Host membrane glycosphingolipids and lipid microdomains facilitate Histoplasma capsulatum internalisation by macrophages.

Recognition and internalisation of intracellular pathogens by host cells is a multifactorial process, involving both stable and transient interactions. The plasticity of the host cell plasma membrane is fundamental in this infectious process. Here, the participation of macrophage lipid microdomains during adhesion and internalisation of the fungal pathogen Histoplasma capsulatum (Hc) was investigated. An increase in membrane lateral organisation, which is a characteristic of lipid microdomains, was observed during the first steps of Hc-macrophage interaction. Cholesterol enrichment in macrophage membranes around Hc contact regions and reduced levels of Hc-macrophage association after cholesterol removal also suggested the participation of lipid microdomains during Hc-macrophage interaction. Using optical tweezers to study cell-to-cell interactions, we showed that cholesterol depletion increased the time required for Hc adhesion. Additionally, fungal internalisation was significantly reduced under these conditions. Moreover, macrophages treated with the ceramide-glucosyltransferase inhibitor (P4r) and macrophages with altered ganglioside synthesis (from B4galnt1-/- mice) showed a deficient ability to interact with Hc. Coincubation of oligo-GM1 and treatment with Cholera toxin Subunit B, which recognises the ganglioside GM1, also reduced Hc association. Although purified GM1 did not alter Hc binding, treatment with P4 significantly increased the time required for Hc binding to macrophages. The content of CD18 was displaced from lipid microdomains in B4galnt1-/- macrophages. In addition, macrophages with reduced CD18 expression (CD18low ) were associated with Hc at levels similar to wild-type cells. Finally, CD11b and CD18 colocalised with GM1 during Hc-macrophage interaction. Our results indicate that lipid rafts and particularly complex gangliosides that reside in lipid rafts stabilise Hc-macrophage adhesion and mediate efficient internalisation during histoplasmosis.

Galectin-3 impacts Cryptococcus neoformans infection through direct antifungal effects.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian ß-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C. neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C. neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C. neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host.

Capsular specific IgM enhances complement-mediated phagocytosis and killing of Cryptococcus neoformans by methamphetamine-treated J774.16 macrophage-like cells.

Methamphetamine (METH) is a powerful and highly addictive stimulant that affects the central nervous system of users in the United States and worldwide, and its consumption is associated to the acquisition of HIV and AIDS-related infections. METH enhances cryptococcosis in mice, an opportunistic infection caused by the encapsulated fungus Cryptococcus neoformans. Due to its ability to survive within macrophages, C. neoformans is an ideal model to study pathogen-macrophage interactions. METH abrogates normal macrophage function, which might contribute to the higher rate and more rapid progression of infections in drug abusers. Hence, we investigated the role of complement and specific IgM to C. neoformans capsular polysaccharide on the function of J774.16 macrophage-like cells after exposure to METH. We found that complement and IgM significantly promotes complement-mediated phagocytosis of C. neoformans by J774.16 cells in comparison to co-incubation with complement alone. IgM enhances the expression of complement receptor 3 on the surface macrophages treated with the drug. Also, IgM-increased macrophage phagocytosis of C. neoformans may be associated with upregulation of GTPase-RhoA, a key regulator of the actin polymerization signaling cascade. Fungal cells incubated with complement and IgM in the presence of METH demonstrated higher number of cells per aggregate, a possible explanation for their enhanced ingestion by phagocytes. IgM increased killing of yeast cells by macrophages by inhibiting the alkalization of the phagosome and stimulating the intracellular production of nitric oxide. Together, our findings suggest that IgM stimulates the effector functions of macrophages against opportunistic pathogens in the setting of drug abuse.

Cryptococcus neoformans: Tripping on Acid in the Phagolysosome.

Cryptococcus neoformans (Cn) is a basidiomycetous pathogenic yeast that is a frequent cause of meningoencephalitis in immunocompromised individuals. Cn is a facultative intracellular pathogen in mammals, insects and amoeba. Cn infection occurs after inhalation of spores or desiccated cells from the environment. After inhalation Cn localizes to the lungs where it can be phagocytosed by alveolar macrophages. Cn is surrounded by a polysaccharide capsule that helps the fungus survive in vivo by interfering with phagocytosis, quenching free radical bursts and shedding polysaccharides that negatively modulates the immune system. After phagocytosis, Cn resides within the phagosome that matures to become a phagolysosome, a process that results in the acidification of the phagolysosomal lumen. Cn replicates at a higher rate inside macrophages than in the extracellular environment, possibly as a result that the phagosomal pH is near that optimal for growth. Cn increases the phagolysosomal pH and modulates the dynamics of Rab GTPases interaction with the phagolysosome. Chemical manipulation of the phagolysosomal pH with drugs can result in direct and indirect killing of Cn and reduced non-lytic exocytosis. Phagolysosomal membrane damage after Cn infection occurs both in vivo and in vitro, and is required for Cn growth and survival. Macrophage treatment with IFN-γ reduces the phagolysosomal damage and increases intracellular killing of Cn. Studies on mice and humans show that treatment with IFN-γ can improve host control of the disease. However, the mechanism by which Cn mediates phagolysosomal membrane damage remains unknown but likely candidates are phospholipases and mechanical damage from an enlarging capsule. Here we review Cn intracellular interaction with a particular emphasis on phagosomal interactions and develop the notion that the extent of damage of the phagosomal membrane is a key determinant of the outcome of the Cn-macrophage interaction.

Neutropenia exacerbates infection by Acinetobacter baumannii clinical isolates in a murine wound model.

The Gram negative coccobacillus Acinetobacter baumannii has become an increasingly prevalent cause of hospital-acquired infections in recent years. The majority of clinical A. baumannii isolates display high-level resistance to antimicrobials, which severely compromises our capacity to care for patients with A. baumannii disease. Neutrophils are of major importance in the host defense against microbial infections. However, the contribution of these cells of innate immunity in host resistance to cutaneous A. baumannii infection has not been directly investigated. Hence, we hypothesized that depletion of neutrophils increases severity of bacterial disease in an experimental A. baumannii murine wound model. In this study, the Ly-6G-specific monoclonal antibody (mAb), 1A8, was used to generate neutropenic mice and the pathogenesis of several A. baumannii clinical isolates on wounded cutaneous tissue was investigated. We demonstrated that neutrophil depletion enhances bacterial burden using colony forming unit determinations. Also, mAb 1A8 reduces global measurements of wound healing in A. baumannii-infected animals. Interestingly, histological analysis of cutaneous tissue excised from A. baumannii-infected animals treated with mAb 1A8 displays enhanced collagen deposition. Furthermore, neutropenia and A. baumannii infection alter pro-inflammatory cytokine release leading to severe microbial disease. Our findings provide a better understanding of the impact of these innate immune cells in controlling A. baumannii skin infections.

Methamphetamine enhances Cryptococcus neoformans pulmonary infection and dissemination to the brain.

UNLABELLED: Methamphetamine (METH) is a major addictive drug of abuse in the United States and worldwide, and its use is linked to HIV acquisition. The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis in patients with AIDS. In addition to functioning as a central nervous system stimulant, METH has diverse effects on host immunity. Using a systemic mouse model of infection and in vitro assays in order to critically assess the impact of METH on C. neoformans pathogenesis, we demonstrate that METH stimulates fungal adhesion, glucuronoxylomannan (GXM) release, and biofilm formation in the lungs. Interestingly, structural analysis of the capsular polysaccharide of METH-exposed cryptococci revealed that METH alters the carbohydrate composition of this virulence factor, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Additionally, we show that METH promotes C. neoformans dissemination from the respiratory tract into the brain parenchyma. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms. IMPORTANCE: Methamphetamine (METH) is a major health threat to our society, as it adversely changes people's behavior, as well as increases the risk for the acquisition of diverse infectious diseases, particularly those that enter through the respiratory tract or skin. This report investigates the effects of METH use on pulmonary infection by the AIDS-related fungus Cryptococcus neoformans. This drug of abuse stimulates colonization and biofilm formation in the lungs, followed by dissemination of the fungus to the central nervous system. Notably, C. neoformans modifies its capsular polysaccharide after METH exposure, highlighting the fungus's ability to adapt to environmental stimuli, a possible explanation for its pathogenesis. The findings may translate into new knowledge and development of therapeutic and public health strategies to deal with the devastating complications of METH abuse.

HER2/ErbB2-induced breast cancer cell migration and invasion require p120 catenin activation of Rac1 and Cdc42.

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.