Glucuronoxylomannan intranasal challenge prior to Cryptococcus neoformans pulmonary infection enhances cerebral cryptococcosis in rodents.

The encapsulated fungus Cryptococcus neoformans is the most common cause of fungal meningitis, with the highest rate of disease in patients with AIDS or immunosuppression. This microbe enters the human body via inhalation of infectious particles. C. neoformans capsular polysaccharide, in which the major component is glucuronoxylomannan (GXM), extensively accumulates in tissues and compromises host immune responses. C. neoformans travels from the lungs to the bloodstream and crosses to the brain via transcytosis, paracytosis, or inside of phagocytes using a "Trojan horse" mechanism. The fungus causes life-threatening meningoencephalitis with high mortality rates. Hence, we investigated the impact of intranasal exogenous GXM administration on C. neoformans infection in C57BL/6 mice. GXM enhances cryptococcal pulmonary infection and facilitates fungal systemic dissemination and brain invasion. Pre-challenge of GXM results in detection of the polysaccharide in lungs, serum, and surprisingly brain, the latter likely reached through the nasal cavity. GXM significantly alters endothelial cell tight junction protein expression in vivo, suggesting significant implications for the C. neoformans mechanisms of brain invasion. Using a microtiter transwell system, we showed that GXM disrupts the trans-endothelial electrical resistance, weakening human brain endothelial cell monolayers co-cultured with pericytes, supportive cells of blood vessels/capillaries found in the blood-brain barrier (BBB) to promote C. neoformans BBB penetration. Our findings should be considered in the development of therapeutics to combat the devastating complications of cryptococcosis that results in an estimated ~200,000 deaths worldwide each year.

Knockout of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation, impairs dendritic spines of hippocampal neurons, and affects memory.

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by three HS 6-O-sulfotransferases (HS6STs). Notably, HS6ST2 is mainly expressed in the brain and HS6ST2 mutations are linked to brain disorders, but the underlying mechanisms remain poorly understood. To determine the role of Hs6st2 in the brain, we carried out a series of molecular and behavioral assessments on Hs6st2 knockout mice. We first carried out strong anion exchange-high performance liquid chromatography and found that knockout of Hs6st2 moderately decreases HS 6-O-sulfation levels in the brain. We then assessed body weights and found that Hs6st2 knockout mice exhibit increased body weight, which is associated with abnormal metabolic pathways. We also performed behavioral tests and found that Hs6st2 knockout mice showed memory deficits, which recapitulate patient clinical symptoms. To determine the molecular mechanisms underlying the memory deficits, we used RNA sequencing to examine transcriptomes in two memory-related brain regions, the hippocampus and cerebral cortex. We found that knockout of Hs6st2 impairs transcriptome in the hippocampus, but only mildly in the cerebral cortex. Furthermore, the transcriptome changes in the hippocampus are enriched in dendrite and synapse pathways. We also found that knockout of Hs6st2 decreases HS levels and impairs dendritic spines in hippocampal CA1 pyramidal neurons. Taken together, our study provides novel molecular and behavioral insights into the role of Hs6st2 in the brain, which facilitates a better understanding of HS6ST2 and HS-linked brain disorders.

Depletion of Alveolar Macrophages Increases Pulmonary Neutrophil Infiltration, Tissue Damage, and Sepsis in a Murine Model of Acinetobacter baumannii Pneumonia.

Acinetobacter baumannii has emerged as an important etiological agent of hospital-related infections, especially nosocomial pneumonia. The virulence factors of this bacterium and their interactions with the cells and molecules of the immune system just recently began to be extensively studied. Here, we investigated the impact of alveolar macrophages on A. baumannii pneumonia using a mouse model of infection and a flexible tissue culture system. We hypothesized that depletion of macrophages would enhance sepsis and severity of A. baumannii disease. We showed that macrophages are important for modulating the antibacterial function of neutrophils and play an important role in eradicating A. baumannii infection in vivo Our findings suggest that in the absence of macrophages in the lungs, A. baumannii replicates significantly, and host proinflammatory cytokines are considerably reduced. Neutrophils are abundantly recruited to pulmonary tissue, releasing high amounts of reactive oxygen species and causing extensive tissue damage. The ability of A. baumannii to form biofilms and resist oxidative stress in the respiratory tract facilitates systemic dissemination and ultimately death of infected C57BL/6 mice. These results provide novel information regarding A. baumannii pathogenesis and may be important for the development of therapies aimed at reducing morbidity and mortality associated with this emerging bacterial pathogen.

Biological depletion of neutrophils attenuates pro-inflammatory markers and the development of the psoriatic phenotype in a murine model of psoriasis.

Although neutrophils are considered a histologic hallmark of psoriasis, their pathophysiologic role in psoriasis remains unclear. We characterized the effects of neutrophil depletion via injection of monoclonal antibody 1A8 on the development of imiquimod (IMQ)-induced psoriatic lesions in a murine model. Lesions were followed with photographs and histologic analysis, revealing reduced psoriasiform scale and epidermal hyperplasia in neutrophil-depleted. ELISA and flow cytometry were used to determine relative levels of cytokines and immune cells. Compared to controls, IMQ-treated neutropenic mice had significantly lower levels of macrophages in tissue samples (P < .05) and displayed significantly lower numbers of CD4+ T-cells (P < .05). Neutropenic animals exhibited lower levels of TNF-α, IFN-γ, and IL-1ß than controls (P < .05). These results show that neutropenia reduces the development of psoriasiform skin lesions and substantially decreases infiltration of pro-inflammatory cytokines and immune cells to IMQ-induced cutaneous lesions, suggesting an active role of neutrophils in maintaining inflammation in psoriasis.

Reduced phagocytosis and killing of Cryptococcus neoformans biofilm-derived cells by J774.16 macrophages is associated with fungal capsular production and surface modification.

Cryptococcus neoformans is an opportunistic encapsulated pathogen that causes life-threatening meningoencephalitis in individuals with immunosuppression. We compared the interactions of C. neoformans planktonic and biofilm-derived cells with J774.16 macrophage-like cells. Planktonic cells are more phagocytized and killed by J774.16 cells than biofilm-derived fungal cells. Biofilm-derived cryptococci possess larger capsule size and release significantly more capsular polysaccharide than planktonic cells in culture. Biofilm-derived fungi exhibited upregulation of genes involved in capsular production. Capsular-specific monoclonal antibody 18B7 demonstrated differential binding to the surface of planktonic and biofilm-derived cryptococci providing a plausible strategy for fungal evasion of macrophages and persistence. Future studies are necessary to elucidate how C. neoformans biofilm-derived cells regulate their virulence factors when interacting with cells of the immune system.

Microbicides Alter the Expression and Function of RND-Type Efflux Pump AdeABC in Biofilm-Associated Cells of Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is a Gram-negative bacterium that causes nosocomial infections worldwide. This microbe's propensity to form biofilms allows it to persist and to survive on clinical abiotic surfaces for long periods. In fact, A. baumannii biofilm formation and its multidrug-resistant nature severely compromise our capacity to care for patients in hospital environments. In contrast, microbicides such as cetrimide (CT) and chlorhexidine (CHX) play important roles in the prevention and treatment of infections. We assessed the efficacy of CT and CHX, either alone or in combination, in eradicating A. baumannii biofilms formed by clinical isolates, by using stainless steel washers to mimic hard abiotic surfaces found in hospital settings. We demonstrated that increasing amounts of each microbicide, alone or in combination, were able to damage and to reduce the viability of A. baumannii biofilms efficaciously. Interestingly, the adeB gene of the resistance-nodulation-cell division (RND) family is predominantly associated with acquired resistance to antimicrobials in A. baumannii. We showed that CT and CHX adversely modified the expression and function of the RND-type efflux pump AdeABC in biofilm-associated A. baumannii cells. Furthermore, we established that these microbicides decreased the negative charges on A. baumannii cell membranes, causing dysregulation of the efflux pump and leading to cell death. Our findings suggest that CT and CHX, alone or in combination, can be used efficaciously for eradication of A. baumannii from hospital surfaces, in order to reduce infections caused by this nosocomial agent.

Sustained Nitric Oxide-Releasing Nanoparticles Induce Cell Death in Candida albicans Yeast and Hyphal Cells, Preventing Biofilm Formation In Vitro and in a Rodent Central Venous Catheter Model.

Candida albicansis a leading nosocomial pathogen. Today, candidal biofilms are a significant cause of catheter infections, and such infections are becoming increasingly responsible for the failure of medical-implanted devices.C. albicansforms biofilms in which fungal cells are encased in an autoproduced extracellular polysaccharide matrix. Consequently, the enclosed fungi are protected from antimicrobial agents and host cells, providing a unique niche conducive to robust microbial growth and a harbor for recurring infections. Here we demonstrate that a recently developed platform comprised of nanoparticles that release therapeutic levels of nitric oxide (NO-np) inhibits candidal biofilm formation, destroys the extracellular polysaccharide matrices of mature fungal biofilms, and hinders biofilm development on surface biomaterials such as the lumen of catheters. We found NO-np to decrease both the metabolic activity of biofilms and the cell viability ofC. albicansin vitroandin vivo Furthermore, flow cytometric analysis found NO-np to induce apoptosis in biofilm yeast cellsin vitro Moreover, NO-np behave synergistically when used in combination with established antifungal drug therapies. Here we propose NO-np as a novel treatment modality, especially in combination with standard antifungals, for the prevention and/or remediation of fungal biofilms on central venous catheters and other medical devices.

Phospholipase B Is Critical for Cryptococcus neoformans Survival in the Central Nervous System.

Cryptococcus neoformans (Cn) is an opportunistic, encapsulated, yeast-like fungus that causes severe meningoencephalitis, especially in countries with high HIV prevalence. In addition to its well-known polysaccharide capsule, Cn has other virulence factors such as phospholipases, a heterogeneous group of enzymes that hydrolyze ester linkages in glycerophospholipids. Phospholipase B (PLB1) has been demonstrated to play a key role in Cn pathogenicity. In this study, we used a PLB1 mutant (plb1) and its reconstituted strain (Rec1) to assess the importance of this enzyme on Cn brain infection in vivo and in vitro. Mice infected with the plb1 strain survive significantly longer, have lower peripheral and central nervous system (CNS) fungal loads, and have fewer and smaller cryptococcomas or biofilm-like brain lesions compared to H99- and Rec1-infected animals. PLB1 causes extensive brain tissue damage and changes microglia morphology during cryptococcal disease, observations which can have important implications in patients with altered mental status or dementia as these manifestations are related to poorer survival outcomes. plb1 cryptococci are significantly more phagocytosed and killed by NR-9460 microglia-like cells. plb1 cells have altered capsular polysaccharide biophysical properties which impair their ability to stimulate glial cell responses or morphological changes. Here, we provide significant evidence demonstrating that Cn PLB1 is an important virulence factor for fungal colonization of and survival in the CNS as well as in the progression of cryptococcal meningoencephalitis. These findings may potentially help fill in a gap of knowledge in our understanding of cerebral cryptococcosis and provide novel research avenues in Cn pathogenesis. IMPORTANCE Cryptococcal meningoencephalitis (CME) is a serious disease caused by infection by the neurotropic fungal pathogen Cryptococcus neoformans. Due to the increasing number of cases in HIV-infected individuals, as well as the limited therapies available, investigation into potential targets for new therapeutics has become critical. Phospholipase B is an enzyme synthesized by Cn that confers virulence to the fungus through capsular enlargement, immunomodulation, and intracellular replication. In this study, we examined the properties of PLB1 by comparing infection of a Cn PLB1 mutant strain with both the wild-type and a PLB1-reconstituted strain. We show that PLB1 augments the survival and proliferation of the fungus in the CNS and strengthens virulence by modulating the immune response and enhancing specific biophysical properties of the fungus. PLB1 expression causes brain tissue damage and impacts glial cell functions, which may be responsible for the dementia observed in patients which may persist even after resolving from CME. The implications of PLB1 inhibition reveal its involvement in Cn infection and suggest that it may be a possible molecular target in the development of antifungal therapies. The results of this study support additional investigation into the mechanism of PLB1 to further understand the intricacies of cerebral Cn infection.

Insulin and IGF-1 elicit robust transcriptional regulation to modulate autophagy in astrocytes.

OBJECTIVE: Insulin is a principal metabolic hormone. It regulates a plethora of metabolic pathways in peripheral tissues. The highly homologous insulin-like growth factor 1 (IGF-1), on the other hand, is important for development and growth. Recent studies have shown that insulin and IGF-1 signaling plays fundamental roles in the brain. Loss of insulin or IGF-1 receptors in astrocytes leads to altered glucose handling, mitochondrial metabolism, neurovascular coupling, and behavioral abnormalities in mice. Here, we aim to investigate molecular mechanisms by which insulin and IGF-1 signaling regulates astrocyte functions. METHODS: IR-flox and IRKO primary astrocytes were treated with 100 nM insulin or IGF-1 for 6 h, and their transcriptomes were analyzed. Astrocytes with either IR deletion, IGF1R deletion or both were used to examine receptor-dependent transcriptional regulations using qPCR. Additional immunoblotting and confocal imaging studies were performed to functionally validate pathways involved in protein homeostasis. RESULTS: Using next-generation RNA sequencing, we show that insulin significantly regulates the expression of over 1,200 genes involved in multiple functional processes in primary astrocytes. Insulin-like growth factor 1 (IGF-1) triggers a similar robust transcriptional regulation in astrocytes. Thus, over 50% of the differentially expressed genes are regulated by both ligands. As expected, these commonly regulated genes are highly enriched in pathways involved in lipid and cholesterol biosynthesis. Additionally, insulin and IGF-1 induce the expression of genes involved in ribosomal biogenesis, while suppressing the expression of genes involved in autophagy, indicating a common role of insulin and IGF-1 on protein homeostasis in astrocytes. Insulin-dependent suppression of autophagy genes, including p62, Ulk1/2, and several Atg genes, is blunted only when both IR and IGF1R are deleted. CONCLUSIONS: In summary, insulin and IGF-1 potently suppress autophagy in astrocytes through transcriptional regulation. Both IR and IGF1R can elicit ligand-dependent transcriptional suppression of autophagy. These results demonstrate an important role of astrocytic insulin/IGF-1 signaling on proteostasis. Impairment of this regulation in insulin resistance and diabetes may contribute to neurological complications related to diabetes.

Understanding the Basis of METH Mouth Using a Rodent Model of Methamphetamine Injection, Sugar Consumption, and Streptococcus mutans Infection.

"METH mouth" is a common consequence of chronic methamphetamine (METH) use, resulting in tooth decay and painful oral tissue inflammation that can progress to complete tooth loss. METH reduces the amount of saliva in the mouth, promoting bacterial growth, tooth decay, and oral tissue damage. This oral condition is worsened by METH users' compulsive behavior, including high rates of consumption of sugary drinks, recurrent tooth grinding, and a lack of frequent oral hygiene. Streptococcus mutans is a Gram-positive bacterium found in the oral cavity and associated with caries in humans. Hence, we developed a murine model of METH administration, sugar intake, and S. mutans infection to mimic METH mouth in humans and to investigate the impact of this drug on tooth colonization. We demonstrated that the combination of METH and sucrose stimulates S. mutans tooth adhesion, growth, and biofilm formation in vivo METH and sucrose increased the expression of S. mutans glycosyltransferases and lactic acid production. Moreover, METH contributes to the low environmental pH and S. mutans sucrose metabolism, providing a plausible mechanism for bacterium-mediated tooth decay. Daily oral rinse treatment with chlorhexidine significantly reduces tooth colonization in METH- and sucrose-treated mice. Furthermore, human saliva inhibits S. mutans colonization and biofilm formation after exposure to either sucrose or the combination of METH and sucrose. These findings suggest that METH might increase the risk of microbial dental disease in users, information that may help in the development of effective public health strategies to deal with this scourge in our society.IMPORTANCE "METH mouth" is characterized by severe tooth decay and gum disease, which often causes teeth to break or fall out. METH users are also prone to colonization by cariogenic bacteria such as Streptococcus mutans In addition, this oral condition is aggravated by METH users' compulsive behavior, including the consumption of beverages with high sugar content, recurrent tooth grinding, and a lack of frequent oral hygiene. We investigated the effects of METH and sugar consumption on S. mutans biofilm formation and tooth colonization. Using a murine model of METH administration, sucrose ingestion, and oral infection, we found that the combination of METH and sucrose increases S. mutans adhesion and biofilm formation on the teeth of C57BL/6 mice. However, daily chlorhexidine-based oral rinse treatment reduces S. mutans tooth colonization. Similarly, METH has been associated with dry mouth or hyposalivation in users. Hence, we assessed the impact of human saliva on biofilm formation and demonstrated that surface preconditioning with saliva substantially reduces S. mutans biofilm formation. Our results are significant because to our knowledge, this is the first basic science study focused on elucidating the fundamentals of METH mouth using a rodent model of prolonged drug injection and S. mutans oral infection. Our findings may have important translational implications for the development of treatments for the management of METH mouth and more effective preventive public health strategies that can be applied to provide effective dental care for METH users in prisons, drug treatment centers, and health clinics.

Acinetobacter baumannii Strains Deficient in the Clp Chaperone-Protease Genes Have Reduced Virulence in a Murine Model of Pneumonia.

Acinetobacter baumannii has emerged as a significant opportunistic Gram-negative pathogen and causative agent of nosocomial pneumonia especially in immunocompromised individuals in intensive care units. Recent advances to understand the contribution and function of A. baumannii virulence factors in its pathogenesis have begun to elucidate how this bacterium interacts with immune cells and its interesting mechanisms for multi-antibiotic resistance. Taking advantage of the availability of the A. baumannii AB5075 transposon mutant library, we investigated the impact of the A. baumannii Clp genes, which encode for a chaperone-protease responsible for the degradation of misfolded proteins, on bacterial virulence in a model of pneumonia using C57BL/6 mice and survival within J774.16 macrophage-like cells. Clp-protease A. baumannii mutants exhibit decreased virulence in rodents, high phagocytic cell-mediated killing and reduced biofilm formation. Capsular staining showed evidence of encapsulation in A. baumannii AB5075 and Clp-mutant strains. Surprisingly, clpA and clpS mutants displayed irregular cell morphology, which may be important in the biofilm structural deficiencies observed in these strains. Interestingly, clpA showed apical-like growth, proliferation normally observed in filamentous fungi. These findings provide new information regarding A. baumannii pathogenesis and may be important for the development of therapies intended at reducing morbidity and mortality associated with this remarkable pathogen.

Combination of acute intravenous methamphetamine injection and LPS challenge facilitate leukocyte infiltration into the central nervous system of C57BL/6 mice.

Methamphetamine (METH) is a stimulant of the central nervous system (CNS) that causes behavioral changes in users. METH is slowly cleared from brain tissue and its chronic use is neurotoxic. METH also alters the cellular and chemical components of inflammation. However, little is known about the effect of a single intravenous dose of METH followed by bacterial lipopolysaccharide (LPS) injection on cellular infiltration and cytokine release in brain tissue. Using a murine model of acute METH administration and flow cytometry, we found that combination of METH and LPS stimulate the infiltration of macrophages (F4/80+cells) and neutrophils (Ly-6G+cells) into the CNS. Histological sections of the brainstem of METH-treated and LPS-challenged C57BL/6 mice demonstrated considerable leukocyte infiltration relative to untreated, LPS, and METH groups. Moreover, rodents treated with LPS alone or combined with METH showed elevated levels of pro-inflammatory cytokines mRNA in brain tissue. Our observations are important because recognizing neuroinflammatory changes after acute METH administration might help us to understand METH-induced neurotoxicity in users.

Fungal serotype-specific differences in bacterial-yeast interactions.

Cryptococcus neoformans (Cn) causes meningoencephalitis in immunocompromised individuals. This encapsulated fungus can be found interacting with environmental microbes in soil contaminated with pigeon excrement. Cn survival within polymicrobial and other challenging communities has been shown to affect the evolution of its virulence factors. We compared the survival of 10 serotype A and D strains after interaction with the soil bacterium, Acinetobacter baumannii (Ab). Although co-incubation with Ab stimulated virulence factors production by strains of both cryptococcal serotypes, on average, serotype A strains displayed significantly higher survival rate, number of metabolically active cells within biofilms, and capsular polysaccharide production and release than serotype D strains. Our findings suggest that interactions of Cn with other microorganisms influence the fungus' regulation and production of virulence factors, important elements needed for the successful colonization of the human host.