Comparative proteome analysis identifies species-specific signature proteins in Aspergillus pathogens.

Aspergillus flavus and Aspergillus fumigatus are important human pathogens that can infect the lung and cornea. During infection, Aspergillus dormant conidia are the primary morphotype that comes in contact with the host. As the conidial surface-associated proteins (CSPs) and the extracellular proteins during the early stages of growth play a crucial role in establishing infection, we profiled and compared these proteins between a clinical strain of A. flavus and a clinical strain of A. fumigatus. We identified nearly 100 CSPs in both Aspergillus, and these non-covalently associated surface proteins were able to stimulate the neutrophils to secrete interleukin IL-8. Mass spectrometry analysis identified more than 200 proteins in the extracellular space during the early stages of conidial growth and germination (early exoproteome). The conidial surface proteins and the early exoproteome of A. fumigatus were enriched with immunoreactive proteins and those with pathogenicity-related functions while that of the A. flavus were primarily enzymes involved in cell wall reorganization and binding. Comparative proteome analysis of the CSPs and the early exoproteome between A. flavus and A. fumigatus enabled the identification of a common core proteome and potential species-specific signature proteins. Transcript analysis of selected proteins indicate that the transcript-protein level correlation does not exist for all proteins and might depend on factors such as membrane-anchor signals and protein half-life. The probable signature proteins of A. flavus and A. fumigatus identified in this study can serve as potential candidates for developing species-specific diagnostic tests. KEY POINTS: • CSPs and exoproteins could differentiate A. flavus and A. fumigatus. • A. fumigatus conidial surface harbored more antigenic proteins than A. flavus. • Identified species-specific signature proteins of A. flavus and A. fumigatus.

Hsa-miR-150-5p inhibits Wnt-ß-catenin signaling in human corneal epithelial stem cells.

Purpose: In our earlier study, we identified hsa-miR-150-5p as a highly expressed miRNA in enriched corneal epithelial stem cells (CESCs). In this study, we aimed to understand the molecular regulatory function of hsa-miR-150-5p in association with the maintenance of stemness in CESCs. Methods: The target mRNAs of hsa-miR-150-5p were predicted and subjected to pathway analysis to identify targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-150-5p mimic, inhibitor, or scrambled sequence using Lipofectamine 3000. The transfected cells were analyzed to determine (i) their colony-forming potential; (ii) the expression levels of stem cell (SC) markers/transcription factors (ABCG2, NANOG, OCT4, KLF4, and ΔNp63), the differentiation marker (Cx43), and the hsa-miR-150-5p predicted targets (JARID2, INHBA, AKT3, and CTNNB1) by qPCR; and (iii) the expression levels of ABCG2, p63α, Cx43, JARID2, AKT3, p-AKT3, ß-catenin, and active ß-catenin by immunofluorescence staining and/or western blotting. Results: The ectopic expression level of hsa-miR-150-5p increased the colony-forming potential (8.29% ± 0.47%, p < 0.001) with the ability to form holoclone-like colonies compared with the control (1.8% ± 0.47%). The mimic-treated cells had higher expression levels of the SC markers but reduced expression levels of Cx43 and the targets of hsa-miR-150-5p that are involved in the Wnt-ß-catenin signaling pathway. The expression levels of ß-catenin and active ß-catenin in the inhibitor-transfected cells were higher than those in the control cells, and the localized nuclear expression indicated the activation of Wnt signaling. Conclusions: Our results indicate a regulatory role for hsa-miR-150-5p in the maintenance of CESCs by inhibiting the Wnt signaling pathway.

Hsa-miR-143-3p inhibits Wnt-ß-catenin and MAPK signaling in human corneal epithelial stem cells.

Our previous study demonstrated hsa-miR-143-3p as one of the highly expressed miRNAs in enriched corneal epithelial stem cells (CESCs). Hence this study aims to elucidate the regulatory role of hsa-miR-143-3p in the maintenance of stemness in CESCs. The target genes of hsa-miR-143-3p were predicted and subjected to pathway analysis to select the targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-143-3p mimic, inhibitor or scrambled sequence using Lipofectamine 3000. The transfected cells were analysed for (i) colony forming potential, (ii) expression of stem cell (SC) markers/ transcription factors (ABCG2, NANOG, OCT4, KLF4, ΔNp63), (iii) differentiation marker (Cx43), (iv) predicted five targets of hsa-miR-143-3p (DVL3, MAPK1, MAPK14, KRAS and KAT6A), (v) MAPK signaling regulators and (vi) Wnt-ß-catenin signaling regulators by qPCR, immunofluorescence staining and/or Western blotting. High expression of hsa-miR-143-3p increased the colony forming potential (10.04 ± 1.35%, p < 0.001) with the ability to form holoclone-like colonies in comparison to control (3.33 ± 0.71%). The mimic treated cells had increased expression of SC markers but reduced expression of Cx43 and hsa-miR-143-3p targets involved in Wnt-ß-catenin and MAPK signaling pathways. The expression of ß-catenin, active ß-catenin and ERK2 in hsa-miR-143-3p inhibitor transfected cells were higher than the control cells and the localized nuclear expression indicated the activation of Wnt and MAPK signaling. Thus, the probable association of hsa-miR-143-3p in the maintenance of CESCs through inhibition of Wnt and MAPK signaling pathways was thus indicated.

Expression of immune response genes in human corneal epithelial cells interacting with Aspergillus flavus conidia.

BACKGROUND: Aspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics. METHODS: Human corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes. RESULTS: Different morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells. CONCLUSIONS: Combining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.

Human Corneal Epithelial Cells Internalize Aspergillus flavus Spores by Actin-Mediated Endocytosis.

Human corneal epithelial (HCE) cells play a significant role in the innate immune response by secreting cytokines and antimicrobial peptides when they encounter fungal pathogens. But the detailed mechanism of attachment and engulfment of the fungal conidia by HCE cells is not well understood. Here, we show the phagocytosis of Aspergillus flavus conidia by RCB2280 cells and primary HCE cultures using confocal microscopy and proteomic analysis of conidium-containing phagosomes. Phalloidin staining showed actin polymerization, leading to an actin ring around engulfed conidia. Cytochalasin D inhibited the actin-mediated endocytosis of the conidia. Immunolabeling of the early endosomal markers CD71 and early endosomal antigen (EEA1) and the late endosomal markers lysosome-associated membrane protein 1 (LAMP1), Rab7, and cathepsin G showed that endosomal proteins were recruited to the site of conidia and showed maturation of the conidium-containing phagosomes. Lysotracker red DND 99 labeling showed the acidification of the phagosomes containing conidia. Phagosome-specific proteome analysis confirmed the recruitment of various phagosomal and endosomal proteins to the conidium-containing phagosomes. These results show that the ocular surface epithelium contributes actively to antifungal defense by the phagocytosis of invading fungal conidia.

Periconia species - An unusual fungal pathogen causing mycotic keratitis.

PURPOSE: The purpose of this study is to determine the epidemiology, risk factors, clinical features, and treatment outcome of molecularly diagnosed Periconia keratitis. METHODS: Clinical records of all culture proven fungal ulcers with molecular identification suggestive of Periconia species who presented to a single tertiary referral center from January 2012 to December 2013 were retrospectively analysed. RESULTS: Among 1356 cases of keratomycosis, 8 (0.6%) patients were affected due to Periconia species. The mean age of presentation was 59 years with males (n = 6; 75%) were more commonly affected than females (n = 2; 25%). Significant history of trauma was present only in one patient. The infiltrate size was less than 5 mm in majority of patients 75% (n = 6). 50% (n = 4) responded to antifungal, 12.5% (n = 1) responded to antibacterial, 12.5% (n = 1) required therapeutic penetrating keratoplasty, 25% (n = 2) lost to follow up after first visit. The mean duration of treatment in healed cases was 20 days. CONCLUSION: This is the first report on Periconia sp causing human corneal ulcer. This study signifies the importance of molecular identification in the diagnosis of rare fungi which will improve our understanding on disease pathology and outcome. Visual prognosis appears good if the infection is diagnosed and topical antifungal interventions started early.

Clinical and demographic study of non-tuberculous mycobacterial ocular infections in South India.

PURPOSE: To describe demographics, risk factors, antibiotic susceptibility, management and outcomes of ocular infections caused by non-tuberculous mycobacteria (NTM). METHODS: A retrospective review of medical case records and microbiology records of patients with ocular infections that were culture positive for non-tuberculous Mycobacteria from January 2014 to December 2018 was done. Antibiotic susceptibility profile was done based on the CLSI guidelines. Laboratory diagnosis for the NTM Species was done by conventional microbiological methods. The species identification was done for stored isolated utilizing polymerase chain reaction targeting 16S rDNA and rpoB gene, followed by DNA sequencing and phylogenetic analysis. RESULTS: Twenty patients with NTM ocular infections were identified during the study period. A majority of cases presented as 12 infectious keratitis (60%) and three suture-related corneal infiltrates (15%). Common risk factors were history of trauma in 9 (45%) patients and history of ocular surgery in 5 (25%) patients. Patients were treated with combination of amikacin and flouroquinolones/chloramphenicol (70%) and surgical interventions were performed in 25% cases. Only twelve isolates were stored and ten isolates were identified as the M. abscessus subsp. abscessus and two isolates as M. abscessus subsp. massiliense by sequencing and phylogenetic analysis. Majority of the NTM were sensitive to amikacin (75%) followed by moxifloxacin, ciprofloxacin, cephotaxime and tobramycin (35%). CONCLUSION: High degree of clinical suspicion, multidrug antibiotic therapy and timely surgical intervention in patients with NTM infections, are advised for better clinical outcomes. Prior ocular trauma, prior ocular surgery and presence of biomaterials were the major predisposing factors. Earlier surgical intervention in cases where abscesses or biomaterials are involved, is necessary for rapid recovery.

Making the decision to donate eyes: Perspectives from the families of the deceased in Madurai, India.

PURPOSE: To identify factors affecting family members' decision whether to donate eye organs. METHODS: A community-based case-control study based on in-home interviews with families of deceased individuals who had or had not donated eye organs, in Madurai district, Tamil Nadu, India. Data collected were knowledge and awareness of eye donations, whether the deceased individual had expressed or pledged willingness to donate, and family members' attitudes and willingness to donate their own eye organs. RESULTS: Seventy-six families of donors and 256 families of non-donors completed the survey. Multivariable analysis showed that the following variables were significantly associated with a donation: age, whether the deceased had registered for eye donation, pre-expressed willingness of deceased to donate, whether family members personally know beneficiaries of eye donations, and higher score on a scale evaluating knowledge and awareness about eye donation. The majority of donors' families (71%) had been encouraged by someone to donate. Among non-donor families, a substantially larger fraction (52.8%) indicated they would have donated had someone reminded or encouraged them to do so, in comparison with those who indicated lack of awareness or knowledge (14.5%). CONCLUSION: Community programs are likely to be effective if they encourage individuals to pledge their eyes or express their willingness to donate their eyes to family members in advance of death; they increase public awareness of the value of eye donation. A friend, family member, neighbor or counselor approaching bereaved families and having a dialogue about eye donation would substantially increase the probability of a decision to donate.

Local Activation of the Alternative Pathway of Complement System in Mycotic Keratitis Patient Tear.

Aspergillus flavus and Fusarium solani are the predominant causative agents of mycotic keratitis in the tropical part of the world. Tear proteins play a major role in the innate immune response against these fungal infections as has been shown by the presence of complement proteins and neutrophil extracellular trap proteins in keratitis patients tear. In this study, we established the presence of the components of the alternate pathway of complement system and their functional state in the tear film of mycotic keratitis patients. The complement proteins namely, C3 and CFH were found only in the open-eye tear of patients but not in control individuals. In vitro analysis showed binding of purified C3b and CFH to fungal spores, which confirmed that the spores can provide a foreign surface for forming the complement complex. Analysis of spore bound tear proteins by mass spectrometry exhibited the presence of known proteins of the alternate pathway complement cascade in keratitis patient tear. Hemolytic assay using rabbit RBC confirmed the presence of a functional alternate pathway of complement cascade in the tear proteome of the patients. The presence of negative regulators, CFH and CFI, in the patient tear indicate that the complement activity is tightly regulated during fungal infection. Mass spectrometry data show vitronectin and clusterin, two known inhibitors of the membrane attack complex only in the patient tear. These data demonstrate the activation of the alternate pathway of complement cascade during the early stages of infection. Interestingly, the production of multiple negative regulators of complement cascade implies the pathogen can effectively evade the host complement system during infection.

MicroRNA Profiling of Highly Enriched Human Corneal Epithelial Stem Cells by Small RNA Sequencing.

The objective of the study was to elucidate the microRNA (miRNA) profile of an enriched human corneal epithelial stem cell (CESC) population in comparison to differentiated central corneal epithelial cells (CCECs) by small RNA sequencing. The CESCs were enriched by differential enzymatic treatment to isolate the basal limbal epithelial cells followed by laser capture microdissection of cells with nucleus to cytoplasm ratio ≥0.7, from donor tissues. Small RNA sequencing was carried out using Illumina NextSeq. 500 platform and the validation of differentially expressed miRNAs by quantitative real-time PCR (qPCR) and locked nucleic acid miRNA in-situ hybridization (LNA-ISH). The sequencing identified 62 miRNAs in CESCs and 611 in CCECs. Six miRNAs: hsa-miR-21-5p, 3168, 143-3p, 10a-5p, 150-5p and 1910-5p were found to be significantly upregulated in enriched CESCs, which was further confirmed by qPCR and LNA-ISH. The expression of hsa-miR-143-3p was exclusive to clusters of limbal basal epithelial cells. The targets of the upregulated miRNAs were predicted to be associated with signaling pathways -Wnt, PI3K-AKT, MAPK and pathways that regulate pluripotency of stem cells, cell migration, growth and proliferation. Further studies are essential to elucidate their functional role in maintenance of stemness. The findings of the study also hypothesize the inherent potential of hsa-miR-143-3p to serve as a biomarker for identifying CESCs.

Dataset for the spore surface proteome and hydrophobin A/RodA proteoforms of A.flavus.

Fungal keratitis is a major sight-threatening corneal infection: and mycotic keratitis is more common in tropical parts of the world including India. Aspergillus flavus and Fusarium are the predominant causative agents of corneal infection. We extracted conidial surface proteins of A. flavus from saprophyte and clinical isolates and analyzed the proteins using high resolution mass spectrometry. The data revealed ecotype specific alteration in surface proteome since the proteome profile of the clinical isolates and saprophyte showed significant differences. Detailed examination of the mass spec data of RodA proteins extracted from polyacrylamide gels revealed the presence of two proteoforms of this protein. We also identified the mechanism of formation of these two isoforms. Detailed analysis of this data and the conclusions derived are described in the article, "Identification of the proteoforms of surface localized Rod A of A. flavus and determination of the mechanism of proteoform generation" [1].

Identification of the proteoforms of surface localized Rod A of Aspergillus flavus and determination of the mechanism of proteoform generation.

Fungal keratitis is a serious, potentially sight-threatening corneal infection that is more prevalent in the tropical parts of the world including India, and A. flavus and Fusarium solani are the predominant etiological agents. The surface of fungal conidia is covered by hydrophobin family proteins, effectively masking the conidial antigens from immune cells. In this study, we report that the outer cell wall layer of A. flavus conidia contain Rod A as well as other hydrophobins, which could be extracted by formic acid. Analysis of these surface proteins by mass spectrometry showed the presence of rodlet forming hydrophobins and other membrane and antigenic proteins. Our analysis revealed that Rod A existed as two proteoforms on the conidial surface. These proteoforms were separated using polyacrylamide gel electrophoresis and the amino acid sequence of these proteoforms was determined by high resolution mass spectrometry. PCR analysis of the mRNA encoding the Rod A showed the retention of intron one, which results in the formation of a truncated proteoform two. This is the first report in which the presence of RodA and its proteoforms and their mechanism of formation has been demonstrated in the corneal pathogenic fungus A. flavus. SIGNIFICANCE: A. flavus is a common fungal pathogen in tropical countries playing a predominant role in causing mycotic keratitis in humans. Surface of fungal conidia is immunologically inert primarily due to the hydrophobin family proteins forming a rodlet layer and masking the conidia from immune cells. In this study we demonstrated the existence two proteoforms of RodA/hydrophobin A and intron retention is shown to be responsible for the formation of one of the proteoforms. In addition, the spore surface proteins of A.flavus corneal isolates and saprophyte are distinctly different, which indicate the spore surface protein profile is ecotype specific. This is the first report showing the presence of two proteoforms of RodA on A.flavus conidial surface and demonstration of the mechanism of formation of the proteoforms.

Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection.

Aspergillus flavus and Fusarium sp. are primary causative agents of keratitis that results in corneal tissue damage leading to vision loss particularly in individuals from the tropical parts of the world. Proteins in the tear film collected from control and keratitis patients was profiled and compared. A total of 1873 proteins from control and 1400 proteins from patient tear were identified by mass spectrometry. While 847 proteins were found to be glycosylated in the patient tear, only 726 were glycosylated in control tear. And, some of the tear proteins showed alterations in their glycosylation pattern after infection. Complement system proteins, proteins specific for neutrophil extracellular traps and proteins involved in would healing were found only in the patient tear. The presence of these innate immune system proteins in the tear film of patients supports the previous data indicating the involvement of neutrophil and complement pathways in antifungal defense. High levels of wound healing proteins in keratitis patient tear implied activation of tissue repair during infection. The early appearance of the host defense proteins and wound healing response indicates that tear proteins could be used as an early marker system for monitoring the progression of pathogenesis. Identification of negative regulators of the above defense pathways in keratitis tear indicates an intricate balance of pro and anti-defense mechanisms operating in fungal infection of the eye. SIGNIFICANCE: Tear proteins from control and mycotic keratitis patients were separated into glycoproteins and non-glycosylated proteins and then identified by mass spectrometry. Tear proteins from keratitis patients showed alteration in the glycosylation pattern indicating the alteration of glycosylation machinery due to infection. Neutrophil extracellular traps specific proteins, complement pathway proteins, as well as wound healing proteins, were found only in patient tear showing the activation of antifungal defense in the patient tear. Negative regulators of these defense pathways were also found in patient tear indicating a fine balance between pathogen clearance and host tissue destruction during fungal infection depending upon the individual specific host - pathogen interaction. This understanding could be used to predict the progression and outcome of infection.

A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis.

Understanding the molecular mechanisms that regulate the corneal epithelial stem cells (CESCs) in maintaining corneal homeostasis remains elusive largely due to the lack of a specific marker for their isolation. This study aims to enrich CESCs from human donor limbal epithelium and to evaluate the level of enrichment based on expression of ΔNp63α, a putative CESC marker. A two-stage enrichment of CESCs was carried out. (a) The limbal basal epithelial cells were isolated by differential enzymatic treatment and five-fold enrichment was achieved from 2% of CESCs present in the total limbal epithelium. The CESCs were quantified on the basis of two parameters-high expression of p63/ABCG2 and nucleus to cytoplasmic (N/C) ratio ≥0.7. (b) Cytospin smears of isolated basal cells were Giemsa stained and cells with N/C ratio ≥0.7 were separated by laser capture microdissection. This strategy resulted in an enrichment of CESCs to 78.57% based on two-parameter analysis using p63 and 76.66% using ABCG2. RT-PCR was carried out for ΔNp63 isoforms (α, ß, and γ) and connexin-43, with GAPDH for normalization. The expression of ΔNp63α was restricted to the enriched population of CESCs in contrast to its absence in limbal basal cells with N/C ratio <0.7 and CCECs. The unique expression of ΔNp63α and 5.9-fold reduced connexin-43 expression in the enriched population of CESCs indicates its high purity. Further analysis of these cells will help in elucidating the molecular mechanisms associated with stemness and also in identifying a specific marker for CESCs.

Data set of Aspergillus flavus induced alterations in tear proteome: Understanding the pathogen-induced host response to fungal infection.

Fungal keratitis is one of the leading causes of blindness in the tropical countries affecting individuals in their most productive age. The host immune response during this infection is poorly understood. We carried out comparative tear proteome analysis of Aspergillus flavus keratitis patients and uninfected controls. Proteome was separated into glycosylated and non-glycosylated fractions using lectin column chromatography before mass spectrometry. The data revealed the major processes activated in the human host in response to fungal infection and reflected in the tear. Extended analysis of this dataset presented here complements the research article entitled "Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection [1]" (Jeyalakhsmi Kandhavelu, Naveen Luke Demonte, Venkatesh Prajna Namperumalsamy, Lalitha Prajna, Chitra Thangavel, Jeya Maheshwari Jayapal, Dharmalingam Kuppamuthu, 2016). The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE:PXD003825.

Topical 5% Natamycin With Oral Ketoconazole in Filamentous Fungal Keratitis: A Randomized Controlled Trial.

PURPOSE: To assess the role of additive oral antifungal therapy in deep keratitis caused by filamentous fungi. DESIGN: A randomized, masked, double-blind clinical trial. METHODS: All patients presenting with culture-positive fungal keratitis with a size measuring 2 to 60 mm2 and involving more than 50% of stromal depth were enrolled in 1 of the 2 treatment arms. Group A received 5% natamycin, whereas Group B was given 200mg of oral ketoconazole twice a day in addition to 5% natamycin. Patients were followed up for 4 weeks. Liver function was assessed at baseline and at exit. Tests for significance included t test to compare the means of continuous variables, chi-square and Fisher's exact tests for comparing categorical variables and Kaplan-Meier procedure to estimate the survival rate. RESULTS: Of the 115 patients enrolled, 108 completed the study. Fifty-eight patients were in group A and 57 in group B. There was no significant difference in baseline characteristics or in ulcer characteristics between the 2 groups. In group A, 68.5% of the patients responded favorably to medical therapy, whereas in group B, 72.2% responded favorably. There was no statistically significant difference in healing between the 2 groups (P = -0.618). All patients had normal liver functions during the study. CONCLUSIONS: Although safe, oral ketoconazole did not add significant benefit to topical natamycin therapy in treating deep fungal keratitis. The efficacy of newer antifungal agents and drug delivery routes needs to be explored.