Increased mRNA expression of key cytokines among suspected cases of Pneumocystis jirovecii infection.

BACKGROUND: Pneumocystis pneumonia (PCP) is a fatal infectious disease caused by Pneumocystis jirovecii (PJP). The major factor relevant to morbidity and mortality seems to be the host inflammatory reaction. The objective of this study was to evaluate the role of IL-2, IL-4, IL-10, and IL-13 cytokine mRNA expression among suspected P. jirovecii infection. METHODS: This was a cross-sectional analytical study undertaken in Aseer region, Saudi Arabia. One hundred suspected PCP cases and 100 healthy controls were included in the study. Basic clinical manifestations, radiological findings, microbiological and immunological findings were extracted from the hospital records from January 2019 to August 2019, Pneumocystis detection was done by immune-fluorescent staining (IFAT, Gomorimethanamine silver staining (GMSS), Giemsa staining, Toluidine blue O (TBO), and Pneumocystis RT-PCR. RESULTS: Increased more than 5 fold, 3 fold, 4 fold, and 7 fold of IL-2, IL-4, IL-10, and IL-13 mRNA expression were observed in PCP cases compared to controls. Higher expression of IL-2 mRNA was connected with crept, wheezing and chest X-ray findings like central perihilar infiltrate, patchy infiltrate, consolidation, hilar lymphadenopathy, pneumothorax, pleural effusion which showed higher expression compared to counterpart (p< 0.0001). Higher expression of IL-4 mRNA was found to be significantly associated with weight loss (p=0.002), dyspnea (p=0.003), crept (p=0.01), and chest X-ray findings (p< 0.0001). Significantly increased expression of IL-10 mRNA was observed to be associated with weight loss, dyspnea, night sweats, wheezing, and different findings of chest X-ray compared to their counterparts, whereas, IL-13 mRNA was observed in cases with fever. Suspected cases of PCP confirmed positive by IFTA with higher IL-2, IL-4, and IL-10 mRNA expression compared to negative cases. RT-PCR confirmed PCP cases had significantly higher expression of IL-2, IL-4, and IL-10 as well as IL-13 mRNA compared to negative cases. Positive detected cases by GMSS showed higher IL-2, IL-10 mRNA expression, while Giemsa showed only higher IL-4 mRNA expression compared to negative cases. CONCLUSION: Confirmed cases of P. jirovecii showed higher IL-2, IL-4, IL-10, and IL-13 mRNA expression comparatively to negative cases. Increased expression of cytokines may be indicative of infection severity and could help in patients' management.

Outbreak-Causing Fungi: Pneumocystis jirovecii.

Pneumocystis jirovecii pneumonia (PCP) is an important cause of morbidity in immunocompromised patients, with a higher mortality in non-HIV than in HIV patients. P. jirovecii is one of the rare transmissible pathogenic fungi and the only one that depends fully on the host to survive and proliferate. Transmissibility among humans is one of the main specificities of P. jirovecii. Hence, the description of multiple outbreaks raises questions regarding preventive care management of the disease, especially in the non-HIV population. Indeed, chemoprophylaxis is well codified in HIV patients but there is a trend for modifications of the recommendations in the non-HIV population. In this review, we aim to discuss the mode of transmission of P. jirovecii, identify published outbreaks of PCP and describe molecular tools available to study these outbreaks. Finally, we discuss public health and infection control implications of PCP outbreaks in hospital setting for in- and outpatients.

Development of substituted pyrido[3,2-d]pyrimidines as potent and selective dihydrofolate reductase inhibitors for pneumocystis pneumonia infection.

Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii (pj) can lead to serious health consequences in patients with an immunocompromised system. Trimethoprim (TMP), used as first-line therapy in combination with sulfamethoxazole, is a selective but only moderately potent pj dihydrofolate reductase (pjDHFR) inhibitor, whereas non-clinical pjDHFR inhibitors, such as, piritrexim and trimetrexate are potent but non-selective pjDHFR inhibitors. To meet the clinical needs for a potent and selective pjDHFR inhibitor for PCP treatment, fourteen 6-substituted pyrido[3,2-d]pyrimidines were developed. Comparison of the amino acid residues in the active site of pjDHFR and human DHFR (hDHFR) revealed prominent amino acid differences which could be exploited to structurally design potent and selective pjDHFR inhibitors. Molecular modeling followed by enzyme assays of the compounds revealed 15 as the best compound of the series with an IC50 of 80 nM and 28-fold selectivity for inhibiting pjDHFR over hDHFR. Compound 15 serves as the lead analog for further structural variations to afford more potent and selective pjDHFR inhibitors.

Serum-based diagnosis of Pneumocystis pneumonia by detection of Pneumocystis jirovecii DNA and 1,3-ß-D-glucan in HIV-infected patients: a retrospective case control study.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP) is one of the most common HIV-related opportunistic infections. The diagnosis of PCP is based on analyses from respiratory tract specimens which may require the invasive procedure of a diagnostic bronchoscopy. The objective of this study was to evaluate the diagnostic potential of Pneumocystis jirovecii PCR in serum combined with the 1,3-ß-D-glucan (betaglucan) test for the diagnosis of PCP in HIV-infected patients. METHODS: This was a retrospective case-control study including serum samples from 26 HIV-infected patients with PCP collected within 5 days prior to the start of PCP treatment, 21 HIV-infected control subjects matched by blood CD4+ cell counts, and 18 blood donors. The serum samples were analyzed for Pneumocystis jirovecii PCR and betaglucan. The reference standard for PCP was based on previously described microbiological and clinical criteria. RESULTS: All patients with PCP had detectabe Pneumocystis jirovecii DNA in serum yielding a sensitivity for the Pneumocystis jirovecii PCR assay in serum of 100%. All blood donors had negative Pneumocystis PCR in serum. The specificity when testing HIV-infected patients was 71%, but with a PCR Cycle threshold (Ct) value of 34 as cut-off the specificity was 90%. At a putative pretest probaility of 20%, the negative and positive predictive value for the Pneumocystis PCR assay in serum was 0.99 and 0.71, respectively. Betaglucan with cut-off level 200 pg/ml combined with a positive Pneumocystis jirovecii PCR result had sensitivity and specificity of 92 and 90%, respectively. The concentration of Pneumocystis jirovecii DNA in serum samples, expressed by the PCR Ct values, correlated inversely to the betaglucan levels in serum. CONCLUSION: In this case-control study including 70% of all HIV-infected patients with PCP treated at Sahlgrenska University Hospital during a time period of 13 years, Pneumocystis PCR analysis on serum samples had a very high sensitivity and negative predictive value for the diagnosis of PCP in HIV-infected patients. A serum-based diagnostic procedure either based on Pneumocystis jirovecii PCR alone or in combination with betaglucan analysis may thus be feasible and would facilitate the care of HIV-infected patients with suspected PCP.

Safety and efficacy of prophylaxis for Pneumocystis jirovecii pneumonia involving trimethoprim-sulfamethoxazole dose reduction in kidney transplantation.

BACKGROUND: Trimethoprim-sulfamethoxazole (TMP-SMX) is the drug of choice for anti-Pneumocystis jirovecii pneumonia (PcP) prophylaxis in kidney transplant recipients (KTR). Post-transplant management balances preventing PcP with managing TMP-SMX-related adverse effects. TMP-SMX dose reduction addresses adverse effects but its implications to incident PcP are unclear. METHODS: We performed a retrospective review of all patients transplanted between 2011 and 2015 prescribed daily single strength TMP-SMX for twelve months post-transplantation as PcP prophylaxis. Actual TMP-SMX dose and duration, adverse effects, number of dose reductions and reasons, and PcP events were captured. Multivariate logistic regression analyses for risk factors associated with dose reduction were performed. RESULTS: Of 438 KTR, 233 (53%) maintained daily TMP-SMX and 205 (47%) sustained ≥1 dose reduction, with the point prevalence of a reduced dose regimen being between 18 and 25%. Median duration for daily TMP-SMX was 8.45/12 months, contributing 4137 patient-months daily TMP-SMX and 1110 patient-months with a reduced dose. PcP did not occur in any patients. There were 84 documented dose reductions for hyperkalemia and 102 for leukopenia, with 12 and 7 patients requiring TMP-SMX cessation. In multivariate analysis, a living donor transplant protected against hyperkalemia (Odds Ratio 0.46, 95% CI 0.26-0.83, p < 0.01) while acute rejection risked leukopenia (Odds Ratio 3.31, 95% CI 1.39-7.90, p = 0.006). CONCLUSIONS: TMP-SMX dose reduction is frequent in the first post-transplant year but PcP does not occur. To limit the need for TMP-SMX dose reduction due to adverse effects, a clinical trial comparing daily to thrice weekly single strength TMP-SMX in de-novo KTR is justified.

Pneumocystis jirovecii-related spontaneous pneumothorax, pneumomediastinum and subcutaneous emphysema in a liver transplant recipient: a case report.

BACKGROUND: Pneumocystis pneumonia (PCP) is a common opportunistic infection caused by Pneumocystis jirovecii. Its incidence at 2 years or more after liver transplant (LT) is < 0.1%. PCP-related spontaneous pneumothorax and/or pneumomediastinum is rare in patients without the human immunodeficiency virus, with an incidence of 0.4-4%. CASE PRESENTATION: A 65-year-old woman who had split-graft deceased-donor LT for primary biliary cirrhosis developed fever, dyspnea and dry coughing at 25 months after transplant. Her immunosuppressants included tacrolimus, mycophenolate mofetil, and prednisolone. PCP infection was confirmed by molecular detection of Pneumocystis jirovecii,in bronchoalveolar lavage. On day-10 trimethoprim-sulphamethoxazole, her chest X-ray showed subcutaneous emphysema bilaterally, right pneumothorax and pneumomediastinum. Computed tomography of the thorax confirmed the presence of right pneumothorax, pneumomediastinum and subcutaneous emphysema. She was managed with 7-day right-sided chest drain and a 21-day course of trimethoprim-sulphamethoxazole before discharge. CONCLUSION: Longer period of PCP prophylaxis should be considered in patients who have a higher risk compared to general LT patients. High index of clinical suspicion, prompt diagnosis and treatment with ongoing patient reassessment to detect and exclude rare, potentially fatal but treatable complications are essential, especially when clinical deterioration has developed.

Circulating and Pulmonary T-cell Populations Driving the Immune Response in Non-HIV Immunocompromised Patients with Pneumocystis jirovecii Pneumonia.

Background: Previous studies in human subjects have mostly been confined to peripheral blood lymphocytes for Pneumocystis infection. We here aimed to compare circulating and pulmonary T-cell populations derived from human immunodeficiency virus (HIV)-uninfected immunocompromised patients with Pneumocystis jirovecii pneumonia (PCP) in order to direct new therapies. Methods: Peripheral blood and bronchoalveolar lavage samples were collected from patients with and without PCP. Populations of Th1/Tc1, Th2/Tc2, Th9/Tc9, and Th17/Tc17 CD4+ and CD8+ T cells were quantified using multiparameter flow cytometry. Results: No significant differences were found between PCP and non-PCP groups in circulating T cells. However, significantly higher proportions of pulmonary Th1 and Tc9 were observed in the PCP than in the non-PCP group. Interestingly, our data indicated that pulmonary Th1 was negatively correlated with disease severity, whereas pulmonary Tc9 displayed a positive correlation in PCP patients. Conclusions: Our findings suggest that pulmonary expansion of Th1 and Tc9 subsets may play protective and detrimental roles in PCP patients, respectively. Thus, these specific T-cell subsets in the lungs may serve as targeted immunotherapies for patients with PCP.

Hypercalcemia Heralding Pneumocystis jirovecii Pneumonia in an HIV-Seronegative Patient with Diffuse Cutaneous Systemic Sclerosis.

Pneumocystis pneumonia (PCP) is a life-threatening fungal infection occurring in immunocompromised patients such as HIV-positive patients with low CD4 cell count or patients under heavy immunosuppressive therapy. We report the case of a 59-year-old male with severe diffuse cutaneous systemic sclerosis presenting with asthenia, dry cough and worsening shortness of breath for the last 15 days. Biological studies were remarkable for PTH-independent severe hypercalcemia with low 25-hydroxyvitamin D and a paradoxically elevated 1,25-dihydroxyvitamin D. Early bronchoalveolar lavage allowed for PCP diagnosis and targeted treatment. We discuss the underlying physiopathology and difficulties regarding prophylaxis and treatment.

Effect of sulfasalazine use on the presence of Pneumocystis organisms in the lung among patients with rheumatoid arthritis: A test-negative design case-control study with PCR tests.

OBJECTIVE: To evaluate the effect of sulfasalazine (SSZ) on the presence of Pneumocystis jirovecii (P. jirovecii) in the lungs of rheumatoid arthritis (RA) patients. METHODS: We retrospectively studied episodes of suspected P. jirovecii pneumonia (PJP) which were examined for P. jirovecii with polymerase chain reaction (PCR). We employed a test negative design case-control study; the cases were episodes of suspected PJP that were positive for PCR, and the controls were episodes of suspected PJP that were negative for PCR. The odds ratio for the positive PCR result associated with SSZ use was estimated by Firth's logistic regression. RESULTS: Between 2003 and 2017, 36 cases and 83 controls were identified. While none of the cases received SSZ before the episode, 18 of the controls received the drug. In the primary analysis involving all the episodes, SSZ use was negatively associated with PCR positivity (adjusted odds ratio, 0.087; confidence interval, <0.001-0.789). The sensitivity analysis, excluding those who received PJP prophylaxis, showed the same association as the primary analysis (adjusted odds ratio 0.085, 95% CI <0.001-0.790). CONCLUSION: This study demonstrated that SSZ use is associated with the absence of P. jirovecii in the lung, suggesting the preventive efficacy of the drug against PJP.

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.

Healthcare related transmission of Pneumocystis pneumonia: From key insights toward comprehensive prevention.

In at risk populations, Pneumocystis pneumonia (PCP) may occur as a solitary event as well as in a cluster- or outbreak setting due to interpatient transmission of Pneumocystis jirovecii. Despite the data and insights obtained from studies of outbreaks of PCP, the development and implementation of comprehensive recommendations for the prevention of healthcare related transmission of P. jirovecii have been delayed. Both optimization of chemoprophylaxis strategies as well as combination with prudent use of isolation precautions are warranted to achieve this goal. The rationale of the available measures for the prevention of PCP should be viewed in the context of what is currently known about the complex biology and epidemiology of P. jirovecii. From there, phased but practical prevention strategies can be deducted to balance the efforts, costs and negative consequences of chemoprophylaxis and isolation precautions with the beneficial effect of preventing healthcare related transmission of P. jirovecii and development of PCP.

An outbreak of Pneumocytis jirovecii pneumonia among liver transplant recipients.

BACKGROUND: An outbreak of Pneumocystis pneumonia (PCP) in liver transplant recipients occurred between 2009 and 2011 at the Beaujon University Hospital just after immediate-release tacrolimus was replaced by extended-release tacrolimus. We conducted a retrospective study to analyze the transmission mode of Pneumocystis, the role of the change in the immunosuppressive regimen, and the factors associated with PCP. PATIENTS AND METHODS: To analyze transmission, we built a transmission map. Two control groups were built. First, to assess the role of the change from tacIR to tacER, cases were matched to controls transplanted before 2009 (tacIR control group). Tacrolimus trough concentrations were compared between the 2 groups. Then, to assess factors associated with PCP, each PCP case was matched to 2 control patients: the one transplanted just before and the one just after (PCPAsFact control group). No PCP prophylaxis was given to any patient. RESULTS: Fifteen cases of PCP were recorded. A contact between a case and a patient who developed PCP afterward was identified in 4 occasions. The comparison of tacrolimus trough concentrations did not conclude to a difference in the exposure to the drug. Lymphopenia was the only factor independently associated with the occurrence of PCP (odds ratio 0.78, 95% confidence interval 0.61-0.99, P = .04). CONCLUSION: Our results suggest that patient-to-patient transmission was not the main mode of transmission of PCP. We found no evidence that the switch from tacIR to tacER led to an overexposure to tacrolimus. Our results suggest the possibility of targeted prophylaxis in immunosuppressed liver transplant recipients.

Clinical course, treatment and outcome of Pneumocystis pneumonia in immunocompromised adults: a retrospective analysis over 17 years.

BACKGROUND: Despite modern intensive care with standardized strategies against acute respiratory distress syndrome (ARDS), Pneumocystis pneumonia (PcP) remains a life-threatening disease with a high mortality rate. Here, we analyzed a large mixed cohort of immunocompromised patients with PcP, with regard to clinical course and treatment, and aimed at identifying predictors of outcome. METHODS: This was a single-center retrospective analysis in a tertiary care institution across 17 years. Diagnosis of PcP required typical clinical features and microbiological confirmation of Pneumocystis jirovecii. Epidemiological, clinical, laboratory and outcome data were collected from patient records. RESULTS: A total of 52,364 specimens from 7504 patients were sent for microbiological assessment (3653 with clinical suspicion of Pneumocystis pneumonia). PcP was confirmed in 240 patients, about half of them HIV positive (52%). The remaining subjects were either solid organ transplant recipients (16.3%) or suffered from malignancy (15.8%) or autoimmune diseases (11.7%). Of note, 95% of patients with PcP were not receiving chemoprophylaxis. Overall in-hospital mortality was 25.4%, increasing to 58% if ICU admission was required. Multivariable regression identified lactate dehydrogenase (LDH) as predictor of in-hospital mortality (adjusted OR 1.17 (95% CI 1.09-1.27), p < 0.0001). Mortality in LDH quartiles increased from 8% to 49%, and a cutoff value of 495 U/L predicted mortality with sensitivity and specificity of 70%. With regard to treatment, 40% of patients received trimethoprim-sulfamethoxazole at doses that were lower than recommended, and these patients had a higher mortality risk (HR 1.80 (95% CI 1.10-3.44), p = 0.02). CONCLUSIONS: PcP remains a life-threatening disease among immunocompromised patients. About half of patients with PcP do not have HIV infection. Initial LDH values might serve as a stratifying tool to identify those patients at high risk of death among patients with HIV and without HIV infection.

Outcomes and prognostic factors of non-HIV patients with pneumocystis jirovecii pneumonia and pulmonary CMV co-infection: A Retrospective Cohort Study.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) and pulmonary cytomegalovirus (CMV) infection are common opportunistic infections among immunocompromised patients. However, few studies have evaluated their co-infection, especially among non-HIV patients. Therefore, we aimed to evaluate the outcomes and prognostic factors among non-HIV patients with PJP according to their CMV infection status. METHODS: This retrospective study evaluated non-HIV patients who were diagnosed with PJP between January 2009 and January2016.The patients were classified and compared according to their pulmonary CMV infection status (positive infection: bronchoalveolar lavage fluid [BALF] CMV DNA loads of >500copies/mL). RESULTS: Among 70 non-HIV patients with PJP, we identified 38 patients (54.3%) with pulmonary CMV infection. There was no significant difference in the mortality rates for the two groups (p = 0.15). Pulmonary CMV infection was significantly more common among patients who were receiving glucocorticoids and immunosuppressants, compared to corticosteroids only (p = 0.02). Pulmonary CMV infection was also significantly associated with severe dyspnea, a lower PaO2/FiO2, and the presence of centrilobular nodules (p = 0.008). Higher CMV DNA loads in the BALF were positively associated with mortality (p = 0.012). CONCLUSIONS: Combined therapy using corticosteroids and other immunosuppressants may be a risk factor for pulmonary CMV co-infection among patients with PJP. In addition, CMV pneumonia should be considered when centrilobular nodules and/or severe hypoxemia are observed in non-HIV patients with PJP. Furthermore, antiviral treatment should be promptly initiated for patients with a high CMV DNA load in BALF, based on their poor prognosis.

The role of CD4 cell count as discriminatory measure to guide chemoprophylaxis against Pneumocystis jirovecii pneumonia in human immunodeficiency virus-negative immunocompromised patients: A systematic review.

BACKGROUND: In recent years, the incidence of Pneumocystis jirovecii pneumonia (PJP) has increased in immunocompromised patients without human immunodeficiency virus (HIV) infection. Chemoprophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) is highly effective in preventing PJP in both HIV-positive and -seronegative patients. In HIV-positive patients, the risk of PJP is strongly correlated with decreased CD4 cell count. The role of CD4 cell count in the pathogenesis of PJP in non-HIV immunocompromised patients is less well studied. For most immunosuppressive conditions, no clear guidelines indicate whether to start TMP-SMX. METHOD: We conducted a systematic literature review with the aim to provide a comprehensive overview on the role of CD4 cell counts in managing the risk of PJP in HIV-seronegative patients. RESULTS: Of the 63 individual studies retrieved, 14 studies report on CD4 cell counts in a variety of immunosuppressive conditions. CD4 cell count were <200/µL in 73.1% of the patients. CONCLUSION: CD4 cell count <200/µL is a sensitive biomarker to identify non-HIV immunocompromised patients who are at risk for PJP. Measuring CD4 cell counts could help clinicians identify patients who may benefit from TMP-SMX prophylaxis.

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.

The Pneumocystis Ace2 transcription factor regulates cell wall-remodeling genes and organism virulence.

Pneumocystis carinii (Pc) ß-glucans are major components of the organism cell wall; yet, the regulation of Pc cell wall genesis and remodeling is not well understood. Ace2 transcription factors, which are present in many fungi, regulate glucanases and other enzymes needed for cell wall remodeling. The cloning and heterologous expression of PcAce2 in ace2Δ Saccharomyces cerevisiae demonstrated that PcAce2 can restore the defective glucanase and endochitinase gene expression of the mutant as well as regulate cell wall ß-glucan biosynthetic genes. Furthermore, when a reconstructed yeast system was used, PcAce2 activated the transcription of the Pneumocystis gsc1 ß-glucan synthetase, confirming the activity of a Pc transcription factor on a native Pneumocystis promoter and gene for the first time. We further observed that Pneumocystis binding to host extracellular matrix proteins and lung epithelial cells induced the phosphorylation (activation) of the PcAce2 transcription factor. Finally, we present a novel method that confirms the role of PcAce2 in modulating organism virulence using ace2Δ Candida glabrata infection in neutropenic mice. Together, these results indicate that the adherence of Pc to lung matrix proteins and epithelial cells leads to the activation of the Ace2 transcription factor, which regulates cell wall degradation and biosynthesis genes that are required for cell wall remodeling.