Jo Rae Wright

Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak.

Genealogy can illuminate the evolutionary path of important human pathogens. In some microbes, strict clonal reproduction predominates, as with the worldwide dissemination of Mycobacterium leprae, the cause of leprosy. In other pathogens, sexual reproduction yields clones with novel attributes, for example, enabling the efficient, oral transmission of the parasite Toxoplasma gondii. However, the roles of clonal or sexual propagation in the origins of many other microbial pathogen outbreaks remain unknown, like the recent fungal meningoencephalitis outbreak on Vancouver Island, Canada, caused by Cryptococcus gattii. Here we show that the C. gattii outbreak isolates comprise two distinct genotypes. The majority of isolates are hypervirulent and have an identical genotype that is unique to the Pacific Northwest. A minority of the isolates are significantly less virulent and share an identical genotype with fertile isolates from an Australian recombining population. Genotypic analysis reveals evidence of sexual reproduction, in which the majority genotype is the predicted offspring. However, instead of the classic a–α sexual cycle, the majority outbreak clone appears to have descended from two α mating-type parents. Analysis of nuclear content revealed a diploid environmental isolate homozygous for the major genotype, an intermediate produced during same-sex mating. These studies demonstrate how cryptic same-sex reproduction can enable expansion of a human pathogen to a new geographical niche and contribute to the ongoing production of infectious spores. This has implications for the emergence of other microbial pathogens and inbreeding in host range expansion in the fungal and other kingdoms.

Surfactant Protein A Enhances Alveolar Macrophage Phagocytosis of Apoptotic Neutrophils

Surfactant protein A (SP-A) is an innate immune molecule that binds foreign organisms that invade the lungs and targets them for phagocytic clearance by the resident pulmonary phagocyte, the alveolar macrophage (AM). We hypothesized that SP-A binds to and enhances macrophage uptake of other nonself particles, specifically apoptotic polymorphonuclear neutrophils (PMNs). PMNs are recruited into the lungs during inflammation, but as inflammation is resolved, PMNs undergo apoptosis and are phagocytosed by AMs. We determined that SP-A increases AM phagocytosis of apoptotic PMNs 280 ± 62% above the no protein control value. The increase is dose dependent, and heat-treated SP-A still enhanced uptake, whereas deglycosylated SP-A had significantly diminished ability to enhance phagocytosis. Surfactant protein D also increased phagocytosis of apoptotic PMNs by ∼125%. However, other proteins that are structurally homologous to SP-A, mannose-binding lectin and complement protein 1q, did not. SP-A enhances phagocytosis via an opsonization-dependent mechanism and binds apoptotic PMNs ∼4-fold more than viable PMNs. Also, binding of SP-A to apoptotic PMNs does not appear to involve SP-A’s lectin domain. These data suggest that the pulmonary collectins SP-A and SP-D facilitate the resolution of inflammation by accelerating apoptotic PMN clearance.

Spores as Infectious Propagules of Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite- or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (∼95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules.

Pulmonary surfactant: a front line of lung host defense

The lung is a uniquely vulnerable organ. Residing at the interface of the body and the environment, the lung is optimized for gas exchange, having a very thin, delicate epithelium, abundant blood flow, and a vast surface area. Inherent in this structure is an enormous immunological burden from pathogens, allergens, and pollutants resident in the 11,000 liters of air inhaled daily. Fortunately, protective immune mechanisms act locally in the lung to facilitate clearance of inhaled pathogens and to modulate inflammatory responses. These defensive mechanisms include both innate (nonantibody-mediated) and adaptive (antibody-mediated) systems. The purpose of this commentary is to review briefly the functions of one unique lung innate immune system, pulmonary surfactant, and to highlight the recent findings of Wu et al. (1) described in this issue of the JCI. Wu and colleagues report a new and intriguing innate immune function of surfactant: direct antimicrobial activity.

Pulmonary Surfactant Proteins A and D Directly Suppress CD3+/CD4+ Cell Function: Evidence for Two Shared Mechanisms

Pulmonary surfactant is a lipoprotein complex that lowers surface tension at the air-liquid interface of the lung and participates in pulmonary host defense. Surfactant proteins (SP), SP-A and SP-D, modulate a variety of immune cell functions, including the production of cytokines and free radicals. Previous studies showed that SP-A and SP-D inhibit lymphocyte proliferation in the presence of accessory cells. The goal of this study was to determine whether SP-A and SP-D directly suppress Th cell function. Both proteins inhibited CD3+/CD4+ lymphocyte proliferation induced by PMA and ionomycin in an IL-2-independent manner. Both proteins decreased the number of cells entering the S and mitotic phases of the cell cycle. Neither SP-A nor SP-D altered cell viability, apoptosis, or secretion of IL-2, IL-4, or IFN-γ when Th cells were treated with PMA and ionomycin. However, both proteins attenuated ionomycin-induced cytosolic free calcium ([Ca2+ ]i), but not thapsigargin-induced changes in [Ca2+]i. In summary, inhibition of T cell proliferation by SP-A and SP-D occurs via two mechanisms, an IL-2-dependent mechanism observed with accessory cell-dependent T cell mitogens and specific Ag, as well as an IL-2-independent mechanism of suppression that potentially involves attenuation of [Ca2+]i.

Surfactant protein A inhibits T cell proliferation via its collagen-like tail and a 210-kDa receptor

Investigation of possible mechanisms to describe the hyporesponsiveness of pulmonary leukocytes has led to the study of pulmonary surfactant and its constituents as immune suppressive agents. Pulmonary surfactant is a phospholipid-protein mixture that reduces surface tension in the lung and prevents collapse of the alveoli. The most abundant protein in this mixture is a hydrophilic molecule termed surfactant-associated protein A (SP-A). Previously, we showed that bovine (b) SP-A can inhibit human T lymphocyte proliferation and interleukin-2 production in vitro. Results presented in this investigation showed that different sources of human SP-A and bSP-A as well as recombinant rat SP-A inhibited human T lymphocyte proliferation in a dose-dependent manner. A structurally similar collagenous protein, C1q, did not block the in vitro inhibitory action of SP-A. The addition of large concentrations of mannan to SP-A-treated cultures also did not disrupt inhibition, suggesting that the effect is not mediated by the carbohydrate recognition domain of SP-A. Use of recombinant mutant SP-As revealed that a 36-amino acid Arg-Gly-Asp (RGD) motif-containing span of the collagen-like domain was responsible for the inhibition of T cell proliferation. A polyclonal antiserum directed against an SP-A receptor (SP-R210) completely blocked the inhibition of T cell proliferation by SP-A. These results emphasize a potential role for SP-A in dampening lymphocyte responses to exogenous stimuli. The data also provide further support for the concept that SP-A maintains a balance between the clearance of inhaled pathogens and protection against collateral immune-mediated damage.

Surfactant proteins A and D specifically stimulate directed actin-based responses in alveolar macrophages

Surfactant protein (SP) A and SP-D are the pulmonary members of the collectin family, structurally related proteins involved in innate immune responses. Here, we have examined the abilities of SP-A, SP-D, mannose-binding protein (MBP), and the complement component C1q to stimulate actin-based cellular functions in rat alveolar macrophages and peripheral blood monocytes. Our goal in this study was to examine the cell specificity of the effects of the collectins to understand further the mechanisms by which SP-A and SP-D stimulate alveolar macrophages. We found that SP-A and SP-D have lung cell-specific effects at physiologically relevant concentrations; they stimulate directional actin polymerization and chemotaxis in alveolar macrophages but not in monocytes. Although C1q and MBP weakly stimulate the rearrangement of actin in both cell types, C1q is chemotactic only for peripheral blood monocytes and MBP does not stimulate chemotaxis of either cell type. Neither C1q nor MBP stimulates actin polymerization in alveolar macrophages. These results support the hypothesis that alveolar macrophages express receptors specific for the pulmonary collectins SP-A and SP-D and provide insight into the potential roles of collectins in the recruitment and maturation of mononuclear phagocytes in the lung.Surfactant protein (SP) A and SP-D are the pulmonary members of the collectin family, structurally related proteins involved in innate immune responses. Here, we have examined the abilities of SP-A, SP-D, mannose-binding protein (MBP), and the complement component C1q to stimulate actin-based cellular functions in rat alveolar macrophages and peripheral blood monocytes. Our goal in this study was to examine the cell specificity of the effects of the collectins to understand further the mechanisms by which SP-A and SP-D stimulate alveolar macrophages. We found that SP-A and SP-D have lung cell-specific effects at physiologically relevant concentrations; they stimulate directional actin polymerization and chemotaxis in alveolar macrophages but not in monocytes. Although C1q and MBP weakly stimulate the rearrangement of actin in both cell types, C1q is chemotactic only for peripheral blood monocytes and MBP does not stimulate chemotaxis of either cell type. Neither C1q nor MBP stimulates actin polymerization in alveolar macrophages. These results support the hypothesis that alveolar macrophages express receptors specific for the pulmonary collectins SP-A and SP-D and provide insight into the potential roles of collectins in the recruitment and maturation of mononuclear phagocytes in the lung.

Identification and Characterization of Human Surfactant Protein A Binding Protein of Mycoplasma pneumoniae

ABSTRACT Mycoplasma pneumoniae infections represent a major primary cause of human respiratory diseases, exacerbate other respiratory disorders, and are associated with extrapulmonary pathologies. Cytadherence is a critical step in mycoplasma colonization, aided by a network of mycoplasma adhesins and cytadherence accessory proteins which mediate binding to host cell receptors. Furthermore, the respiratory mucosa is enriched with extracellular matrix components, including surfactant proteins, fibronectin, and mucin, which provide additional in vivo targets for mycoplasma parasitism. In this study we describe interactions between M. pneumoniae and human surfactant protein-A (hSP-A). Initially, we found that viable M. pneumoniae cells bound to immobilized hSP-A in a dose- and calcium (Ca2+)-dependent manner. Mild trypsin treatment of intact mycoplasmas reduced binding markedly (80 to 90%) implicating a surface-associated mycoplasma protein(s). Using hSP-A-coupled Sepharose affinity chromatography and polyacrylamide gel electrophoresis, we identified a 65-kDa hSP-A binding protein of M. pneumoniae. The presence of Ca2+ enhanced binding of the 65-kDa protein to hSP-A, which was reduced by the divalent cation-chelating agent, EDTA. The 65-kDa hSP-A binding protein of M. pneumoniae was identified by sequence analysis as a novel protein (MPN372) possessing a putative S1-like subunit of pertussis toxin at the amino terminus (amino acids 1 to 226), with the remaining amino acids (227 to 591) exhibiting no homology with other subunits of pertussis toxin, other known toxins, or any reported proteins. Recombinant MPN372 (MPN372) bound to hSP-A in a dose-dependent manner, which was markedly reduced by preincubation with mouse recombinant MPN372 antisera. Also, adherence of viable M. pneumoniae cells to hSP-A was inhibited by recombinant MPN372 antisera, demonstrating that MPN372, a previously designated hypothetical protein, is surface exposed and mediates mycoplasma attachment to hSP-A.

Surfactant protein A enhances alveolar macrophage phagocytosis of a live, mucoid strain of P. aeruginosa.

In this study, we investigate the interaction between surfactant protein A (SP-A) and a live, mucoid strain of Pseudomonas aeruginosa and identify a mechanism of clearance of this organism by alveolar macrophages. (125)I-labeled SP-A bound live, but not heat-killed, P. aeruginosa organisms in a concentration-dependent manner. Unlabeled SP-A bound live bacteria, protein isolated from whole organisms, and specific proteins of the P. aeruginosa outer membrane. The binding of SP-A to P. aeruginosa and outer membrane components was inhibited by either EDTA or mannose. Phagocytosis assays with fluorescent microscopy demonstrated that the percentage of macrophages with internalized FITC-labeled P. aeruginosa was increased 1.8-fold (19 vs. 35%) by pretreating the live bacteria with SP-A. This finding was confirmed by direct visualization of ingested bacteria by electron microscopy. Adhering macrophages to SP-A-coated surfaces attenuated the increased uptake of P. aeruginosa pretreated with SP-A, suggesting that SP-A acts as an opsonin to stimulate macrophage phagocytosis of this strain of P. aeruginosa.

Host Defense Functions of Pulmonary Surfactant

Surfactant is a complex of lipids and proteins that reduces surface tension at the air/liquid interface of the lung and regulates immune cell function. Surfactant immune function is primarily attributed to two proteins: SP-A and SP-D. SP-A and SP-D are members of a protein family known as ‘collectins’, which are distinguished by their N-terminal collagen-like region and their C-terminal lectin domain. The lectin domain binds preferentially to sugars on the surface of pathogens and thereby opsonizes them for uptake by phagocytes. The collectins also modulate the functions of cells of the adaptive immune network including dendritic cells and T lymphocytes. In addition, recent studies show that bacterial products degrade surfactant. In summary, surfactant plays an important role in lung host defense. Surfactant degradation or inactivation may contribute to enhanced susceptibility to lung inflammation and infection.