Carmona, Eva M.

ABSTRACT Pneumocystis cyst life forms contain abundant β-glucan carbohydrates, synthesized using β-1,3 and β-1,6 glucan synthase enzymes and the donor uridine diphosphate (UDP)-glucose. In yeast, phosphoglucomutase (PGM) plays a crucial role in carbohydrate metabolism by interconverting glucose 1-phosphate and glucose 6-phosphate, a vital step in UDP pools for β-glucan cell wall formation. This pathway has not yet been defined in Pneumocystis . Herein, we surveyed the Pneumocystis jirovecii and Pneumocystis murina genomes, which predicted a homolog of the Saccharomyces cerevisiae major PGM enzyme. Furthermore, we show that PjPgm2p and PmPgm2p function similarly to the yeast counterpart. When both Pneumocystis pgm2 homologs are heterologously expressed in S. cerevisiae pgm2Δ cells, both genes can restore growth and sedimentation rates to wild-type levels. Additionally, we demonstrate that yeast pgm2Δ cell lysates expressing the two Pneumocystis pgm2 transcripts individually can restore PGM activities significantly altered in the yeast pgm2Δ strain. The addition of lithium, a competitive inhibitor of yeast PGM activity, significantly reduces PGM activity. Next, we tested the effects of lithium on P. murina viability ex vivo and found the compound displays significant anti- Pneumocystis activity. Finally, we demonstrate that a para-aryl derivative (ISFP10) with known inhibitory activity against the Aspergillus fumigatus PGM protein and exhibiting 50-fold selectivity over the human PGM enzyme homolog can also significantly reduce Pmpgm2 activity in vitro . Collectively, our data genetically and functionally validate phosphoglucomutases in both P. jirovecii and P. murina and suggest the potential of this protein as a selective therapeutic target for individuals with Pneumocystis pneumonia.

ABSTRACT Pneumocystis jirovecii, the fungus that causes Pneumocystis jirovecii pneumonia (PJP), is a leading cause of morbidity and mortality in immunocompromised individuals. We have previously shown that lung epithelial cells can bind Pneumocystis spp. β-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. β-glucans activation of the inflammatory signaling cascade in macrophages can be pharmacodynamically inhibited with the EphA2 receptor small-molecule inhibitor ALW-II-41-27. In vitro , when ALW-II-41-27 is administrated via intraperitoneal to mice prior to the administration of highly proinflammatory Saccharomyces cerevisiae β-glucans in the lung, a significant reduction in TNF-alpha release was noted in the ALW-II-41-27 pre-treated group. Taken together, our data suggest that targeting host lung macrophage activation via EphA2 receptor-fungal β-glucans interactions with ALW-II-41-27 or other EphA2 receptor kinase targeting inhibitors might be an attractive and viable strategy to reduce detrimental lung inflammation associated with PJP.

Abstract Background Definition of the microbiology of pleural space infection has been challenging due to the poor yield of conventional culture, a challenge that has led to recommendations for broad spectrum antimicrobial therapy for these infections. Since August 2019, we have offered a 16S ribosomal RNA gene PCR assay followed by Sanger or next generation sequencing, or reported as negative, depending on the PCR signal strength (e.g., crossing point) in clinical practice at Mayo Clinic. Methods Here, we describe the microbiologic and clinical findings of 57 pleural fluids testing positive by 16S rRNA gene PCR/sequencing from August 2020 to January 2023. Demographic data and clinical features, and results of pleural fluid and blood cultures were recorded. Analysis was performed using SAS software version 9.4 (SAS Inc). Results Median patient age was 63 years (range 5-83 years) and 75% were men. Pleural space infection was community-acquired in 84%. The most frequent symptom was dyspnea (80%), followed by chest pain (52%). 89% of patients had received antibiotics in the two weeks preceding specimen collection. Radiological consolidation was observed in 63% and pleural fluid was loculated in 58%. Pleural fluid and blood cultures were positive in 32 and 11%, respectively. The microbiology found with the sequencing assay is shown in the table. 23/57 (40%) detections were polymicrobial. Fusobacterium nucleatum group and/or Streptococcus intermedius were detected in 31/57 (54%) cases [including 28/48 (58%) in the subset with community-acquired infection], with additional facultative and/or anaerobic species in various combinations also found in 17/31 (55%) of these cases. Conclusion Based on analysis of pleural fluid using a targeted metagenomic sequencing assay in clinical practice, the most frequent organism profile involved in community-acquired pleural space infection is a combination of Fusobacterium nucleatum group and/or Streptococcus intermedius, with or without other normal microbiota, a grouping for which we propose the term FuSion (Fusobacterium nucleatum group, Streptococcus intermedius and oral normal microbiota). Disclosures Eva Carmona, MD, boehringer ingelheim: Advisor/Consultant Øyvind Kommedal, n/a, Pathogenomix inc.: Advisor/Consultant|Pathogenomix inc.: Stocks/Bonds Robin Patel, MD, Abbott Laboratories: Advisor/Consultant|Adaptive Phage Therapeutics: Grant/Research Support|Adaptive Phage Therapeutics: Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics.|BIOFIRE: Grant/Research Support|CARB-X: Advisor/Consultant|ContraFect: Grant/Research Support|Day Zero Diagnostics: Advisor/Consultant|HealthTrackRx: Advisor/Consultant|Mammoth Biosciences: Advisor/Consultant|Netflix: Advisor/Consultant|Oxford Nanopore Technologies: Advisor/Consultant|PhAST: Advisor/Consultant|See details: Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic|See details: continued, patent on an anti-biofilm substance issued|TenNor Therapeutics Limited: Grant/Research Support|Torus Biosystems: Advisor/Consultant|Trellis Bioscience, Inc.: Advisor/Consultant

Pneumocystis sp. are fungal pathogens and members of the Ascomycota phylum. Immunocompetent individuals can readily eliminate the fungus, whereas immunocompromised individuals can develop Pneumocystis jirovecii pneumonia (PJP). Currently, over 500,000 cases occur worldwide, and the organism is listed on the recently released WHO fungal priority pathogens list. Overall, the number of PJP cases over the last few decades in developed countries with the use of highly effective antiretroviral therapy has decreased, but the cases of non-HIV individuals using immunosuppressive therapies have significantly increased. Even with relatively effective current anti-Pneumocystis therapies, the mortality rate remains 30–60% in non-HIV patients and 10–20% during initial episodes of PJP in HIV/AIDS patients. Although the role of alveolar macrophages is well studied and established, there is also well-established and emerging evidence regarding the role of epithelial cells in the immune response to fungi. This mini review provides a brief overview summarizing the innate immune response of the lung epithelium and various continuously cultured mammalian cell lines to Pneumocystis.

Introduction. C-type lectin receptors (CLRs) are prominently expressed on myeloid cells where they perform multiple functions including serving as pattern recognition receptors (PRRs) to drive innate as well as adaptive immunity to pathogens. Depending on the presence of a tyrosine-based signalling motif, CLR-microbial pathogen engagement may result in either anti- or pro-inflammatory signalling. Impact statement. In this manuscript, we report our laboratory study of two novel CLRs that recognize Pneumocystis murina cell wall homogenates (CWH) and a purified Pneumocystis carinii cell wall fraction (CWF). Aim. To study the potential of newly generated hFc-CLR fusions on binding to Pneumocystis murina CWHs and P. carinii CWFs and subsequent downstream inflammatory signalling analysis. Methods. Newly generated hFc-CLR fusion CLEC4A and CLEC12B were screened against P. murina CWHs and P. carinii CWFs preparations via modified ELISA. Immunofluorescence assay (IFA) was utilized to visualize hFc-CLR fusion binding against intact fixed fungal life forms to verify results. Quantitative PCR (q-PCR) analysis of lung mRNA from the mouse immunosuppressed Pneumocystis pneumonia (PCP) model versus uninfected mice was employed to detect possible changes in the respective Clec4a and Clec12b transcripts. Lastly, siRNA technology of both CLRs was conducted to determine effects on downstream inflammatory events in mouse macrophages stimulated in the presence of P. carinii CWFs. Results. We determined that both CLEC4A and CLEC12B hFc-CLRs displayed significant binding with P. murina CWHs and P. carinii CWFs. Binding events showed significant binding to both curdlan and laminarin, both polysaccharides containing β-(1,3) glucans as well as N-acetylglucosamine (GlcNAc) residues and modest yet non-significant binding to the negative control carbohydrate dextran. IFA with both CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of both CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that both CLRs were significantly up regulated during infection. Lastly, siRNA of both CLRs in the mouse RAW macrophage cell line was conducted and results demonstrated that silencing of Clec4a resulted in no significant changes in TNF-alpha generation in P. carinii CWF stimulated macrophages. On the contrary, silencing of Clec12b CLR resulted in significant decreases in TNF-alpha in RAW cells stimulated with the same CWF. Conclusion. The data presented here provide new members of the CLRs family recognizing Pneumocystis. Future studies using CLEC4A and/or CLEC12B deficient mice in the PCP mouse model should provide further insights into the host immunological response to Pneumocystis.

Abstract Background In the United States, rare disease (RD) is defined as a condition that affects fewer than 200,000 individuals. Collectively, RD affects an estimated 30 million Americans. A significant portion of RD has an underlying genetic cause; however, this may go undiagnosed. To better serve these patients, the Mayo Clinic Program for Rare and Undiagnosed Diseases (PRaUD) was created under the auspices of the Center for Individualized Medicine (CIM) aiming to integrate genomics into subspecialty practice including targeted genetic testing, research, and education. Methods Patients were identified by subspecialty healthcare providers from 11 clinical divisions/departments. Targeted multi-gene panels or custom exome/genome-based panels were utilized. To support the goals of PRaUD, a new clinical service model, the Genetic Testing and Counseling (GTAC) unit, was established to improve access and increase efficiency for genetic test facilitation. The GTAC unit includes genetic counselors, genetic counseling assistants, genetic nurses, and a medical geneticist. Patients receive abbreviated point-of-care genetic counseling and testing through a partnership with subspecialty providers. Results Implementation of PRaUD began in 2018 and GTAC unit launched in 2020 to support program expansion. Currently, 29 RD clinical indications are included in 11 specialty divisions/departments with over 142 referring providers. To date, 1,152 patients have been evaluated with an overall solve rate of 14.1% and as high as 66.7% depending on the phenotype. Noteworthy, 42.7% of the solved or likely solved patients underwent changes in medical management and outcome based on genetic test results. Conclusion Implementation of PRaUD and GTAC have enabled subspecialty practices advance expertise in RD where genetic counselors have not historically been embedded in practice. Democratizing access to genetic testing and counseling can broaden the reach of patients with RD and increase the diagnostic yield of such indications leading to better medical management as well as expanding research opportunities.

Abstract Background Acute respiratory exacerbations in idiopathic pulmonary fibrosis (IPF), often triggered by infection, are life threatening events characterized by rapid deterioration of lung function and associated with poor prognosis. Since microbial antigens modulate immunity by activating different pattern recognition receptors (PRR) expressed by innate immune cells (including B-cells), we sought to investigate how the activation of human B-cells through PRR stimulation by different microbial (CpG and β-glucan) and non-microbial (Mitochondrial DAMPs; MTDs) antigens resulted in the release of an inflammatory/fibrotic milieu that contributed to the pathogenesis of fibrosis in patients with IPF. Results Our results show that an inflammatory milieu is induced by stimulating circulating B-cells with both noninfectious (MTDs) and infectious antigens (CpG and β-glucan) commonly found in bacteria and fungi. Moreover, we show that the inflammatory milieu produced was specific to the inducing antigen, and that the microbial antigens (CpG and β-glucan) triggered distinct signaling pathways in B-cells. Specifically, CpG induced mTOR-activation while β-glucan was mTOR-independent and activated Src and p38. Furthermore, our results also show that B-cell aggregates are present within fibrotic areas of the lung in IPF patients and activated B-cells induce fibroblast migration, which was modified by antifibrotics (nintedanib and pirfenidone). Conclusion Our results highlight the potential contribution of activated B-cells through PRRs to the proinflammatory and profibrotic milieu seen in patients with idiopathic pulmonary fibrosis.

Abstract We hypothesize that B-lymphocytes are conducive to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a progressive and fatal interstitial lung disease. B-lymphocytes aggregate in the lungs of IPF patients to form tertiary lymphoid follicles. How these follicles are formed and their role in the pathogenesis of IPF is not known. Hence, we sought to characterize how the activation of B-lymphocytes from IPF patients may contribute to the disease. Methods IPF patients defined by ATS guidelines were identified and recruited under local IRB #14-004714. B-lymphocytes were then isolated by negative selection. Cytokines and immunoglobulins from plasma or B-lymphocyte supernatants were measured by ELISA and signaling pathways were elucidated from whole-cell lysates by immunoblotting. Our results show that B-lymphocyte aggregates are present within fibrotic areas of the lung of IPF patients and that activated B-lymphocytes induce fibroblast migration. This effect was abrogated by nintedanib inhibition of B-lymphocyte activation. Furthermore, microbial antigen (β-glucan and CpG) activation of B-lymphocytes through pattern recognition receptors (PRRs; TLR9 and Dectin-1) contributed to the proinflammatory and profibrotic milieu seen in IPF patients. Stimulation of TLR9 and Dectin-1 resulted in activation of mTOR-dependent and -independent pathways, respectively. Rapamycin decreased TLR9 signaling, but not Dectin-1 mediated pathways. Nintedanib abrogated B-cell activation, IgM production, and the profibrotic milieu by interfering with both the mTOR-dependent and -independent pathways. In conclusion, our findings implicate activated B-lymphocytes in IPF pathogenesis highlighting the role of PRRs in this disease.

ObjectiveAutoreactive B cells are responsible for antineutrophil cytoplasmic antibody (ANCA) production in ANCA‐associated vasculitis (AAV). Rituximab (RTX) depletes circulating B cells, including autoreactive B cells. We aimed to evaluate changes and associations with relapse of the circulating autoreactive B cell pool following therapeutic B cell depletion in AAV.MethodsSequential flow cytometry was performed on 148 samples of peripheral blood mononuclear cells from 23 patients with proteinase 3 (PR3)–ANCA–positive AAV who were treated with RTX for remission induction and monitored after stopping therapy during long‐term follow‐up in a prospective clinical trial. PR3 was used as a ligand to target autoreactive PR3‐specific (PR3+) B cells. B cell recurrence was considered as the first blood sample with ≥10 B cells/μl after RTX treatment.ResultsAt B cell recurrence, PR3+ B cell frequency among B cells was higher than baseline (P < 0.01). Within both PR3+ and total B cells, frequencies of transitional and naive subsets were higher at B cell recurrence than at baseline, while memory subsets were lower (P < 0.001 for all comparisons). At B cell recurrence, frequencies of B cells and subsets did not differ between patients who experienced relapse and patients who remained in remission. In contrast, the plasmablast frequency within the PR3+ B cell pool was higher in patients who experienced relapse and associated with a shorter time to relapse. Frequencies of PR3+ plasmablasts higher than baseline were more likely to be found in patients who experienced relapse within the following 12 months compared to those in sustained remission (P < 0.05).ConclusionThe composition of the autoreactive B cell pool varies significantly following RTX treatment in AAV, and early plasmablast enrichment within the autoreactive pool is associated with future relapses.