Pulmonary dimorphic fungal infections among HIV/AIDS non-TB patients with chronic cough in Kampala, Uganda.

INTRODUCTION: Dimorphic fungi cause infection following the inhalation of spores into the pulmonary system. In the lower respiratory tract, the conidia transform into yeasts, which are engulfed by alveolar macrophages and may be destroyed without disease manifestation. However, in some immunocompromised individuals, they may persist and cause active fungal disease characterized by formation of granulomas in the infected tissues, which may mimic Mycobacterium tuberculosis (MTB). OBJECTIVE: To determine the prevalence of pulmonary dimorphic fungal infections among HIV/AIDS patients with non-TB chronic cough at Mulago National Referral and Teaching Hospital in Kampala, Uganda. METHODS: Sputum samples were collected from 175 consented HIV/AIDS patients attending the immuno-suppression syndrome (ISS) clinic at the hospital. Upon Xpert MTB/RIF sputum testing, 21 patients tested positive for MTB, and these were excluded from further analysis. The other 154 sputum negative samples were then subjected to PCR for dimorphic fungi at MBN Clinical Laboratories. Singleplex PCR was used to detect the target sequences in selected respective genes of each dimorphic fungal species of interest. DNA amplicons were detected based on gel electrophoresis. RESULTS: Dimorphic fungi were detected in 16.2% (25/154) of the studied population. Of these 9.1% (14/154) had Blastomyces dermatitidis and 7.1% (11/154) had Talaromyces marneffei. The remaining 84% of the studied participants had no dimorphic fungi. Histoplasma capsulatum, Coccidioides immitis and Paracoccidioides brasiliensis were not detected in any of the participants. CONCLUSION: Dimorphic fungi (B. dermatitidis and T. marneffei) were found in 16.2% of the HIV/AIDS patients with non-TB chronic cough in Kampala, Uganda. We recommend routine testing for these pathogens among HIV/AIDS patients with chronic cough.

Endophytic Fungus Talaromyces sp. MR1 Promotes the Growth and Cadmium Uptake of Arabidopsis thaliana L. Under Cadmium Stress.

Endophytes play essential roles in plant growth under metal(loid)s stress. An endophytic fungus strain MR1 was isolated from the roots of Miscanthus floridulus collected from a lead-zinc mining area (Huayuan, China), which could produce indole-3-acetic acid and have Cadmium (Cd) tolerance. Further 18S rRNA sequencing analysis showed that it was highly similar (99.83%) to Talaromyces pinophilus. In pot experiments, we explored the effects of strain MR1 on the growth and Cd uptake of a wide-type Arabidopsis thaliana under low (LC) and high (HC) Cd concentrations. The results showed that MR1 effectively increased the dry weight of aboveground and underground tissues by 25.95-107.21% in both LC and HC groups. Due to MR1 inoculation, the Cd content in the underground tissues was significantly (p < 0.05) decreased by 39.28% under low Cd concentration, while it was significantly (p < 0.05) increased by 28.28% under high Cd concentration. Besides, MR1 inoculations significantly (p < 0.05) increased the total content of removed Cd (17.080 µg) and BCF (0.064) by 129.77% and 153.95% under high Cd concentration. Therefore, we speculated that MR1 might be selected as the effective microbial agent to increase crop yield and control Cd content in the crop in light Cd-contaminated soil. Besides, MR1 could potentially enhance the phytoremediation efficiency of extremely Cd-contaminated soil.

Identification of glutathione metabolic genes from a dimorphic fungus Talaromyces marneffei and their gene expression patterns under different environmental conditions.

Talaromyces marneffei is a human fungal pathogen that causes endemic opportunistic infections, especially in Southeast Asia. The key virulence factors of T. marneffei are the ability to survive host-derived heat and oxidative stress, and the ability to convert morphology from environmental mold to fission yeast forms during infection. Glutathione metabolism plays an essential role in stress response and cellular development in multiple organisms. However, the role of the glutathione system in T. marneffei is elusive. Here, we identified the genes encoding principal enzymes associated with glutathione metabolism in T. marneffei, including glutathione biosynthetic enzymes (Gcs1 and Gcs2), glutathione peroxidase (Gpx1), glutathione reductase (Glr1), and a family of glutathione S-transferase (Gst). Sequence homology search revealed an extended family of the TmGst proteins, consisting of 20 TmGsts that could be divided into several classes. Expression analysis revealed that cells in conidia, mold, and yeast phases exhibited distinct expression profiles of glutathione-related genes. Also, TmGst genes were highly upregulated in response to hydrogen peroxide and xenobiotic exposure. Altogether, our findings suggest that T. marneffei transcriptionally regulates the glutathione genes under stress conditions in a cell-type-specific manner. This study could aid in understanding the role of glutathione in thermal-induced dimorphism and stress response.

Integration of metabolomics and transcriptomics analyses reveals sphingosine-1-phosphate-mediated S1PR2/PI3K/Akt pathway involved in Talaromyces marneffei infection of macrophages.

Talaromycosis is a fatal mycosis caused by the thermally dimorphic fungus Talaromyces marneffei (T. marneffei). The pathogenic mechanisms of talaromycosis are still poorly understood. This work combined metabolomics, transcriptomics, and verification experiments in vivo and in vitro to detect metabolic profiles and differentially expressed genes (DEGs) in T. marneffei infected and uninfected macrophages to explore possible pathogenesis and underlying mechanisms. A total of 256 differential metabolites (117 up-regulated and 148 down-regulated) and 1320 DEGs (1286 up-regulated and 34 down-regulated) were identified between the two groups. Integrative metabolomics and transcriptomics analysis showed sphingolipid signaling pathway is the most influential. Verification experiments showed that compared with the control group, the production of sphingosine-1-phosphate (S1P) and the expression of the S1PR1, S1PR2, phosphor-PI3K, and phosphor-Akt genes involved in the sphingolipid signaling pathway have significantly increased in the T. marneffei infection group (p < 0.05). T. marneffei activates the S1PR2/PI3K/Akt pathways in J774A.1 macrophage, regulation of the S1P singling might serve as a promising therapeutic strategy for talaromycosis.

Case report: Hemophagocytic lymphohistiocytosis in a child with primary immunodeficiency infected with Talaromyces marneffei.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune-mediated disease that affects patients with known genetic defects and is increasingly found among those with autoimmune diseases and persistent infections. Talaromyces marneffei (TM) is a human opportunistic fungus that commonly infects immunodeficient or immunosuppressed individuals. Few TM-associated secondary HLH cases resulting from autoimmune deficiency have been reported previously. The current case study describes a pediatric patient hospitalized with recurrent fever and lymphadenopathy. The child had abnormal blood cell classification, and microscopy revealed mature granulocytes that phagocytized fungal spores. It was speculated that the patient was infected with TM. The pathogen was detected earlier than the blood culture and confirmed by metagenomic next-generation sequencing. Whole-exome sequencing revealed that the patient had complex mutations associated with immunodeficiency. This included a mutation in exon 3 of the CD40LG gene, c.346G>A, which may be linked to hyper-IgM syndrome, a primary immunodeficiency disease with immunoglobulin conversion recombination defects that could explain the patient's increased susceptibility to serious opportunistic infections. In addition, a heterozygous frameshift variant, c.820dup (p.Asp274GlyfsTer61), was detected in exon 6 of CARD9, a key gene associated with fungal immune surveillance. After 4 days of fungal treatment, the abnormal blood cell clusters disappeared, but other infections occurred in succession for 6 months after rehabilitation. The patient was followed with the aim of providing subsequent immunotherapy. This study found that infection can trigger HLH in HIV-negative individuals, highlighting the importance of early definitive identification of the causative agent and investigation of potential immunodeficiency.

Overexpression of CD86 enhances the ability of THP-1 macrophages to defend against Talaromyces marneffei.

BACKGROUND: Macrophages are the first line of defense against Talaromyces marneffei. CD86 is a surface molecule expressed on antigen-presenting cells, such as macrophages, that provide costimulatory signals necessary for T cell activation and survival. In a prior study, it was shown that as infection progressed, CD86 expression levels in macrophages considerably declined while CD86 concentrations in the supernatant significantly increased. Additionally, M1 macrophage polarization was insufficient and switched to M2 macrophage polarization. Besides costimulation, however, additional roles of CD86 are not known or well-studies. Therefore, we hypothesized that upregulating CD86 on macrophages might promote T. marneffei defense. METHODS: A lentivirus vector, called Lenti-CD86, was used to infect THP-1 cells to overexpress secretory CD86. Through killing assay, nitric oxide detection, and cytokine detection, the capacity of THP-1 macrophages to phagocytose and kill T. marneffei was examined. RESULTS: In the current study, Lenti-CD86 transfection of THP-1 cells resulted in a signifant expression of CD86. Additionally, the THP-1 macrophages stably transfected with Lenti-CD86 showed higher nitric oxide and IL-1ß production, faster polarization, and stronger phagocytosis and killing capabilities than the non-transfected or control virus transfected cells. CONCLUSION: Our study shows that lentivirus-mediated CD86 overexpression improves THP-1 macrophages' capacity to phagocytose and eliminate T. marneffei.

Talaromyces marneffei Infections in 8 Chinese Children with Inborn Errors of Immunity.

PURPOSE: Talaromyces marneffei (TM) is an opportunistic fungus leading to multi-organ damages and poor prognosis in immunocompromised individuals. TM infections in children are rare and our knowledge to TM infection is insufficient. To investigate the clinical characteristics of TM-infected children and to explore the underlying mechanisms for host against TM, we analysed TM-infected patients diagnosed in our hospital. METHODS: Eight patients with TM infections have been identified in Shenzhen Children's Hospital during 2017-2021. Clinical data were collected from medical records. Immunological features were evaluated by flow cytometry. Literatures were also reviewed to summarize the reported inborn errors of immunity (IEIs) with TM infections. RESULTS: All 8 children were HIV-negative. The most common symptom of TM infections was fever (8/8), followed by weight loss (7/8), pneumonia (7/8), hepatomegaly (7/8), splenomegaly (6/8), anemia (6/8), lymphadenopathy (5/8), thrombocytopenia (3/8), diarrhea (3/8), rashes or skin lesions (3/8), and osteolytic lesions (1/8). Five children died during the follow-ups. CD3+ T cells were decreased in 6 patients. Eight patients had reduced natural killer cells. All patients went gene sequencing and were finally diagnosed as IEIs, including STAT1 gain-of-function, IL-2 receptor common gamma chain deficiency, adenosine deaminase deficiency, CD40 ligand deficiency, and STAT3 deficiency. Another 4 types of IEIs (CARD9, IFN-γ receptor 1, RelB, and NFKB2 deficiency), have been reported with TM infections based on literature review. CONCLUSION: TM infections resulted in systemic injuries and high mortality. The spectrum of IEIs underlying TM infections indicated that T cell-mediated immunity, IFN-γ, IL-17 signalings and NF-κB pathways were important for host responses against TM infection. In reverse, for HIV-negative children without other secondary immunodeficiencies, IEIs should be considered in TM-infected children.

A mass in the upper abdomen derived from Talaromyces marneffei infected lymphadenopathy: a case report.

BACKGROUND: An upper abdominal mass without tenderness often indicates a benign or malignant tumor once liver or spleen hyperplasia has been excluded. A lymphadenopathic mass from Talaromyces marneffei infection is rare. CASE PRESENTATION: We report the case of a 29-year-old human immunodeficiency virus (HIV) infected man who presented with an upper abdominal mass and without any symptoms related with infection. Histopathology and next-generation sequencing (NGS) following biopsy of the mass confirmed T. marneffei-infected lymphadenopathy, and the patient was successfully treated with amphotericin B and itraconazole. CONCLUSIONS: This case report suggests that potential fungal infection should be considered during the diagnostic workup of a mass in clinical practice.

Talaromyces marneffei promotes M2-like polarization of human macrophages by downregulating SOCS3 expression and activating the TLR9 pathway.

Little is known about how Talaromyces marneffei, a thermally dimorphic fungus that causes substantial morbidity and mortality in Southeast Asia, evades the human immune system. Polarization of macrophages into fungal-inhibiting M1-like and fungal-promoting M2-like types has been shown to play an important role in the innate immune response against fungal pathogens. This mechanism has not been defined for T. marneffei. Here, we demonstrated that T. marneffei promotes its survival in human macrophages by inducing them toward M2-like polarization. Our investigations of the mechanism revealed that T. marneffei infection led to SOCS3 protein degradation by inducing tyrosine phosphorylation, thereby relieving the inhibitory effect of SOCS3 on p-STAT6, a key factor for M2-like polarization. Our SOCS3-overexpression experiments showed that SOCS3 is a positive regulator of M1-like polarization and plays an important role in limiting M2-like polarization. Furthermore, we found that inhibition of the TLR9 pathway partially blocked T. marneffei-induced M2-like polarization and significantly enhanced the killing activity of macrophages against T. marneffei. Collectively, these results reveal a novel mechanism by which T. marneffei evades the immune response of human macrophages.

Intertwined Biosynthesis of Skyrin and Rugulosin A Underlies the Formation of Cage-Structured Bisanthraquinones.

Skyrin and rugulosin A are bioactive bisanthraquinones found in many fungi, with the former suggested as a precursor of hypericin (a diversely bioactive phytochemical) and the latter characterized by its distinct cage-like structure. However, their biosynthetic pathways remain mysterious, although they have been characterized for over six decades. Here, we present the rug gene cluster that governs simultaneously the biosynthesis of skyrin and rugulosin A in Talaromyces sp. YE3016, a fungal endophyte residing in Aconitum carmichaeli. A combination of genome sequencing, gene inactivation, heterologous expression, and biotransformation tests allowed the identification of the gene function, biosynthetic precursor, and enzymatic sets involved in their molecular architecture constructions. In particular, skyrin was demonstrated to form from the 5,5'-dimerization of emodin radicals catalyzed by RugG, a cytochrome P450 monooxygenase evidenced to be potentially applicable for the (chemo)enzymatic synthesis of dimeric polyphenols. The fungal aldo-keto reductase RugH was shown to be capable of hijacking the closest skyrin precursor (CSP) immediately after the emodin radical coupling, catalyzing the ketone reduction of CSP to inactivate its tautomerization into skyrin and thus allowing for the spontaneous intramolecular Michael addition to cyclize the ketone-reduced form of CSP into rugulosin A, a representative of diverse cage-structured bisanthraquinones. Collectively, the work updates our understanding of bisanthraquinone biosynthesis and paves the way for synthetic biology accesses to skyrin, rugulosin A, and their siblings.

Cysteine Engineering of an Endo-polygalacturonase from Talaromyces leycettanus JCM 12802 to Improve Its Thermostability.

Thermostable enzymes have many advantages for industrial applications. Therefore, in this study, computer-aided design technology was used to improve the thermostability of a highly active endo-polygalacturonase from Talaromyces leycettanus JCM12802 at an optimal temperature of 70 °C. The melting temperature and specific activity of the obtained mutant T316C/G344C were increased by 10 °C and 36.5%, respectively, compared with the wild-type enzyme. The crystal structure of the T316C/G344C mutant showed no formation of a disulfide bond between the introduced cysteines, indicating a different mechanism than the conventional mechanism underlying improved enzyme thermostability. The cysteine substitutions directly formed a new alkyl hydrophobic interaction and caused conformational changes in the side chains of the adjacent residues Asn315 and Thr343, which in turn caused a local reconstruction of hydrogen bonds. This method greatly improved the thermostability of the enzyme without affecting its activity; thus, our findings are of great significance for both theoretical research and practical applications.

Penicillium and Talaromyces Species as Postharvest Pathogens of Pear Fruit (Pyrus communis) in Serbia.

Pears are one of the oldest and the third most important fruit species grown in temperate regions. They are consumed because of their nutritional and health benefits, in fresh form or as various processed products. This article resolves the etiology of the Penicillium-like mold symptoms on pear fruits in Serbia. Samples of pear fruits with blue mold and other Penicillium-like mold symptoms were collected in Serbia from 2016 to 2019, from four storages. The recovered isolates were identified and characterized according to a polyphasic approach. Morphological and physiological analyses were performed on three media and five temperatures, respectively. Four loci (internal transcribed spacer, beta-tubulin, calmodulin, and DNA-dependent RNA polymerase II second largest subunit) were used for sequencing, genetic identification, and phylogenetic analyses. The results of the identification by conventional and molecular methods were in agreement, and they revealed that the obtained isolates belong to five species: Penicillium crustosum, P. expansum, P. italicum, Talaromyces minioluteus, and T. rugulosus. In a pathogenicity test, P. crustosum, P. expansum, T. minioluteus, and T. rugulosus produced decay on artificially inoculated pear fruits, and P. italicum induced tissue response lesions. The results of this study are the first reports of T. minioluteus and T. rugulosus as postharvest pear pathogens. Also, these are the first world records of T. minioluteus, T. rugulosus, and P. italicum on fruits of European pear. Furthermore, this is the first finding of P. crustosum, P. expansum, P. italicum, T. minioluteus, and T. rugulosus on pear fruit in Serbia.

Gastrointestinal manifestations of Talaromyces marneffei infection in an HIV-infected patient rapidly verified by metagenomic next-generation sequencing: a case report.

BACKGROUND: The manifestation of Talaromyces marneffei infection in some HIV-infected patients may be atypical. Cases with gastrointestinal involvement have rarely been reported. It is hard to make a diagnosis when patients are lacking the characteristic rash and positive blood culture. CASE PRESENTATION: Here, we described a patient living with HIV who complained of fever and abdominal pain, and was rapidly diagnosed with Talaromyces marneffei infection by metagenomic next-generation sequencing (mNGS) using formalin-fixation and paraffin-embedded (FFPE) samples of omentum majus tissue. We also reviewed reported related cases. CONCLUSIONS: Talaromyces marneffei is an unusual cause of clinical presentations involving obvious abdominal pain and lower gastrointestinal bleeding, but can be included in the differential diagnosis. As an important diagnostic tool, the significance of mNGS using FFPE samples of lesions provides a more targeted diagnosis.

Pneumonia caused by Talaromyces marneffei in an epidermal growth factor receptor (EGFR) mutation-positive advanced lung adenocarcinoma patient: a case report.

Talaromyces marneffei (T. marneffei) is a pathogenic, thermally dimorphic fungus that can cause invasive infection and significant morbidity in immunocompromised patients, especially those with human immunodeficiency virus (HIV) or other immune defects. Currently, T. marneffei infection is understood to be not limited only to immunodeficient patients, as cases of immunocompromised patients or immunocompetent patients associated with T. marneffei infection have been increasingly reported in recent years. The exact mechanism is not yet clear. This study reports a case of an advanced lung adenocarcinoma patient with T. marneffei infection. The patient is a 59-year-old female with a 3-month history of coughing, expectoration, and progressive dyspnea. Computed tomography (CT) scans showed a mass in the left lower lung, multiple plaques and nodules in both lungs, and left pleural effusion. The patient was diagnosed with T. marneffei infection, as T. marneffei was found in both the bronchoalveolar lavage fluid (BALF) and the sputum. According to the pathology of the left lung lesion by transbronchial lung biopsy (TBLB) and contrast-enhanced brain magnetic resonance imaging (MRI), the patient was diagnosed with epidermal growth factor receptor (EGFR) mutation-positive stage Ⅳ lung adenocarcinoma (T4N3M1c). She received intravenous liposomal amphotericin B and oral itraconazole as anti-fungal treatments, meanwhile, icotinib was used as an anti-tumor treatment. Following treatment, CT re-examination showed that the mass was remarkably absorbed, and some of the lung nodules had disappeared. No relapse of T. marneffei infection was found during the follow-up. This case indicates that patients with malignant lung tumors may possibly become infected with T. marneffei. Sequential treatment of amphotericin liposome B followed by itraconazole is effective for lung cancer patients with T. marneffei infection.

Unusual disseminated Talaromyces marneffei infection mimicking lymphoma in a non-immunosuppressed patient in East China: a case report and review of the literature.

BACKGROUND: Talaromyces marneffei infection is an important opportunistic infection associated with acquired immune deficiency syndrome (AIDS). However, it is unusual in patients with non-AIDS and other non-immunosuppressed conditions. We report a case of delayed diagnosis of disseminated T. marneffei infection in non-AIDS, non-immunosuppressive and non-endemic conditions. CASE PRESENTATION: We describe a previously healthy 24-year-old man who complained of a 3-month history of intermittent diarrhea and a recent week of uncontrollable high fever. The HIV antibody test was negative. Enhanced abdominal computed tomography (CT) and integrated 18F-2-deoxy-2-fluoro-D-glucose position emission tomography/computed tomography (FDG PET/CT) both suspected malignant lymphoma. However, a large number of yeast-like cells were found in macrophages in cervical lymph node samples by hematoxylin and eosin stain and silver hexamine stain. Subsequent blood culture suggested T. marneffei infection. Metagenomic Next Generation Sequencing (mNGS) results suggested T. marneffei as the dominant pathogen. Unfortunately, the patient continued to develop acute liver failure and died due to adverse events associated with amphotericin B. CONCLUSIONS: Early diagnosis in HIV-negative patients who are otherwise not immunosuppressed and endemic poses a serious challenge. T. marneffei infection is an FDG-avid nonmalignant condition that may lead to false-positive FDG PET/CT scans. Nevertheless, integrated FDG PET/CT is necessary in patients with fever of unknown origin in the early period to perform earlier biopsy for histopathology and culture in highly avid sites and to avoid delays in diagnosis and treatment.

Methylcitrate cycle gene MCD is essential for the virulence of Talaromyces marneffei.

Talaromyces marneffei (T. marneffei), which used to be known as Penicillium marneffei, is the causative agent of the fatal systemic mycosis known as talaromycosis. For the purpose of understanding the role of methylcitrate cycle in the virulence of T. marneffei, we generated MCD deletion (ΔMCD) and complementation (ΔMCD+) mutants of T. marneffei. Growth in different carbon sources showed that ΔMCD cannot grow on propionate media and grew slowly on the valerate, valine, methionine, isoleucine, cholesterol, and YNB (carbon free) media. The macrophage killing assay showed that ΔMCD was attenuated in macrophages of mice in vitro, especially at the presence of propionate. Finally, virulence studies in a murine infection experiment revealed attenuated virulence of the ΔMCD, which indicates MCD is essential for T. marneffei virulence in the host. This experiment laid the foundation for the further study of the specific mechanisms underlying the methylcitrate cycle of T. marneffei and may provide suitable targets for new antifungals.

A unique aspartyl protease gene expansion in Talaromyces marneffei plays a role in growth inside host phagocytes.

Aspartyl proteases are a widely represented class of proteolytic enzymes found in eukaryotes and retroviruses. They have been associated with pathogenicity in a range of disease-causing microorganisms. The dimorphic human-pathogenic fungus Talaromyces marneffei has a large expansion of these proteases identified through genomic analyses. Here we characterize the expansion of these genes (pop - paralogue of pep) and their role in T. marneffei using computational and molecular approaches. Many of the genes in this monophyletic family show copy number variation and positive selection despite the preservation of functional regions and possible redundancy. We show that the expression profile of these genes differs and some are expressed during intracellular growth in the host. Several of these proteins have distinctive localization as well as both additive and epistatic effects on the formation of yeast cells during macrophage infections. The data suggest that this is a recently evolved aspartyl protease gene family which affects intracellular growth and contributes to the pathogenicity of T. marneffei.

A new polyketide purpurogenic acid: the activated production of polyketides by the diethyl sulphate mutagenesis of marine-derived Penicillium purpurogenum G59.

A new polyketide, purpurogenic acid (1), and two known polyketides, (-)-mitorubrin (2) and (-)-mitorubrinol (3), were isolated from a fungal mutant derived from the diethyl sulphate (DES) mutagenesis of marine-derived Penicillium purpurogenum G59. The planar structure of new 1 was elucidated by spectroscopic methods and the absolute configuration was assigned on the basis of [α]D and CD data. In our preliminary MTT assay, 1 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells with the inhibition rates of 52.7, 78.8, 38.4 and 35.3% at the 100 µg/mL, respectively.