocompetent CARD9-/- animals infected with Pneumocystis. Taken together, our data demonstrate that CARD9 has a critical function in innate immune responses against Pneumocystis.

Pneumocystis carinii Major Surface Glycoprotein Dampens Macrophage Inflammatory Responses to Fungal ß-Glucan.

BACKGROUND: Pneumocystis major surface glycoprotein (Msg) is a 120-kD surface protein complex on the organism with importance in adhesion and immune recognition. In this study, we show that Msg significantly impairs tumor necrosis factor (TNF)-α secretion by macrophages induced by Saccharomyces cerevisiae and Pneumocystis carinii (Pc) ß-glucans. METHODS: Major surface glycoprotein was shown to greatly reduce ß-glucan-induced Dectin-1 immunoreceptor tyrosine-based activating motif (ITAM) phosphorylation. Major surface glycoprotein also down regulated Dectin-1 receptor messenger ribonucleic acid (mRNA) expression in the macrophages. It is interesting that Msg incubation with macrophages resulted in significant mRNA upregulation of both C-type lectin receptors (CLR) Mincle and MCL in Msg protein presence alone but to even greater amounts in the presence of Pc ß-glucan. RESULTS: The silencing of MCL and Mincle resulted in TNF-α secretions similar to that of macrophages treated with Pneumocystis ß-glucan alone, which is suggestive of an inhibitory role for these 2 CLRs in Msg-suppressive effects on host cell immune response. CONCLUSIONS: Taken together, these data indicate that the Pneumocystis Msg surface protein complex can act to suppress host macrophage inflammatory responses to the proinflammatory ß -glucan components of the organisms.

Detection of anti-Pneumocystis jirovecii antibodies in human serum using a recombinant synthetic multi-epitope kexin-based antigen.

Interest in the detection of specific anti-Pneumocystis jirovecii antibodies has emerged as less-invasive alternative diagnostic approaches. Here is presented the performance of an ELISA based on a recombinant synthetic multi-epitope kexin 1 (Kex1) antigen of P. jirovecii, previously developed. Results showed that IgM anti-Kex1 levels were found significantly increased in patients with Pneumocystis pneumonia (PcP) compared with non-PcP cases (p < 0.001), allowing a diagnostic performance of PcP with a 70.8% sensitivity and a 75.0% specificity. These results suggest that this Kex1-based ELISA is a promising tool toward the serodiagnosis of PcP when the standard methods are difficult to perform.

C-Type Lectin Receptors in Antifungal Immunity.

Most fungal species are harmless to humans and some exist as commensals on mucocutaneous surfaces. Yet many fungi are opportunistic pathogens, causing life-threatening invasive infections when the immune system becomes compromised. The fungal cell wall contains conserved pathogen-associated molecular patterns (PAMPs), which allow the immune system to distinguish between self (endogenous molecular patterns) and foreign material. Sensing of invasive microbial pathogens is achieved through recognition of PAMPs by pattern recognition receptors (PRRs). One of the predominant fungal-sensing PRRs is the C-type lectin receptor (CLR) family. These receptors bind to structures present on the fungal cell wall, eliciting various innate immune responses as well as shaping adaptive immunity. In this chapter, we specifically focus on the four major human fungal pathogens, Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and Pneumocystis jirovecii, reviewing our current understanding of the CLRs that are involved in their recognition and protection of the host.

The Interaction of Pneumocystis with the C-Type Lectin Receptor Mincle Exerts a Significant Role in Host Defense against Infection.

Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality within immunocompromised patients. In this study, we examined the potential role of macrophage-inducible C-type lectin (Mincle) for host defense against Pneumocystis Binding assays implementing soluble Mincle carbohydrate recognition domain fusion proteins demonstrated binding to intact Pneumocystis carinii as well as to organism homogenates, and they purified major surface glycoprotein/glycoprotein A derived from the organism. Additional experiments showed that rats with PCP expressed increased Mincle mRNA levels. Mouse macrophages overexpressing Mincle displayed increased binding to P. carinii life forms and enhanced protein tyrosine phosphorylation. The binding of P. carinii to Mincle resulted in activation of FcRγ-mediated cell signaling. RNA silencing of Mincle in mouse macrophages resulted in decreased activation of Syk kinase after P. carinii challenge, critical in downstream inflammatory signaling. Mincle-deficient CD4-depleted (Mincle-/-) mice showed a significant defect in organism clearance from the lungs with higher organism burdens and altered lung cytokine responses during Pneumocystis murina pneumonia. Interestingly, Mincle-/- mice did not demonstrate worsened survival during PCP compared with wild-type mice, despite the markedly increased organism burdens. This may be related to increased expression of anti-inflammatory factors such as IL-1Ra during infection in the Mincle-/- mice. Of note, the P. murina-infected Mincle-/- mice demonstrated increased expression of known C-type lectin receptors Dectin-1, Dectin-2, and MCL compared with infected wild-type mice. Taken together, these data support a significant role for Mincle in Pneumocystis modulating host defense during infection.

Sulfonamide desensitization in solid organ transplant recipients: A protocol-driven approach during the index transplant hospitalization.

Trimethoprim-sulfamethoxazole (TMP-SMX) is the first-line agent for Pneumocystis jiroveci pneumonia (PJP) prophylaxis for solid organ transplant (SOT) recipients because of its efficacy for this indication, extended antimicrobial coverage, and favorable cost. Reported sulfonamide allergy is not uncommon and often results in TMP-SMX avoidance. Desensitization offers an efficacious and cost-effective alternative to TMP-SMX avoidance. Herein, we reviewed our experience with desensitization during the index transplant hospitalization among 52 SOT recipients with history of a non-anaphylactic sulfonamide allergy. Of those enrolled in the desensitization protocol, 92% (48/52) completed the protocol, with nearly 80% (41/52) still on TMP-SMX at 3 months without adverse reaction. Eleven patients discontinued TMP-SMX (7 for allergic reactions and 4 for non-allergic reasons) and switched to pentamidine. A cost savings of $575 per desensitization was calculated based on annual wholesale drug prices, for a total savings of $23 575. Additionally, the protocol did not delay discharge in any patient nor was it associated with any severe allergic reactions. These findings suggest TMP-SMX desensitization is safe and effective in SOT recipients with a history of non-anaphylactic, non-life-threatening sulfonamide hypersensitivity.

Dectin-2 Is a C-Type Lectin Receptor that Recognizes Pneumocystis and Participates in Innate Immune Responses.

Pneumocystis is an important fungal pathogen that causes life-threatening pneumonia in patients with AIDS and malignancy. Lung fungal pathogens are recognized by C-type lectin receptors (CLRs), which bind specific ligands and stimulate innate immune responses. The CLR Dectin-1 was previously shown to mediate immune responses to Pneumocystis spp. For this reason, we investigated a potential role for Dectin-2. Rats with Pneumocystis pneumonia (PCP) exhibited elevated Dectin-2 mRNA levels. Soluble Dectin-2 carbohydrate-recognition domain fusion protein showed binding to intact Pneumocystis carinii (Pc) and to native Pneumocystis major surface glycoprotein/glycoprotein A (Msg/gpA). RAW macrophage cells expressing V5-tagged Dectin-2 displayed enhanced binding to Pc and increased protein tyrosine phosphorylation. Furthermore, the binding of Pc to Dectin-2 resulted in Fc receptor-γ-mediated intracellular signaling. Alveolar macrophages from Dectin-2-deficient mice (Dectin-2-/-) showed significant decreases in phospho-Syk activation after challenge with Pc cell wall components. Stimulation of Dectin-2-/- alveolar macrophages with Pc components showed significant decreases in the proinflammatory cytokines IL-6 and TNF-α. Finally, during infection with Pneumocystis murina, Dectin-2-/- mice displayed downregulated mRNA expression profiles of other CLRs implicated in fungal immunity. Although Dectin-2-/- alveolar macrophages had reduced proinflammatory cytokine release in vitro, Dectin-2-/- deficiency did not reduce the overall resistance of these mice in the PCP model, and organism burdens were statistically similar in the long-term immunocompromised and short-term immunocompetent PCP models. These results suggest that Dectin-2 participates in the initial innate immune signaling response to Pneumocystis, but its deficiency does not impair resistance to the organism.

Comparative Analysis of the Wako ß-Glucan Test and the Fungitell Assay for Diagnosis of Candidemia and Pneumocystis jirovecii Pneumonia.

(1→3)-ß-d-Glucan (BDG) is a biomarker for invasive fungal disease. Until now, all BDG data in the Western Hemisphere were obtained using the Fungitell assay (FA). How it compares to the Wako ß-glucan test (GT), which was recently launched in Europe, is largely unknown. We conducted a case-control study to compare the two assays in serum samples from 120 candidemia and 63 Pneumocystis jirovecii pneumonia (PCP) patients. Two hundred patients with bacteremia or negative blood cultures served as candidemia control group. In patients with candidemia the median BDG values of the FA and the GT were 351 and 8.4 pg/ml, respectively. With both assays, the BDG levels in candidemia were significantly higher than those measured in the control group (P < 0.001). The sensitivity, specificity, and positive and negative predictive values for the diagnosis of candidemia were 86.7%, 85.0%, 6.0%, and 99.8% for the FA and 42.5%, 98.0%, 19.0%, and 99.4% for the GT, respectively. In PCP patients the median BDG values of the FA and the GT were 963 and 57.7 pg/ml, respectively. The sensitivities for PCP diagnosis were 100% for the FA and 88.9% for the GT. In practical terms, the GT proved to be robust and applicable for testing single samples, whereas for economic reasons the FA required the samples to be tested in batch. The sensitivity of the FA is superior to that of the GT. However, the GT is a valuable alternative to the FA, especially for patients with suspected PCP and in laboratories with low sample throughput.

Prophylaxis and treatment of Pneumocystis Jirovecii pneumonia after solid organ transplantation.

Pneumocystis jirovecii pneumonia (PJP) is an opportunistic infection diagnosed in immunocompromized patients. After solid organ transplantation, early infection has decreased as a result of effective prophylaxis, but late infections and even outbreaks caused by interpatient transmission of pneumocystis by air are present in the SOT community. Different risk factors for PJP have been described and several indications for PJP prophylaxis have to be considered by clinicians in patients even years after transplantation. Diagnosis of PJP is confirmed by microscopy and immunofluorescence staining of bronchial fluid but PCR as well as serum ß-D-Glucan analysis have become increasingly valuable diagnostic tools. Treatment of choice is Trimethoprim/sulfamethoxazole and early treatment improves prognosis. However, mortality of PJP in solid organ transplant patients is still high and many aspects including the optimal management of immunosuppression during PJP treatment require further investigations.

Lymphopenia is associated with late onset Pneumocystis jirovecii pneumonia in solid organ transplantation.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) affected 5%-15% of solid organ transplant (SOT) recipients prior to universal prophylaxis, classically with trimethoprim-sulfamethoxazole (TMP-SMX). Guidelines generally recommend 6-12 months of prophylaxis post-SOT, yet optimal duration and robust PJP risk stratification have not been established. METHODS: A retrospective, single-center, case-control study of PJP among SOT recipients from January 1998 to December 2013 was conducted. Cases had positive PJ direct fluorescent antibody assay of respiratory specimens. Controls were matched 4:1 by nearest date of SOT. Univariate testing and multivariate logistic regressions were performed. RESULTS: Fifteen cases were identified among 5505 SOT recipients (0.27% rate) and analyzed vs 60 controls. PJP occurred on average 6.1 years (range 0.9-13.8) post-SOT; no case was receiving PJP prophylaxis at diagnosis. Most were treated with reduced immunosuppression and TMP-SMX plus steroids (80%). Six patients (40%) required critical care; 3 (20%) died. There were no significant demographic differences, though cases tended to be older at SOT (54 vs 48 years, P = .1). In univariate analysis, prior viral infection was more common among cases (67% vs 37%, P = .08). Lower absolute lymphocyte count (ALC) at diagnosis date was strongly associated with PJP (400 vs 1230 × 106  cells/µL, P < .001); odds of infection were high with ALC ≤ 500 × 106 cells (OR 18.7, P < .01). CONCLUSION: Pneumocystis jirovecii pneumonia is a rare, late complication of SOT with significant morbidity and mortality. Severe lymphopenia may be useful in identifying SOT recipients who warrant continued or reinstated PJP prophylaxis.

Overcoming Hurdles to Development of a Vaccine against Pneumocystis jirovecii.

Development of Pneumocystis pneumonia (PCP) is a common problem among immunosuppressed individuals. There are windows of opportunity in which vaccination would be beneficial, but to date, no vaccines have made it to clinical trials. Significant hurdles to vaccine development include host range specificity, making it difficult to translate from animal models to humans. Discovery of cross-reactive epitopes is critical to moving vaccine candidates from preclinical animal studies to clinical trials.

Immunization with Pneumocystis Cross-Reactive Antigen 1 (Pca1) Protects Mice against Pneumocystis Pneumonia and Generates Antibody to Pneumocystis jirovecii.

Pneumocystis pneumonia (PcP) is a life-threatening infection that affects immunocompromised individuals. Nearly half of all PcP cases occur in those prescribed effective chemoprophylaxis, suggesting that additional preventive methods are needed. To this end, we have identified a unique mouse Pneumocystis surface protein, designated Pneumocystis cross-reactive antigen 1 (Pca1), as a potential vaccine candidate. Mice were immunized with a recombinant fusion protein containing Pca1. Subsequently, CD4+ T cells were depleted, and the mice were exposed to Pneumocystis murina Pca1 immunization completely protected nearly all mice, similar to immunization with whole Pneumocystis organisms. In contrast, all immunized negative-control mice developed PcP. Unexpectedly, Pca1 immunization generated cross-reactive antibody that recognized Pneumocystis jirovecii and Pneumocystis carinii Potential orthologs of Pca1 have been identified in P. jirovecii Such cross-reactivity is rare, and our findings suggest that Pca1 is a conserved antigen and potential vaccine target. The evaluation of Pca1-elicited antibodies in the prevention of PcP in humans deserves further investigation.

Vaccine-Induced Immunogenicity and Protection Against Pneumocystis Pneumonia in a Nonhuman Primate Model of HIV and Pneumocystis Coinfection.

BACKGROUND: The ubiquitous opportunistic pathogen Pneumocystis jirovecii causes pneumonia in immunocompromised individuals, including human immunodeficiency virus (HIV)-infected individuals, and pulmonary colonization with P. jirovecii is believed to be a cofactor in the development of chronic obstructive pulmonary disease. There is no vaccine for P. jirovecii; however, most adults are seropositive, indicating natural immune priming to this pathogen. We have shown that humoral response to a recombinant subunit of the P. jirovecii protease kexin (KEX1) correlates with protection from P. jirovecii colonization and pneumonia. METHODS: Here we evaluated the immunogenicity and protective capacity of the recombinant KEX1 peptide vaccine in a preclinical, nonhuman primate model of HIV-induced immunosuppression and Pneumocystis coinfection. RESULTS: Immunization with KEX1 induced a robust humoral response remained at protective levels despite chronic simian immunodeficiency virus/HIV-induced immunosuppression. KEX1-immunized macaques were protected from Pneumocystis pneumonia, compared with mock-immunized animals (P= .047), following immunosuppression and subsequent natural, airborne exposure to Pneumocystis CONCLUSIONS: These data support the concept that stimulation of preexisting immunological memory to Pneumocystis with a recombinant KEX1 vaccine prior to immunosuppression induces durable memory responses and protection in the context of chronic, complex immunosuppression.

Synthetic p55 tandem DNA vaccine against Pneumocystis carinii in rats.

Pneumocystis spp. are opportunistic fungal pathogens that are closely associated with severe pneumonia and pulmonary complications in patients with impaired immunity. In this study, the antigenic epitopes of the gene encoding the 55 kDa antigen fragment of Pneumocystis (p55), which may play an important role in Pneumocystis pneumonia, were analyzed. A gene containing tandem variants of the p55 antigen was synthesized and named the tandem antigen gene (TAG). TAG's potential as a DNA vaccine was assessed in immunosuppressed rats. Immunization with p55-TAG DNA vaccine significantly reduced both the pathogen burden and lung-weight to body-weight ratios. Additionally, p55-TAG vaccination in immunosuppressed rats elicited both cell-mediated and humoral immunity.

Evidence for Proinflammatory ß-1,6 Glucans in the Pneumocystis carinii Cell Wall.

Inflammation is a major cause of respiratory impairment during Pneumocystis pneumonia. Studies support a significant role for cell wall ß-glucans in stimulating inflammatory responses. Fungal ß-glucans are comprised of d-glucose homopolymers containing ß-1,3-linked glucose backbones with ß-1,6-linked glucose side chains. Prior studies in Pneumocystis carinii have characterized ß-1,3 glucan components of the organism. However, recent investigations in other organisms support important roles for ß-1,6 glucans, predominantly in mediating host cellular activation. Accordingly, we sought to characterize ß-1,6 glucans in the cell wall of Pneumocystis and to establish their activity in lung cell inflammation. Immune staining revealed specific ß-1,6 localization in P. carinii cyst walls. Homology-based cloning facilitated characterization of a functional P. carinii kre6 (Pckre6) ß-1,6 glucan synthase in Pneumocystis that, when expressed in kre6-deficient Saccharomyces cerevisiae, restored cell wall stability. Recently synthesized ß-1,6 glucan synthase inhibitors decreased the ability of isolated P. carinii preparations to generate ß-1,6 carbohydrate. In addition, isolated ß-1,6 glucan fractions from Pneumocystis elicited vigorous tumor necrosis factor alpha (TNF-α) responses from macrophages. These inflammatory responses were significantly dampened by inhibition of host cell plasma membrane microdomain function. Together, these studies indicate that ß-1,6 glucans are present in the P. carinii cell wall and contribute to lung cell inflammatory activation during infection.

Myeloid-derived suppressor cells impair alveolar macrophages through PD-1 receptor ligation during Pneumocystis pneumonia.

Myeloid-derived suppressor cells (MDSCs) were recently found to accumulate in the lungs during Pneumocystis pneumonia (PcP). Adoptive transfer of these cells caused lung damage in recipient mice, suggesting that MDSC accumulation is a mechanism of pathogenesis in PcP. In this study, the phagocytic activity of alveolar macrophages (AMs) was found to decrease by 40% when they were incubated with MDSCs from Pneumocystis-infected mice compared to those incubated with Gr-1(+) cells from the bone marrow of uninfected mice. The expression of the PU.1 gene in AMs incubated with MDSCs also was decreased. This PU.1 downregulation was due mainly to decreased histone 3 acetylation and increased DNA methylation caused by MDSCs. MDSCs were found to express high levels of PD-L1, and alveolar macrophages (AMs) were found to express high levels of PD-1 during PcP. Furthermore, PD-1 expression in AMs from uninfected mice was increased by 18-fold when they were incubated with MDSCs compared to those incubated with Gr-1(+) cells from the bone marrow of uninfected mice. The adverse effects of MDSCs on AMs were diminished when the MDSCs were pretreated with anti-PD-L1 antibody, suggesting that MDSCs disable AMs through PD-1/PD-L1 ligation during PcP.

A novel intronic splice site deletion of the IL-2 receptor common gamma chain results in expression of a dysfunctional protein and T-cell-positive X-linked Severe combined immunodeficiency.

X-linked severe combined immunodeficiency is caused by mutations in the IL-2 receptor common gamma chain and classically presents in the first 6 months of life with predisposition to bacterial, viral and fungal infections. In most instances, affected individuals are lymphopenic with near complete absence of T cells and NK cells. We report a boy who presented at 12 months of age with Pneumocystis jiroveci pneumonia and a family history consistent with X-linked recessive inheritance. He had a normal lymphocyte count including the presence of T cells and a broad T-cell-receptor diversity, as well as normal surface expression of the common gamma chain (CD132) protein. He however had profound hypogammaglobulinaemia, and IL-2-induced STAT5 phosphorylation was absent. Sequencing of IL-2RG demonstrated a 12-base pair intronic deletion close to the canonical splice site of exon 5, which resulted in a variety of truncated IL2RG mRNA species. A review of the literature identified 4 other patients with T-cell-positive X-SCID, with the current patient being the first associated with an mRNA splicing defect. This case raises the question of how a dysfunctional protein incapable of mediating STAT5 phosphorylation might nonetheless support T-cell development. Possible explanations are that STAT5-mediated signal transduction may be less relevant to IL7-receptor-mediated T-cell development than are other IL7R-induced intracellular transduction pathways or that a low level of STAT5 phosphorylation, undetectable in the laboratory, may be sufficient to support some T-cell development.