[Show abstract][Hide abstract] ABSTRACT: Acinetobacter baumannii (Ab) is a common cause of community-acquired pneumonia (CAP) in chronic alcoholics in tropical and sub-tropical climates and associated with a >50% mortality rate. Using a murine model of alcohol (EtOH) administration, we demonstrated that EtOH enhances Ab-mediated pneumonia leading to systemic infection. Although EtOH did not affect neutrophil recruitment to the lungs of treated mice, it decreased phagocytosis and killing of bacteria by these leukocytes leading to increased microbial burden and severity of disease. Moreover, we determined that mice that received EtOH prior to Ab infection were immunologically impaired, which was reflected in increased pulmonary inflammation, sequential dissemination to the liver and kidneys, and decreased survival. Furthermore, immunosuppression by EtOH was associated with deregulation of cytokine production in the organs of infected mice. This study establishes that EtOH impairs immunity in vivo exacerbating Ab infection and disease progression. The ability of Ab to cause disease in alcoholics warrants the study of its virulence mechanisms and host interactions.
PLoS ONE 04/2014; 9(4):e95707. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acinetobacter baumannii (Ab) is a frequent cause of hospital-acquired pneumonia and has recently increased in incidence as the causative agent of severe disease in troops wounded in Afghanistan and Iraq. Clinical approaches are limited since Ab strains isolated from patients are extremely resistant to current antimicrobials. Ab can survive desiccation and during outbreaks has been recovered from various sites in the patients' environment. To better understand its prevalence in hospital-settings, we used a stainless steel washer (SSW) platform to investigate Ab biofilm formation in abiotic surfaces. Scanning electron microscopy demonstrated that Ab forms strong biofilms on stainless steel surfaces. This platform was combined with a colorimetric 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay to observe the metabolic activity of bacterial cells and facilitate the manipulation and comparison of multiple Ab clinical strains. A strong correlation between XTT and CFU assays was demonstrated. To complement the cell viability assays, Ab biofilm mass was measured by crystal violet staining. Furthermore, the effect of commonly-used disinfectants and environmental stressors on Ab biofilms and planktonic cells was compared and characterized. Biofilms on SSWs were significantly more resistant than their planktonic counterparts providing additional evidence that may allow us to understand the high prevalence of this microbe in hospital-settings. Our results validate that SSWs are a simple, versatile, and innovative method to study Ab biofilms in vitro.
[Show abstract][Hide abstract] ABSTRACT: Acinetobacter baumannii is an opportunistic Gram-negative bacterium that affects critically ill hospitalized patients with breaches in skin integrity and airway protection leading to significant morbidity and mortality. Considering the paucity of well-established animal models of immunosuppression to study A. baumannii pathogenesis, we set out to characterize a murine model of immunosuppression using the alkylating agent cyclophosphamide (CYP). We hypothesize that CYP-induced immunosuppression would increase the susceptibility of C57BL/6 to develop A. baumannii-mediated pneumonia followed by systemic disease. We demonstrated that CYP intensified A. baumannii-mediated pulmonary disease, abrogated normal immune cell function and lead to altered pro-inflammatory cytokine release. The development of an animal model that mimics A. baumannii infection onset in immunosuppressed individuals is crucial for generating novel approaches to patient care and improving public health strategies to decrease susceptibility of infection for individuals at risk.
Journal of Medical Microbiology 09/2013; · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. Methicillin resistant Staphylococcus aureus has become a major health threat in the US. Staphylococcal enterotoxin B is a potent superantigen that contributes to its virulence. High mortality and frequent failure of therapy despite available antibiotics have stimulated research efforts to develop adjunctive therapies.Methods. Treatment benefits of SEB specific mAb-20B1 were investigated in mice in a sepsis, superficial skin, as well as deep tissue infection model.Results. Mice challenged with a SEB producing MRSA-strain developed fatal sepsis, extensive tissue skin infection and abscess forming deep seeded thigh muscle infection. Animals pre-immunized against SEB, or treated passively with mAb-20B1 exhibited enhanced survival in the sepsis model, whereas decrease of bacterial burden was observed in the superficial skin and deep tissue model. Mab-20B1 bound to SEB in the infected tissue and decreased abscess formation as well as pro-inflammatory cytokine levels, lymphocyte proliferation and neutrophil recruitment.Conclusions. The SEB neutralizing mAb-20B1 constitutes an effective therapy in MRSA infection. Administration of mAb-20B1 protects from lethal sepsis and reduces invasion of skin tissue and deep abscess formation. The mAb penetrates well into the abscess and binds to SEB. It affects outcome by modulating the pro-inflammatory host response.
The Journal of Infectious Diseases 08/2013; · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acinetobacter baumannii (Ab) is a common cause of community-acquired pneumonia (CAP) in chronic alcoholics in tropical and sub-tropical climates and associated with a > 50% mortality rate. We demonstrated that exposure of J774.16 macrophage-like cells to physiological alcohol (EtOH) concentrations decreased phagocytosis and killing of Ab. EtOH-mediated macrophage phagocytosis dysfunction may be associated with reduced expression of GTPase-RhoA, a key regulator of the actin polymerization signaling cascade. EtOH inhibited nitric oxide (NO) generation via inducible NO-synthase inactivation, which enhanced Ab survival within macrophages. Additionally, EtOH alters cytokine production resulting in a dysregulated immune response. This study is a proof of principle which establishes that EtOH might exacerbate Ab infection and be an important factor enhancing CAP in individuals at risk.
[Show abstract][Hide abstract] ABSTRACT: Methamphetamine (METH) is a drug of abuse that is a potent and highly addictive central nervous system (CNS) stimulant. The blood brain barrier (BBB) is a unique interface that in part functions to prevent microbial invasion of the CNS. The effects of METH on brain vasculature have not been studied extensively. We hypothesized that METH alters the BBB integrity increasing susceptibility to CNS infection. Using a murine model of METH administration, we demonstrated that METH alters BBB integrity and modifies the expression of tight junction and adhesion molecules. Additionally, we showed that BBB disruption accelerates transmigration of the neurotropic fungus Cryptococcus neoformans (Cn) into the brain parenchyma after systemic infection. Furthermore, METH-treated mice displayed increased mortality compared to untreated animals. Our findings provide novel evidence of the impact of METH use on the integrity of the cells that comprise the BBB, the cells that protect the brain from infection.
The Journal of Infectious Diseases 03/2013; · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Methamphetamine (METH) is a potent and highly addictive central nervous system (CNS) stimulant. Additionally, METH adversely impacts immunological responses, which might contribute to the higher rate and more rapid progression of certain infections in drug abusers. However no studies have shown the impact of METH on inflammation within specific organs, cellular participation and cytokine production. Using a murine model of METH administration, we demonstrated that METH modifies, with variable degrees, leukocyte recruitment and alters cellular mediators in the lungs, liver, spleen and kidneys of mice. Our findings demonstrate the pleotropic effects of METH on the immune response within diverse tissues. These alterations have profound implications on tissue homeostasis and the capacity of the host to respond to diverse insults, including invading pathogens.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT 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.
[Show abstract][Hide abstract] ABSTRACT: Autophagy is used by eukaryotes in bulk cellular material recycling and in immunity to intracellular pathogens. We evaluated the role of macrophage autophagy in the response to Cryptococcus neoformans and Candida albicans, two important opportunistic fungal pathogens. The autophagosome marker LC3 (microtubule-associated protein 1 light chain 3 alpha) was present in most macrophage vacuoles containing C. albicans. In contrast, LC3 was found in only a few vacuoles containing C. neoformans previously opsonized with antibody but never after complement-mediated phagocytosis. Disruption of host autophagy in vitro by RNA interference against ATG5 (autophagy-related 5) decreased the phagocytosis of C. albicans and the fungistatic activity of J774.16 macrophage-like cells against both fungi, independent of the opsonin used. ATG5-knockout bone marrow-derived macrophages (BMMs) also had decreased fungistatic activity against C. neoformans when activated. In contrast, nonactivated ATG5-knockout BMMs actually restricted C. neoformans growth more efficiently, suggesting that macrophage autophagy plays different roles against C. neoformans, depending on the macrophage type and activation. Interference with autophagy in J774.16 cells also decreased nonlytic exocytosis of C. neoformans, increased interleukin-6 secretion, and decreased gamma interferon-induced protein 10 secretion. Mice with a conditionally knocked out ATG5 gene in myeloid cells showed increased susceptibility to intravenous C. albicans infection. In contrast, these mice manifested no increased susceptibility to C. neoformans, as measured by survival, but had fewer alternatively activated macrophages and less inflammation in the lungs after intratracheal infection than control mice. These results demonstrate the complex roles of macrophage autophagy in restricting intracellular parasitism by fungi and reveal connections with nonlytic exocytosis, humoral immunity, and cytokine signaling.
Infection and immunity 06/2012; 80(9):3065-76. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wound healing is a complex process, coordinated by various biological factors. In immunocompromised states wound healing can be interrupted as a result of decreased numbers of immune cells, impairing the production of effector molecules such as nitric oxide (NO). Therefore, topical NO-releasing platforms, such as diethylenetriamine (DETA NONOate), have been investigated to enhance wound healing. Recently, we demonstrated a nanoparticle platform that releases NO (NO-NPs) in a sustained manner, accelerating wound healing in both uninfected and infected murine wound models. Here, NO-NPs were investigated and compared to DETA NONOate in an immunocompromised wound model using non-obese, diabetic, severe combined immunodeficiency mice. NO-NP treatment accelerated wound closure as compared to controls and DETA NONOate treatment. In addition, histological assessment revealed that wounds treated with NO-NPs had less inflammation, more collagen deposition, and more blood vessel formation as compared to other groups, consistent with our previous data in immunocompetent animals. These data suggest that NO-NPs may serve as a novel wound-healing therapy in the setting of immunocompromised states associated with impaired wound healing. FROM THE CLINICAL EDITOR: Wound healing in an immunocompromised host is often incomplete and is a source of major concern in such conditions. This work demonstrates in a murine model that in these settings NO releasing nanoparticles significantly enhance wound healing.
Nanomedicine: nanotechnology, biology, and medicine 03/2012; 8(8):1364-71. · 6.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wound healing is a complex process that involves coordinated interactions between diverse immunological and biological systems. Long-term wounds remain a challenging clinical problem, affecting approximately 6 million patients per year, with a high economic impact. To exacerbate the problem, these wounds render the individual susceptible to life-threatening microbial infections. Because current therapeutic strategies have proved suboptimal, it is imperative to focus on new therapeutic approaches and the development of technologies for both short- and long-term wound management. In recent years, nitric oxide (NO) has emerged as a critical molecule in wound healing, with NO levels increasing rapidly after skin damage and gradually decreasing as the healing process progresses. In this study, we examined the effects of a novel NO-releasing nanoparticle technology on wound healing in mice. The results show that the NO nanoparticles (NO-np) significantly accelerated wound healing. NO-np modified leukocyte migration and increased tumor growth factor-β production in the wound area, which subsequently promoted angiogenesis to enhance the healing process. By using human dermal fibroblasts, we demonstrate that NO-np increased fibroblast migration and collagen deposition in wounded tissue. Together, these data show that NO-releasing nanoparticles have the ability to modulate and accelerate wound healing in a pleiotropic manner.
American Journal Of Pathology 02/2012; 180(4):1465-73. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) is a critical component of host defense against invading pathogens; however, its therapeutic utility is limited due to a lack of practical delivery systems. Recently, a NO-releasing nanoparticulate platform (NO-np) was shown to have in vitro broad-spectrum antimicrobial activity and in vivo pre-clinical efficacy in a dermal abscess model. To extend these findings, both topical (TP) and intralesional (IL) NO-np administration was evaluated in a MRSA intramuscular murine abscess model and compared with vancomycin. All treatment arms accelerated abscess clearance clinically, histologically, and by microbiological assays on both days 4 and 7 following infection. However, abscesses treated with NO-np via either route demonstrated a more substantial, statistically significant decrease in bacterial survival based on colony forming unit assays and histologically revealed less inflammatory cell infiltration and preserved muscular architecture. These data suggest that the NO-np may be an effective addition to our armament for deep soft tissue infections.
[Show abstract][Hide abstract] ABSTRACT: Candida albicans is a leading fungal cause of burn infections in hospital settings, and sepsis is one of the principle causes of death after a severe burn. The prevalence of invasive candidiasis in burn cases varies widely, but it accounts for 3-23% of severe infection with a mortality rate ranging from 14 to 70%. Therefore, it is imperative that we develop innovative therapeutics to which this fungus is unlikely to evolve resistance, thus curtailing the associated morbidity and mortality and ultimately improving our capacity to treat these infections. An inexpensive and stable nitric oxide (NO)-releasing nanoparticle (NO-np) platform has been recently developed. NO is known to have direct antifungal activity, modulate host immune responses and significantly regulate wound healing. In this study, we hypothesized that NO-np would be an effective therapy in the treatment of C. albicans burn infections. Using a murine burn model, NO-np demonstrated antifungal activity against C. albicans in vivo, most likely by arresting its growth and morphogenesis as demonstrated in vitro. NO-np demonstrated effective antimicrobial activity against yeast and filamentous forms of the fungus. Moreover, we showed that NO-np significantly accelerated the rate of wound healing in cutaneous burn infections when compared to controls. The histological evaluation of the affected tissue revealed that NO-np treatment modified leukocyte infiltration, minimized the fungal burden, and reduced collagen degradation, thus providing potential mechanisms for the therapeutics' biological activity. Together, these data suggest that NO-np have the potential to serve as a novel topical antifungal which can be used for the treatment of cutaneous burn infections and wounds.
Frontiers in Microbiology 01/2012; 3:193. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Does the age of a microbial cell affect its virulence factors? To our knowledge, this question has not been addressed previously, but the answer is of great relevance for chronic infections where microbial cells persist and age in hosts. Cryptococcus neoformans is an encapsulated human-pathogenic fungus notorious for causing chronic infections where cells of variable age persist in tissue. The major virulence factor for C. neoformans is a polysaccharide (PS) capsule. To understand how chronological age could impact the cryptococcal capsule properties, we compared the elastic properties, permeabilities, zeta potentials, and glycosidic compositions of capsules from young and old cells and found significant differences in all parameters measured. Changes in capsular properties were paralleled by changes in PS molecular mass and density, as well as modified antigenic density and antiphagocytic properties. Remarkably, chronological aging under stationary-phase growth conditions was associated with the expression of α-1,3-glucans in the capsule, indicating a new structural capsular component. Our results establish that cryptococcal capsules are highly dynamic structures that change dramatically with chronological aging under prolonged stationary-phase growth conditions. Changes associated with cellular aging in chronic infections could contribute to the remarkable capacity of this fungus to persist in tissues by generating phenotypically and antigenically different capsules.
Infection and immunity 12/2011; 79(12):4990-5000. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microplusin is an antimicrobial peptide isolated from the cattle tick Rhipicephalus (Boophilus) microplus. Its copper-chelating ability is putatively responsible for its bacteriostatic activity against Micrococcus luteus as microplusin inhibits respiration in this species, which is a copper-dependent process. Microplusin is also active against Cryptococcus neoformans (MIC(50) = 0.09 μM), the etiologic agent of cryptococcosis. Here, we show that microplusin is fungistatic to C. neoformans and this inhibitory effect is abrogated by copper supplementation. Notably, microplusin drastically altered the respiratory profile of C. neoformans. In addition, microplusin affects important virulence factors of this fungus. We observed that microplusin completely inhibited fungal melanization, and this effect correlates with the inhibition of the related enzyme laccase. Also, microplusin significantly inhibited the capsule size of C. neoformans. Our studies reveal, for the first time, a copper-chelating antimicrobial peptide that inhibits respiration and growth of C. neoformans and modifies two major virulence factors: melanization and formation of a polysaccharide capsule. These features suggest that microplusin, or other copper-chelation approaches, may be a promising therapeutic for cryptococcosis.
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO) plays a vital role in mammalian host defense through a variety of mechanisms. In particular, NO can oxidize to form reactive nitrogen species or interact with protein thiols and metal centers, blocking essential microbial processes. S-nitrosoglutathione (GSNO), a potent NO donor formed by the interaction of NO with intracellular glutathione (GSH), is a major factor in this pathway and is considered one of the strongest naturally occurring nitrosating agent. We previously described the broad-spectrum antimicrobial activity of a nanoparticulate platform capable of controlled and sustained release of NO (NO-np). Interestingly, in vivo efficacy of the NO-np surpassed in vitro data generated. We hypothesized that the enhanced activity was in part achieved via the interaction between the generated NO and available GSH, forming GSNO. In the current study, we investigated the efficiency of NO-np to form GSNO in the presence of GSH was evaluated, and assessed the antimicrobial activity of the formed GSNO against methicillin resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. When GSH was combined with NO-np, GSNO was rapidly produced and significant concentrations of GSNO were maintained for >24h. The GSNO generated was more effective compared to NO-np alone against all bacterial strains examined, with P. aeruginosa being the most sensitive and K. pneumoniae the most resistant. We conclude that the combination of NO-np with GSH is an effective means of generating GSNO, and presents a novel approach to potent antimicrobial therapy.
[Show abstract][Hide abstract] ABSTRACT: The rapidly evolving crisis of antibiotic resistance among microorganisms has contributed to the rise of patient morbidity and mortality from nosocomial and community-acquired infections. Therefore, innovative antimicrobial technology targeting mechanisms to which the bacteria are unlikely to evolve resistance is urgently needed. We have previously described a nitric oxide-releasing nanoparticle (NO-np) with efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii in vitro and in murine wound and abscess models. Although the prior findings suggest that the NO-np can be a useful therapeutic for skin and soft tissue infections, the antimicrobial spectrum of NO-np has yet to be fully elucidated. In the current study, we investigated the efficacy of a NO-np against several Gram-positive and -negative clinical isolates. We found that the NO-np were uniformly active against all of the Streptococcus pyogenes, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa clinical isolates examined, including strains that were both sensitive and resistant to commonly used antibiotics. We concluded that the NO-np have the potential to serve as a novel broad spectrum antimicrobial agent.
[Show abstract][Hide abstract] ABSTRACT: The Gram-negative coccobacillus Acinetobacter baumannii (Ab) has become an increasingly prevalent cause of hospital-acquired infections during the last two decades primarily resulting in pneumonia and complicated infections, including wound infections in troops injured in Afghanistan and Iraq. Moreover, the majority of clinical Ab isolates display high-level resistance to commonly utilized antimicrobial drugs, which severely compromises our capacity to care for patients with Ab disease. Thus, radically new approaches are urgently needed. This review focuses on novel therapies that can challenge the evolving ability of Ab to develop resistance and cause disease.
[Show abstract][Hide abstract] ABSTRACT: Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.
The Journal of clinical investigation 03/2011; 121(4):1471-83. · 15.39 Impact Factor