Characterization of a novel yeast phase-specific antigen expressed during in vitro thermal phase transition of Talaromyces marneffei

Abstract

Talaromyces marneffei is a dimorphic fungus that has emerged as an opportunistic pathogen particularly in individuals with HIV/AIDS. Since its dimorphism has been associated with its virulence, the transition from mold to yeast-like cells might be important for fungal pathogenesis, including its survival inside of phagocytic host cells. We investigated the expression of yeast antigen of T. marneffei using a yeast-specific monoclonal antibody (MAb) 4D1 during phase transition. We found that MAb 4D1 recognizes and binds to antigenic epitopes on the surface of yeast cells. Antibody to antigenic determinant binding was associated with time of exposure, mold to yeast conversion, and mammalian temperature. We also demonstrated that MAb 4D1 binds to and recognizes conidia to yeast cells' transition inside of a human monocyte-like THP-1 cells line. Our studies are important because we demonstrated that MAb 4D1 can be used as a tool to study T. marneffei virulence, furthering the understanding of the therapeutic potential of passive immunity in this fungal pathogenesis.

Figure 1

The specific immunoreactivity of MAb 4D1 against yeast phase antigen of T. marneffei. (a) Indirect ELISA. (b) Corresponding bright fields and indirect IFA showing immunoreactivity of MAb 4D1 against T. marneffei mold and yeast culture, the arrow indicated the fission yeast cell. TM; Talaromyces marneffei, SS; Sporothrix schenckii, HC; Histoplasma capsulatum, CA; Candida albicans, CK; Candida krusei, CG; Candida glabrata, CN; Cryptococcus neoformans, AF; Aspergillus fumigatus, PC; Penicillium citrinum, PI; Pythium insidiosum, PB; Pseudallescheria boydii.

Figure 2

Biochemical characteristics of T. marneffei cytoplasmic yeast antigen or TM CYA. (a) SDS-PAGE showing the antigenic profile of the TM CYA stained with Coomassie blue. (b) Native TM CYA recognized by MAb 4D1 with a molecular weight ranging 50–150 kDa with the diffuse binding characteristic of “broad high molecular mass smear” (c) TM CYA recognized by HRP-GNA with the molecular weight approximately 72 kDa. (M: Pre-staining molecular weight marker; kDa).

Figure 3

The effect of N-linked deglycosylation of TM CYA on antigenic recognition by MAb 4D1. (a) Native recognition patterns of TM CYA by MAb 4D1. (b) Recognition patterns of TM CYA by MAb 4D1 after digested with PNGase F at 90 min. (c) Complete recognition patterns of TM CYA by MAb 4D1 after digestion with PNGase F at 180 min. (d) T. marneffei cytoplasmic mycelial antigen or TM CMA used as a negative control.

Figure 4

The effect of proteinase K treated TM CYA showing altered antigenic recognition by MAb 4D1. (a) Native recognition patterns of TM CYA by MAb 4D1. (b) Recognition patterns of TM CYA by MAb 4D1 after digestion with proteinase K at 30 min. (c) Recognition patterns of TM CYA by MAb 4D1 after digestion with proteinase K at 60 min. (d) Proteinase K treated T. marneffei cytoplasmic mycelial antigen or TM CMA used as a negative control.

Figure 5

Immunoreactivity of MAb 4D1 against cytoplasmic antigen from T. marneffei subjected to BHI culture temperature shift at (a) 37 °C, and (b) 25 °C after 0, 24, 30, 36, 42, 48, 54, and 60 h, respectively. (c) Culture temperature shift from 25 to 37 °C, and from 37 to 25 °C, with the arrow indicating shifting time point.

Figure 6

The morphological transition of T. marneffei yeast cell in 1% proteose peptone. (a1) T. marneffei yeast cell after 48 h of conidial inoculation in 1% proteose peptone stained with lactophenol cotton blue, the arrow indicating the fission yeast cell.(a2,a3) Corresponding bright fields and indirect IFA showing the immunoreactivity of MAb 4D1 against T. marneffei yeast cells cultured in 1% proteose peptone. (b) The percentages of MAb 4D1 surface labeled fluorescent positive yeast cells, each bar represents the mean ± SD of 3 sets of independent determinations. An asterisk (*) indicates a statistically significant difference (p < 0.05) between transit time of 24 to 48 h and 96 to 108 h.

Figure 7

Reactivity of anti-yeast specific MAb 4D1 against T. marneffei yeast cells’ uptake by THP-1 cells at different time points as studied by flow cytometer. (a) The scatter plot panel illustrates the distribution patterns of the fungal particles analyzed according to forward scatter and side scatter of each P.I. time point. Whereas, the histograms demonstrate the relative fluorescence intensity shift of MAb 4D1 positive yeast cell of each P.I. time point. (b) The averages % of positive yeast cells ± SD of 3 sets of independent experiments was shown for each time point. An asterisk (*) indicates a statistically significant difference (p < 0.05) between transit times at initial expression between 12 to 24 h and maximal expression of 24 to 36 h (P.I. post of internalization).

Figure 8

Internalization of T. marneffei conidia by THP-1 cells and intracellular germination to give rise to fission yeast cells. THP-1 cells were incubated with FITC-labeled T. marneffei conidia for 2 h, and then washed to remove unbound conidia. The THP-1 cells were further incubated for an additional time points 0, 8, 12, 24 and 36 h. At each time point, T. marneffei yeast cells were labeled with MAb 4D1 and Alexaflor 555 conjugated goat anti-mouse IgG antibody. From left to right: fluorescence image showing the green channel (FITC labeled conidia); fluorescence image of the red channel (MAb 4D1 positive yeast cells); THP-1 nuclei were stained with DAPI (blue); a merged channel showing the overlapping of triple images. Bars, 5 µm.

Figure 9

Pro-inflammatory cytokine release from THP-1 macrophages co-cultured with conidia from T. marneffei at different time points. (a) Interleukin 1β (IL-1β), (b) tumor necrosis factor alpha (TNF-α) and (c) interleukin 6 (IL-6). Measurements of TNF-α, IL-6, and IL-1β levels were achieved using supernatants pooled from three sets of experiments and expressed as mean ± SD. An asterisk (*) indicates a significant difference (p < 0.05) from supernatants at 8, 24 and 48 h compared with 0 h.