Fig 1.
Cbp1 is conserved among closely related thermally dimorphic fungi that are intracellular pathogens of humans.
A. Protein alignments of Hc G217B Cbp1 and closely related homologs. The sequences of the Histoplasma homologs (G217B, G186AR, G184A, H88, and H143) highlighted in green, Paracoccidioides homologs (Pb03, Pb18, Pb01, PbCNH, and Pb300) highlighted in blue, and the Emergomyces and Emmonsia homologs (Es. Africanus, E. crescens, Es. orientalis, Es. pasteurianus_1, and Es. pasteurianus_2) highlighted in yellow are shown. The start of the mature peptide as determined by mass spectrometry of the Hc G217B, Hc G186AR, Hc H88, Pb03, Es. africanus, E. crescens, Es. orientalis, Es. pasteurianus_1, and Es. pasteurianus_2 homologs is indicated with a black box for each. The six cysteine residues that form intramolecular disulfide bonds are indicated, as well as the percent identity and similarity to the Hc G217B, the full length of the immature protein, and the molecular weight and pI of the mature peptides. B. Species tree that tracks the acquisition and loss events of Cbp1 in Ajellomycetaceae species that are human fungal pathogens.
Fig 2.
Crystal structures of H88 and Pb03 Cbp1 reveal a novel “binocular” fold.
A. Structures of Pb03 and H88 Cbp1 as determined by crystallographic methods are shown in native dimer form and as a monomer. B. Asymmetric unit of H88 Cbp1 shows a dimer of dimers that interact though the C-terminal helical bundles. The two dimers in the H88 asymmetric unit are aligned and the RMSD score was determined to be 0.755 angstroms. C. Structural alignments of Pb03 and H88 Cbp1 structures reveal a highly similar fold with an RMSD value of 1.686 angstroms. D. Prior experiments in our laboratory determined that the purple residues are required for secretion, the orange residues are absolutely required for lysis, and the yellow residues contribute to maximal lysis [32]. When these residues are modeled on the Pb03 or H88 crystal structure (top two rows), the purple residues face inwards and the orange and yellow residues are predicted to be on the surface of the protein facing outwards. In contrast, modeling the purple, orange, and yellow residues on the NMR structure does not result in a consistent pattern.
Table 1.
Data collection and refinement statistics.
Fig 3.
Emergomyces species contain Cbp1 homologs in their genomes that can be expressed and secreted by the Hc G217B strain but are insufficient to confer macrophage lysis.
A. InstantBlue stained SDS-PAGE gel of culture supernatants of Hc G186AR, Hc H88, Hc G217B, Hc G217B cbp1 mutant, and Hc G217B cbp1 mutant expressing the G217B, P. americana (Pb03), E. crescens, Es. orientalis, Es. pasteurianus_1, Es. pasteurianus_2, or Es. africanus Cbp1. White asterisk denotes the bands that were excised for mass spectrometric analysis to confirm their identity as the appropriate Cbp1. Molecular weight standards (kD) are marked on the left. B. Hc cbp1 mutant isolates expressing the Emergomyces Cbp1 homologs were used to infect BMDMs at an MOI of 1 as described in the Materials and Methods. “WT” indicates the Hc G217B ura5 mutant carrying the URA5 gene on a plasmid. LDH release was used to quantify percent host cell lysis. C. Intracellular growth of Hc cbp1 mutant isolates expressing the Emergomyces Cbp1 homologs was determined from BMDM infection as described in the Materials and Methods. “WT” indicates the Hc G217B ura5 mutant carrying the URA5 gene on a plasmid. At 48 hpi, CFU counts were terminated for the WT Hc infection because the extent of host-cell lysis made it difficult to distinguish between intracellular and extracellular growth of Hc. D. InstantBlue stained SDS-PAGE gel of culture supernatants of G217B Hc cbp1 mutant expressing the chimeric constructs that convert key residues from E. crescens Cbp1 into their counterpart residue from G217B or Pb03 Cbp1 in the N-terminus, the C-terminal loops, both the N-terminus and C-terminal loops, or a 1st helix swap. White asterisk denotes the bands that were excised for mass spectrometric analysis to confirm their identity as the appropriate chimeric Cbp1. E. Hc cbp1-mutant isolates expressing E. crescens chimeric Cbp1 homologs were used to infect BMDMs and LDH release was quantified. F. Intracellular growth of Hc cbp1 mutant isolates expressing the E. crescens chimeric Cbp1 homologs was determined from BMDM infection as described in the Materials and Methods. “WT” indicates the Hc G217B ura5 mutant carrying the URA5 gene on a plasmid. At 48 hpi, CFU counts were terminated for the WT Hc infection because the extent of host-cell lysis made it difficult to distinguish between intracellular and extracellular growth of Hc. For LDH analyses, asterisk indicated p-value <0.05 relative to WT according to a t-test. For CFU analyses, asterisk indicates p-value <0.05 according to t-test relative to WT whereas dagger indicates p<0.05 relative to the cbp1 mutant.
Fig 4.
Es. africanus and Es. pasteurianus yeast can replicate intracellularly in macrophages but are unable to cause lysis.
A. Es. africanus and Es. pasteurianus yeast were used to infect BMDMs at an MOI of 1 as described in the Materials and Methods and the macrophage monolayers were stained with Rapi-Diff Stain II Set to qualitatively measure monolayer clearance at 0, 72 and 120 hours post infection. B. DIC images of BMDMs infected with wildtype Es. africanus at an MOI of 1 at one day post-infection (left) or five days post infection (right). The scale bar represents 100 μm for the top images and 10 μm for the bottom (zoomed-in) images. Arrowheads indicate intracellular yeasts, which are marked on d1 but not on d5 when the macrophage is filled with yeast cells.
Fig 5.
Hc and Pb03 Cbp1 enter the macrophage cytosol during infection despite lack of general permeabilization of the Hc-containing phagosome.
A. BMDMs were mock-infected (uninf) or infected with either the “WT” strain (Hc G217B ura5△ carrying a URA5 vector control), the cbp1 mutant (Hc G217B ura5△ cbp1△) carrying either the vector control or G217B Cbp1 with 3XFLAG (G217B Cbp1 3XFLAG), Pb03 Cbp1 with 3XFLAG (Pb03 Cbp1 3XFLAG), WT G217B ura5△ carrying Yps-3 3XFLAG (Yps-3 3XFLAG), or the Hc G217B ura5△ yps3△ mutant (yps3△). Macrophage lysates were subjected to fractionation to separate cytosolic and membrane fractions, followed by SDS-PAGE and Western blotting using anti-Calnexin (marking the endoplasmic reticulum), anti-α-Tubulin (marking the cytosol), anti-FLAG or anti-Cbp1 antibodies. B. BMDMs were either mock-infected or infected at an MOI of 2 with Hc G217B ura5△ cbp1△ expressing the WT untagged Cbp1(WT) or the cbp1 mutant carrying the Pb03 Cbp1 tagged with 3XFLAG. At 48 hours post infection, cells were fixed, stained with DAPI and Calcofluor White, and subjected to indirect immunofluorescence with the FLAG antibody. Scale bar represents 10 μm. C. BMDMs were either mock-infected or infected with WT Hc (Hc G217B ura5△ carrying a URA5 vector control) or Hc expressing β-Lactamase at an MOI of 2 for 24 hours or infected with a WT Legionella pneumophila (flaA△, BlaM-RalF) or the dotA mutant (dotA△, BlaM-RalF, flaA△) at an MOI of 100 for 4 hours. The BMDMs were then loaded with the CCF4-AM FRET probe and then monitored for probe cleavage as denoted by a shift from green to blue by flow cytometry. D. The percentage of the total cell population that showed blue fluorescence for each sample type in panel C is displayed.
Fig 6.
Hc G217B Cbp1 forms a complex with Yps-3.
A. Hc culture supernatants from the Hc G217B ura5- strain carrying a plasmid expressing 2xstrep enhanced GFP (2xstrep-eGFP) or Yps3-1xstrep, and the Hc G217B cbp1 mutant expressing either G217B 1xstrep-Cbp1 or Pb03 2xstrep Cbp1 were subjected to StrepTactin affinity purification. Eluates were analyzed by SDS-PAGE followed by silver staining. The pulldowns of Yps-3-1xstrep and 2xstrep-eGFP were confirmed with SDS-PAGE followed with Western blot analysis with both anti-strep and anti-Cbp1 antibodies. B. Hc culture supernatant from the Yps-3-6XHis strain was subjected to SDS-PAGE followed by Western blotting with either anti-His or anti-Cbp1 antibodies. The left-hand panel shows input, flow-through (FT) and elution from the HisPur resin whereas the right-hand panel shows the fractions containing both Yps3 and Cbp1 from a size exclusion column (SEC). C. Purified Yps3-6XHis and purified G217B Cbp1 were either subjected separately to SDS-PAGE and Western blotting (lanes 1 and 2) or mixed first at defined molar ratios and then isolated by Ni-NTA pulldown of Yps-3-6XHis (lanes 3 and 4). D. BMDMs were either mock-infected (uninf) or infected at an MOI = 1 with G217B ura5- transformed with a URA5 vector (WT), the cbp1 mutant transformed with a URA5 vector, G217B yps-3Δ transformed with a URA5 vector, or the G217B yps-3Δ mutant transformed with wild-type YPS-3. LDH release was calculated at multiple timepoints post-infection. E. Intracellular growth of WT, cbp1Δ, yps-3Δ, yps-3Δ+YPS-3 yeast was determined from BMDM infection as described in the Materials and Methods. CFU counts were terminated at 48 hpi for the WT Hc infection and 72 hpi for the yps-3Δ infection because the extent of host-cell lysis made it difficult to distinguish between intracellular and extracellular growth of Hc. F. Kaplan-Meier survival curves of C57BL/6 mice mock infected (uninf, n = 3) or infected with 1x106 Hc yeast (n = 11–12). Asterisk denotes p-value <0.05 by log rank test.
Fig 7.
Model figure showing the conservation of Cbp1 sequence, structure, and lytic function in Ajellomycetacea species that are human fungal pathogens.
Based on predicted protein homology and syntenic regions of the gene, homologs of Cbp1 were identified in the genomes Histoplasma, Emergomyces, Emmonsia, and Paracoccidioides species but not Blastomyces, correlating with its largely extracellular lifestyle relative to the other organisms. A novel structural fold was conserved between the Histoplasma and Paracoccidioides Cbp1 homologs despite divergence of their primary amino acid sequence. The Paracoccidioides Cbp1 homolog is capable of causing lysis of infected macrophages when expressed heterologously in G217B Hc. In constrast, none of the newly identified Emergomyces or Emmonsia homologs could trigger macrophage lysis when expressed heterologously in G217B Hc. These results are consistent with our observation that Emergomyces species are unable to lyse out of BMDMs.
Table 2.
Primers used in this study.