A Role for <i>LHC1</i> in Higher Order Structure and Complement Binding of the <i>Cryptococcus neoformans</i> Capsule

<div><p>Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus <i>Cryptococcus neoformans</i>. In the present study, we demonstrate an unusual role for a secreted <u>l</u>actono<u>h</u>ydrolase of <i><u>C</u>. neoformans</i>, <i>LHC1</i> in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and <i>lhc1</i>Δ strains by dynamic and static light scattering suggested a role for the <i>LHC1</i> locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in <i>LHC1</i>-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen.</p></div

wt and <i>lhc1</i>Δ mutant strains of <i>C. neoformans</i> differ in antibody binding and antibody mediated phagocytosis by a J774.16 macrophage-like cell line.

<p>(A) Indicated strains were prepared and stained with indicated monoclonal antibodies as described in Material and Methods. (B) Puncta from cells labeled as in A were quantified from 50 cells. (C) Indicated strains were opsonized with the indicated antibody and then incubated with J774.16 cell monolayers and phagocytic index determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004037#s4" target="_blank">Materials and Methods</a> (N = 4). (D) Fungal Killing Assay: Cells treated as in C, except that incubation was continued for 81 hours and fungal burden assayed by CFU after macrophage lysis (N = 4). *** p<0.001.</p

Scheme of working model of capsule modification by Lhc1.

<p>(A) Data from particle analysis of DMSO-solubilized PS and cryo-electron microscopy suggests hydrolytic, Lhc1-dependent processing of two populations of particles during capsule induction. Hydrolysis of outer branching units within the PS surface structure reduces both the size and overall branching as the wt particle adopts a surface having reduced hydration, radius of hydration and increased zeta potential. (B) Model of outer capsular synthesis. In the absence of Lhc1, unprocessed PS units with increased branching and increased surface hydration provide sites for antibody and complement binding, resulting in increased phagocytosis. Action of Lhc1 serves to provide a less hydrated, more compact capsule, resulting in better exclusion of innate immune products, reducing phagocytosis and increasing virulence potential. Events are not intended to be sequential; PS synthesis and <i>LHC1</i>-dependent remodeling likely are simultaneous. Representation does not intend to imply that PS particles are symmetrical or have regularly-spaced branches.</p

Molecular parameters of capsular PS samples performed by static and dynamic light scattering analysis.

1<p>The refractive index as a function of concentration (dn/dc) in units of mL/g, average-molecular mass (M_w), radius of gyration (R_g), hydrodynamic radius (R_h), polydispersity, shape factor (R_g/R_h), mass density (M_w/R_g), and 2^nd virial coefficient (A₂) of capsular PS samples. M_w, R_g data are represented as mean +/− SD of 2 measurements. R_h and polydispersity data are represented as mean +/− SE of 10 measurements.</p

Identification of a capsular-adherent putative lactonohydrolase from <i>C. neoformans</i> and role in virulence-related phenotypes.

<p>(A) SDS-PAGE of DMSO-solubilized capsular proteins adsorbed on DEAE-agarose. (B) Clustal-W comparison of proteins sequences of closest matches of the 50 kDa Lhc1 sequence. Indicated strains were assayed for (D) laccase by melanin formation and, (C) capsule by India Ink microscopy. (E) Capsule radius of India ink-stained cells was determined in 100 cells of the indicated strains. (F) Capsule of <i>LHC1</i> strains during brain infection. Indicated strains (1×10⁶) were inoculated intravenously and when moribund, mice were sacrificed and brains excised, sectioned and stained with H&E as described in methods. Bar = 5 microns.</p

Deletion of <i>LHC1</i> results in increased C3 binding from human or mouse serum and reduced virulence in a mouse model.

<p><b>Human studies, A–D</b>: (A) Indicated fungal strains were incubated with human serum (left panel) or heat killed serum (right panel) and subjected to phagocytosis using a J774.16 cell line according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004037#s4" target="_blank">Materials and Methods</a>. (B) Strains indicated in B were incubated in the presence of serum and monocytes from a healthy volunteer and percent killing over 4 h measured according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004037#s4" target="_blank">Materials and Methods</a>. (C, D) Indicated strains were incubated in the presence of human serum in the presence (+EGTA) or absence (−EGTA) of EGTA and analyzed by flow cytometry (C) or visualized by fluorescence microscopy (D) using an anti-human C3 antibody. For microscopic evaluation of C3 deposition, FITC-labeled goat anti-human C3 was added and samples resuspended in mounting medium, placed on glass slides and examined under oil immersion at 1000× for C3 deposition (FITC) or using differential interference contrast (DIC). <b>Mouse studies, E–G:</b> (E) Indicated strains were incubated with mouse serum (left panel) or heat killed serum (right panel) and subjected to phagocytosis using a J774.16 cell line according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004037#s4" target="_blank">Materials and Methods</a>. (F) Indicated strains were incubated in the presence of mouse serum in the presence (+EGTA) or absence (−EGTA) of EGTA and analyzed by flow cytometry using an anti-mouse C3 antibody. (G) Cumulative mortality in CBA/J mice inoculated intravenously with 1×10⁴ cells of the indicated strains (F-left panel). <i>p</i><0.01 for the comparison of the <i>lhc1</i><b>Δ</b> mutant versus either wt or the <i>LHC1</i> complemented strain (F-right panel). Cumulative mortality of mice inoculated the same as in F-left panel, except mice were treated with cobra venom factor to deplete serum complement according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004037#s4" target="_blank">Materials and Methods</a> (p = NS).</p

<i>C. neoformans</i> possesses a <i>LHC1</i>-dependent lactonohydrolase activity that is localized to the capsule and expressed during human infection.

<p>(A) Scheme of lactonohydrolase-dependent hydrolysis of D-pantolactone. (B) Indicated cells were grown in asparagine media (0.2% glucose) for 3 days, centrifuged and washed 3× in 10 mM Tris buffer, pH 7.0, incubated in the presence of 100 mM D-pantolactone at 37°C for the indicated times and the reaction terminated with methanol. Aliquots were then assayed for lactone (black arrow) or the hydrolyzed acid (red arrow) as determined using chemical standards. (C) Western blot analysis of polyclonal anti-Lhc1 reactivity against <i>C. neoformans</i> protein extracts. Protein supernatants of cell lysates (cytosol) or detergent-solubilized cell pellets (cell wall/capsule) from the indicated strains were prepared and western blot performed using polyclonal serum against the Lhc1 protein as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004037#s4" target="_blank">Materials and Methods</a>. (D) A <i>C. neoformans lhc1</i><b>Δ</b> strain complemented with a vector expressing Lhc1-mCherry (+Lhc1) or empty vector alone (−Lhc1) was grown in YPD (uninduced) or induced on 1∶10 SAB (capsule induced) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004037#s4" target="_blank">Materials and Methods</a>. (E) Sections of a brain autopsy specimen were obtained from a 30 y.o. female who died of severe and diffuse <i>C. neoformans</i> infection and stained with a mouse affinity-purified antibody against a maltose-binding protein-Lhc1 fusion protein (α-Lhc1 Ab) or anti-MBP polyclonal serum (α-MBP Ab) followed by incubation with a secondary Alexa Fluor 594 goat anti-rabbit IgG (AF494) according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004037#s4" target="_blank">Materials and Methods</a>. Panels represent fluorescent antibody treated (AF594) or differential interference contrast microscopy (DIC) images. Cells were washed and mounted in anti-fade medium and imaged in an epifluorescence microscope using a 63× 1.4 NA objective. The exposure conditions were identical for each sample and are representative of 25 cells visualized. Bar = 5 microns.</p

Paradoxical Immune Responses in Non-HIV Cryptococcal Meningitis

<div><p>The fungus <i>Cryptococcus</i> is a major cause of meningoencephalitis in HIV-infected as well as HIV-uninfected individuals with mortalities in developed countries of 20% and 30%, respectively. In HIV-related disease, defects in T-cell immunity are paramount, whereas there is little understanding of mechanisms of susceptibility in non-HIV related disease, especially that occurring in previously healthy adults. The present description is the first detailed immunological study of non-HIV-infected patients including those with severe central nervous system (s-CNS) disease to 1) identify mechanisms of susceptibility as well as 2) understand mechanisms underlying severe disease. Despite the expectation that, as in HIV, T-cell immunity would be deficient in such patients, cerebrospinal fluid (CSF) immunophenotyping, T-cell activation studies, soluble cytokine mapping and tissue cellular phenotyping demonstrated that patients with s-CNS disease had effective microbiological control, but displayed strong intrathecal expansion and activation of cells of both the innate and adaptive immunity including HLA-DR+ CD4+ and CD8+ cells and NK cells. These expanded CSF T cells were enriched for cryptococcal-antigen specific CD4+ cells and expressed high levels of IFN-γ as well as a lack of elevated CSF levels of typical T-cell specific Th2 cytokines -- IL-4 and IL-13. This inflammatory response was accompanied by elevated levels of CSF NFL, a marker of axonal damage, consistent with ongoing neurological damage. However, while tissue macrophage recruitment to the site of infection was intact, polarization studies of brain biopsy and autopsy specimens demonstrated an M2 macrophage polarization and poor phagocytosis of fungal cells. These studies thus expand the paradigm for cryptococcal disease susceptibility to include a prominent role for macrophage activation defects and suggest a spectrum of disease whereby severe neurological disease is characterized by immune-mediated host cell damage.</p></div

Brain immunofluorescence of autopsy specimens from patients who died of s-CNS cryptococcosis.

<p>Fluorescent macrophage marker (CD68) and fungal marker calcofluor white (CFW) was used to stain cells within the peri-vascular brain parenchyma. Magnification at 10x with inset and scale bar set at 100 μm for 10x images.</p

<i>Ex vivo</i> cryptococcal antigenic stimulation by pulsed autologous mature dendritic cells (mDCs) co-cultured with T lymphocytes from CSF or blood demonstrates compartmentalization of immune responses in a subgroup of 8 s-CNS and 5 non-CNS patients.

<p>(A) Sum of cytokine-producing IFN-γ+, TNF-α+, and IFN-γ+/TNF-α+ CD4+ and CD8+ T lymphocytes (after mDC presentation of MP and Crypto). Events are normalized to fluorescent beads. B) IFN-γ production of activated CD4+ and CD8+ T lymphocytes after Crypto presentation by mDCs. Tc = T lymphocyte; Crypto = glass bead-fractured, heat-killed <i>C</i>. <i>neoformans</i> strain H99; MP = <i>C</i>. <i>neoformans</i> mannoprotein; No Ag = no antigen (un-loaded mDCs). Open circles and bars are representative of non-CNS (Pulmonary) cases, filled circles and bars of s-CNS (CNS) cases. Error bars specify minimum to maximum values. *0.01≤p<0.05; **0.001≤p<0.01; ***0.0001≤p<0.001.</p