ABSTRACT
Localized provoked vulvodynia (LPV) is the most common cause of chronic dyspareunia in premenopausal women, characterized by pain with light touch to the vulvar vestibule surrounding the vaginal opening. The devastating impact of LPV includes sexual dysfunction, infertility, depression, and even suicide. Yet, its etiology is unclear. No effective medical therapy exists; surgical removal of the painful vestibule is the last resort. In LPV, the vestibule expresses a unique inflammatory profile with elevated levels of pro-nociceptive proinflammatory mediators prostaglandin E2 (PGE2) and interleukin-6 (IL-6), which are linked to lower mechanical sensitivity thresholds. Specialized pro-resolving mediators (SPMs), lipids produced endogenously within the body, hold promise as an LPV treatment by resolving inflammation without impairing host defense. Ten of 13 commercially available SPMs reduced IL-6 and PGE2 production by vulvar fibroblasts, administered either before or after inflammatory stimulation. Using a murine vulvar pain model, coupling proinflammatory mediator quantification with mechanical sensitivity threshold determination, topical treatment with the SPM, maresin 1, decreased sensitivity and suppressed PGE2 levels. Docosahexaenoic acid, a precursor of maresin 1, was also effective in reducing PGE2 in vulvar fibroblasts and rapidly restored mouse sensitivity thresholds. Overall, SPMs and their precursors may be a safe and efficacious for LPV. Perspective: Vulvodynia, like many pain conditions, is difficult to treat because disease origins are incompletely understood. Here, we applied our knowledge of more recently discovered vulvodynia disease mechanisms to screen novel therapeutics. We identified several specialized pro-resolving mediators as likely potent and safe for treating LPV with potential for broader application.
Subject(s)
Dinoprostone , Docosahexaenoic Acids/pharmacology , Fibroblasts/drug effects , Inflammation/drug therapy , Interleukin-6 , Nociception/drug effects , Vulvodynia/drug therapy , Animals , Disease Models, Animal , Female , MiceABSTRACT
Staphylococcus aureus is an opportunistic pathogen with a clinical spectrum ranging from asymptomatic skin colonization to invasive infections. While traditional antibiotic therapies can be effective against S. aureus, the increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. Photodynamic inactivation (PDI) is an innovative and promising alternative to antibiotics. While progress has been made in our understanding of the bacterial response to PDI, major gaps remain in our knowledge of PDI tolerance, the global cellular response, and adaptive genomic mutations acquired as a result of PDI. To address these gaps, S. aureus HG003 and isogenic mutants with mutations in agr, mutS, mutL, and mutY exposed to single or multiple doses of PDI were assessed for survival and tolerance and examined by global transcriptome and genome analyses to identify regulatory and genetic adaptations that contribute to tolerance. Pathways in inorganic ion transport, oxidative response, DNA replication recombination and repair, and cell wall and membrane biogenesis were identified in a global cellular response to PDI. Tolerance to PDI was associated with superoxide dismutase and the S. aureus global methylhydroquinone (MHQ)-quinone transcriptome network. Genome analysis of PDI-tolerant HG003 identified a nonsynonymous mutation in the quinone binding domain of the transcriptional repressor QsrR, which mediates quinone sensing and oxidant response. Acquisition of a heritable QsrR mutation through repeated PDI treatment demonstrates selective adaption of S. aureus to PDI. PDI tolerance of a qsrR gene deletion in HG003 confirmed that QsrR regulates the S. aureus response to PDI.IMPORTANCEStaphylococcus aureus can cause disease at most body sites, with illness ranging from asymptomatic infection to death. The increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. S. aureus acquires resistance to antibiotics through multiple mechanisms, often by genetic variation that alters antimicrobial targets. Photodynamic inactivation (PDI), which employs a combination of a nontoxic dye and low-intensity visible light, is a promising alternative to antibiotics that effectively eradicates S. aureus in human infections when antibiotics are no longer effective. In this study, we demonstrate that repeated exposure to PDI results in resistance of S. aureus to further PDI treatment and identify the underlying bacterial mechanisms that contribute to resistance. This work supports further analysis of these mechanisms and refinement of this novel technology as an adjunctive treatment for S. aureus infections.
Subject(s)
Genome, Bacterial/radiation effects , Light , Microbial Viability/radiation effects , Staphylococcus aureus/genetics , Staphylococcus aureus/radiation effects , Gene Expression Profiling , Humans , Photochemotherapy , Staphylococcal Infections/microbiology , Staphylococcal Infections/therapyABSTRACT
Educating the next generation of physicians is a key means of communicating and disseminating impactful immunologic scientific knowledge, and its practical application to human disease. We present our perspective, using as our model a first-year medical school course entitled Host Defense. As the name suggests, immunology is the overarching principle that links the multiple subjects in the course. We address a range of immunologically relevant topics, including innate and adaptive immunity, vaccines, inflammation, allergy, tumor immunotherapy, transplantation, and autoimmunity. These topics are integrated with the fields of infectious diseases, pathology, clinical laboratory testing, and public health, to illustrate how the basic science discoveries in immunology are relevant to clinical practice. The course objectives are not only to deliver "first principles" and molecular mechanisms, but also to connect these principles with the clinical world of diagnosis and therapy. We detail the different methodologies used to achieve these objectives and to reach today's medical students. This provides a framework for course structure and execution designed to engage both the novice and the more "immunologically experienced" learner. The framework includes classical didactic components and personalized instructor access, aligned with current approaches to self-directed learning and using digital media. We also address some of the challenges of assembling a course like Host Defense in the context of an academic medical center with multiple scientific, educational, and clinical missions. This perspective is not meant be proscriptive, but rather to outline our experiences on the strategies tried, while describing their advantages and drawbacks in teaching immunology.
Subject(s)
Allergy and Immunology/education , Education, Medical , Education, Distance , Humans , Internet , Students, MedicalABSTRACT
BACKGROUND AND OBJECTIVES: Hospital-acquired infections (HAIs) and multidrug resistant bacteria pose a significant threat to the U.S. healthcare system. With a dearth of new antibiotic approvals, novel antimicrobial strategies are required to help solve this problem. Violet-blue visible light (400-470 nm) has been shown to elicit strong antimicrobial effects toward many pathogens, including representatives of the ESKAPE bacterial pathogens, which have a high propensity to cause HAIs. However, phototherapeutic solutions to prevention or treating infections are currently limited by efficient and nonobtrusive light-delivery mechanisms. STUDY DESIGN/MATERIALS AND METHODS: Here, we investigate the in vitro antimicrobial properties of flexible Corning® light-diffusing fiber (LDF) toward members of the ESKAPE pathogens in a variety of growth states and in the context of biological materials. Bacteria were grown on agar surfaces, in liquid culture and on abiotic surfaces. We also explored the effects of 405 nm light within the presence of lung surfactant, human serum, and on eukaryotic cells. Pathogens tested include Enterococcus spp, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Staphylococcus epidermidis, Streptococcus pyogenes, Candida albicans, and Escherichia coli. RESULTS: Overall, the LDF delivery of 405 nm violet-blue light exerted a significant degree of microbicidal activity against a wide range of pathogens under diverse experimental conditions. CONCLUSIONS: The results exemplify the fiber's promise as a non-traditional approach for the prevention and/or therapeutic intervention of HAIs. Lasers Surg. Med. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.
Subject(s)
Candida albicans/radiation effects , Cross Infection/prevention & control , Disinfection/methods , Gram-Negative Bacteria/radiation effects , Gram-Positive Bacteria/radiation effects , Lasers, Semiconductor/therapeutic use , Optical Fibers , Candidiasis/prevention & control , Disinfection/instrumentation , Gram-Negative Bacterial Infections/prevention & control , Gram-Positive Bacterial Infections/prevention & control , Humans , Low-Level Light Therapy , Microbial Sensitivity TestsABSTRACT
Introduction: New options are needed to improve wound healing while preventing excessive scar formation. Temporary primary dressings are important options in topical wound management that allow the natural healing process. Methods: We evaluated a novel primary dressing consisting of a biosynthetic, variable porosity, matrix-containing gelatin and Aloe Vera extract and a derivative dressing coated with the anti-scarring agent salinomycin for their ability to promote cell growth, reduce myofibroblast formation, and regulate cytokine production. In addition, salinomycin-coated primary dressings were tested for antimicrobial activity. Results: Both primary wound dressings permitted cell growth and attenuated TGFß-induced scar-forming myofibroblast formation. The primary wound dressings also reduced IL-6 production by 50%, IL-8 by 20%, MCP-1 by 75%, and GRO by 60% in human mesenchymal stem cells treated with TGFß. Salinomycin coating of the dressing showed antimicrobial activity by preventing Staphylococcus aureus growth. Conclusions: Both primary wound dressings support the growth of human fibroblasts and stem cells, as well as reduce inflammatory cytokine production, demonstrating their potential to serve as temporary wound dressings.
ABSTRACT
OBJECTIVES: Localized provoked vulvodynia (LPV) afflicts approximately 8% of women in the United States and represents a huge financial, physical, and psychological burden. Women with LPV experience intense pain localized to the vulvar vestibule (area immediately surrounding vaginal opening). We have identified mechanisms involved in the development of LPV whereby vulvar fibroblasts respond to proinflammatory stimuli to perpetuate an inflammatory response that causes pain. However, these mechanisms are not fully elucidated. Therefore, we explored the role of toll-like receptors (TLRs), a class of innate immune receptors that rapidly respond to microbial assaults. MATERIALS AND METHODS: To determine whether TLRs are expressed by vulvar fibroblasts and whether these contribute to proinflammatory mediator production and pain in LPV, we examined TLR expression and innate immune responses in fibroblasts derived from painful vestibular regions compared with nonpainful external vulvar regions. RESULTS: Human vulvar fibroblasts express functional TLRs that trigger production of inflammatory mediators associated with chronic pain. We focused on the TLR-7-imiquimod proinflammatory interaction, because imiquimod, a ligand of TLR-7, may exacerbate pain in women during treatment of human papillomavirus-associated disease. CONCLUSIONS: Human vulvar fibroblasts express a broad spectrum of TLRs (a new finding). A significantly higher TLR-mediated proinflammatory response was observed in LPV case vestibular fibroblasts, and with respect to the imiquimod-TLR 7 interaction, development of chronic vestibular pain and inflammation may be a possible sequelae of treatment of vulvar human papillomavirus-associated disease. Suppressing enhanced TLR-associated innate immune responses to a spectrum of pathogen-associated molecular patterns may represent a new/effective therapeutic approach for vulvodynia.
Subject(s)
Aminoquinolines/metabolism , Fibroblasts/immunology , Immunity, Innate , Inflammation Mediators/metabolism , Signal Transduction , Toll-Like Receptor 7/analysis , Vulvodynia/chemically induced , Cells, Cultured , Female , Fibroblasts/metabolism , Gene Expression Profiling , Humans , Imiquimod , Toll-Like Receptor 7/genetics , Vulvodynia/pathologyABSTRACT
Pneumocystis pneumonia (PcP) is a life-threatening infection that affects immunocompromised individuals. Nearly half of all PcP cases occur in those prescribed effective chemoprophylaxis, suggesting that additional preventive methods are needed. To this end, we have identified a unique mouse Pneumocystis surface protein, designated Pneumocystis cross-reactive antigen 1 (Pca1), as a potential vaccine candidate. Mice were immunized with a recombinant fusion protein containing Pca1. Subsequently, CD4+ T cells were depleted, and the mice were exposed to Pneumocystis murina Pca1 immunization completely protected nearly all mice, similar to immunization with whole Pneumocystis organisms. In contrast, all immunized negative-control mice developed PcP. Unexpectedly, Pca1 immunization generated cross-reactive antibody that recognized Pneumocystis jirovecii and Pneumocystis carinii Potential orthologs of Pca1 have been identified in P. jirovecii Such cross-reactivity is rare, and our findings suggest that Pca1 is a conserved antigen and potential vaccine target. The evaluation of Pca1-elicited antibodies in the prevention of PcP in humans deserves further investigation.
Subject(s)
Antigens, Fungal/immunology , Fungal Proteins/immunology , Pneumocystis carinii/immunology , Pneumocystis/immunology , Pneumonia, Pneumocystis/immunology , Animals , Antibodies, Fungal/immunology , Antibody Specificity/immunology , Antigens, Fungal/administration & dosage , Antigens, Fungal/genetics , Cross Reactions , Fungal Proteins/administration & dosage , Fungal Proteins/genetics , Fungal Vaccines/administration & dosage , Fungal Vaccines/immunology , Immunization , Mice , Pneumocystis/genetics , Pneumocystis carinii/genetics , Pneumonia, Pneumocystis/prevention & controlABSTRACT
Exposure to radiation, particularly a large or total-body dose, weakens the immune system through loss of bone marrow precursor cells, as well as diminished populations of circulating and tissue-resident immune cells. One such population is the skin-resident immune cells. Changes in the skin environment can be of particular importance as the skin is also host to a number of commensal organisms, including Candida albicans , a species of fungus that causes opportunistic infections in immunocompromised patients. In a previous study, we found that a 6 Gy sublethal dose of radiation in mice caused a reduction of cutaneous dendritic cells, indicating that the skin may have a poorer response to infection after irradiation. In this study, the same 6 Gy sublethal radiation dose led to a weakened response to a C. ablicans cutaneous infection, which resulted in systemic dissemination from the ear skin to the kidneys. However, this impaired response was mitigated through the use of interleukin-12 (IL-12) administered to the skin after irradiation. Concomitantly with this loss of local control of infection, we also observed a reduction of CD4+ and CD8+ T cells in the skin, as well as the reduced expression of IFN-γ, CXCL9 and IL-9, which influence T-cell infiltration and function in infected skin. These changes suggest a mechanism by which an impaired immune environment in the skin after a sublethal dose of radiation increases susceptibility to an opportunistic fungal infection. Thus, in the event of radiation exposure, it is important to include antifungal agents, or possibly IL-12, in the treatment regimen, particularly if wounds are involved that result in loss of the skin's physical barrier function.
Subject(s)
Candida albicans/physiology , Skin/microbiology , Skin/radiation effects , Whole-Body Irradiation , Animals , Candida albicans/radiation effects , Cytokines/metabolism , Granulocytes/immunology , Granulocytes/radiation effects , Interleukin-12/pharmacology , Kidney/microbiology , Kidney/radiation effects , Mice , Skin/drug effects , Skin/immunologyABSTRACT
Chronic vulvar pain is alarmingly common in women of reproductive age and is often accompanied by psychological distress, sexual dysfunction, and a significant reduction in quality of life. Localized provoked vulvodynia (LPV) is associated with intense vulvar pain concentrated in the vulvar vestibule (area surrounding vaginal opening). To date, the origins of vulvodynia are poorly understood, and treatment for LPV manages pain symptoms, but does not resolve the root causes of disease. Until recently, no definitive disease mechanisms had been identified; our work indicates LPV has inflammatory origins, although additional studies are needed to understand LPV pain. Bradykinin signaling is one of the most potent inducers of inflammatory pain and is a candidate contributor to LPV. We report that bradykinin receptors are expressed at elevated levels in LPV patient versus healthy control vestibular fibroblasts, and patient vestibular fibroblasts produce elevated levels of proinflammatory mediators with bradykinin stimulation. Inhibiting expression of one or both bradykinin receptors significantly reduces proinflammatory mediator production. Finally, we determined that bradykinin activates nuclear factor (NF)κB signaling (a major inflammatory pathway), whereas inhibition of NFκB successfully ablates this response. These data suggest that therapeutic agents targeting bradykinin sensing and/or NFκB may represent new, more specific options for LPV therapy. PERSPECTIVE: There is an unmet need for the development of more effective vulvodynia therapies. As we explore the mechanisms by which human vulvar fibroblasts respond to proinflammatory/propain stimuli, we move closer to understanding the origins of chronic vulvar pain and identifying new therapeutic targets, knowledge that could significantly improve patient care.
Subject(s)
Bradykinin/metabolism , Pelvic Pain/metabolism , Signal Transduction/physiology , Adult , Bradykinin/analogs & derivatives , Bradykinin/genetics , Bradykinin/pharmacology , Bradykinin Receptor Antagonists/pharmacology , Case-Control Studies , Cells, Cultured , Chronic Pain , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Interleukin-6/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Pelvic Pain/drug therapy , Pelvic Pain/pathology , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Bradykinin/genetics , Receptors, Bradykinin/metabolismABSTRACT
Fibroblast strains were derived from 2 regions of the lower genital tract of localized provoked vulvodynia (LPV) cases and pain-free controls. Sixteen strains were derived from 4 cases and 4 controls, age and race matched, after presampling mechanical pain threshold assessments. Strains were challenged with 6 separate stimuli: live yeast species (Candida albicans, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae), yeast extract (zymosan), or inactive vehicle. Production of prostaglandin E2 (PGE2) and interleukin 6 (IL-6) were proinflammatory response measures. Highest IL-6 and PGE2 occurred with vestibular strains after C albicans, C glabrata, and zymosan challenges, resulting in the ability to significantly predict IL-6 and PGE2 production by genital tract location. After C albicans and C glabrata challenge of all 16 fibroblast strains, adjusting for dual sampling of subjects, PGE2 and IL-6 production significantly predicted the presampling pain threshold from the genital tract site of sampling. At the same location of pain assessment and fibroblast sampling, in situ immunohistochemical (IHC)(+) fibroblasts for IL-6 and Cox-2 were quantified microscopically. The correlation between IL-6 production and IL-6 IHC(+) was statistically significant; however, biological significance is unknown because of the small number of IHC(+) IL-6 fibroblasts identified. A low fibroblast IL-6 IHC(+) count may result from most IL-6 produced by fibroblasts existing in a secreted extracellular state. Enhanced, site-specific, innate immune responsiveness to yeast pathogens by fibroblasts may be an early step in LPV pathogenesis. Fibroblast strain testing may offer an attractive and objective marker of LPV pathology in women with vulvodynia of inflammatory origin.
Subject(s)
Candida/isolation & purification , Candida/metabolism , Vulvodynia/microbiology , Vulvodynia/pathology , Adult , Candida/genetics , Cyclooxygenase 2/metabolism , Dinoprostone/metabolism , Female , Fibroblasts/metabolism , Humans , Inflammation/physiopathology , Interleukin-6/metabolism , Linear Models , Pain MeasurementABSTRACT
OBJECTIVE: Our goal was to gain a better understanding of the inflammatory pathways affected during localized vulvodynia, a poorly understood, common, and debilitating condition characterized by chronic pain of the vulvar vestibule. STUDY DESIGN: In a control matched study, primary human fibroblast strains were generated from biopsies collected from localized provoked vulvodynia (LPV) cases and from age- and race-matched controls. We then examined intracellular mechanisms by which these fibroblasts recognize pathogenic Candida albicans; >70% of vulvodynia patients report the occurrence of prior chronic Candida infections, which is accompanied by localized inflammation and elevated production of proinflammatory/pain-associated interleukin (IL)-6 and prostaglandin E2 (PGE2). We focused on examining the signaling pathways involved in recognition of yeast components that are present and abundant during chronic infection. RESULTS: Dectin-1, a surface receptor that binds C albicans cell wall glucan, was significantly elevated in vestibular vs external vulvar cells (from areas without pain) in both cases and controls, while its abundance was highest in LPV cases. Blocking Dectin-1 signaling significantly reduced pain-associated IL-6 and PGE2 production during the response to C albicans. Furthermore, LPV patient vestibular cells produced inflammatory mediators in response to low numbers of C albicans cells, while external vulvar fibroblasts were nonresponsive. Inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (proinflammatory transcription factor) nearly abrogated IL-6 and PGE2 production induced by C albicans, in keeping with observations that Dectin-1 signals through the nuclear factor kappa-light-chain-enhancer of activated B cells pathway. CONCLUSION: These findings implicate that a fibroblast-mediated proinflammatory response to C albicans contributes to the induction of pain in LPV cases. Targeting this response may be an ideal strategy for the development of new vulvodynia therapies.
Subject(s)
Vulvodynia/physiopathology , Adult , Candidiasis, Vulvovaginal/physiopathology , Dinoprostone/metabolism , Female , Fibroblasts/physiology , Humans , Inflammation/physiopathology , Interleukin-6/metabolism , Lectins, C-Type/metabolism , NF-kappa B/metabolism , Pain/etiology , Pain/physiopathology , Real-Time Polymerase Chain Reaction , Vulvodynia/microbiologyABSTRACT
BACKGROUND AND OBJECTIVE: The primary therapy for deep tissue abscesses is drainage accompanied by systemic antimicrobial treatment. However, the long antibiotic course required increases the probability of acquired resistance, and the high incidence of polymicrobial infections in abscesses complicates treatment choices. Photodynamic therapy (PDT) is effective against multiple classes of organisms, including those displaying drug resistance, and may serve as a useful adjunct to the standard of care by reduction of abscess microbial burden following drainage. STUDY DESIGN/MATERIALS AND METHODS: Aspirates were obtained from 32 patients who underwent image-guided percutaneous drainage of the abscess cavity. The majority of the specimens (24/32) were abdominal, with the remainder from liver and lung. Conventional microbiological techniques and nucleotide sequence analysis of rRNA gene fragments were used to characterize microbial populations from abscess aspirates. We evaluated the sensitivity of microorganisms to methylene blue-sensitized PDT in vitro both within the context of an abscess aspirate and as individual isolates. RESULTS: Most isolates were bacterial, with the fungus Candida tropicalis also isolated from two specimens. We examined the sensitivity of these microorganisms to methylene blue-PDT. Complete elimination of culturable microorganisms was achieved in three different aspirates, and significant killing (P < 0.0001) was observed in all individual microbial isolates tested compared to controls. CONCLUSIONS: These results and the technical feasibility of advancing optical fibers through catheters at the time of drainage motivate further work on including PDT as a therapeutic option during abscess treatment.
Subject(s)
Abscess/drug therapy , Candidiasis/drug therapy , Gram-Negative Bacterial Infections/drug therapy , Gram-Positive Bacterial Infections/drug therapy , Methylene Blue/therapeutic use , Photochemotherapy , Photosensitizing Agents/therapeutic use , Abdominal Abscess/drug therapy , Abdominal Abscess/microbiology , Abdominal Abscess/surgery , Abscess/microbiology , Abscess/surgery , Adolescent , Adult , Aged , Aged, 80 and over , Candida tropicalis/isolation & purification , Candidiasis/microbiology , Candidiasis/surgery , Combined Modality Therapy , Drainage/methods , Feasibility Studies , Female , Gram-Negative Bacterial Infections/microbiology , Gram-Negative Bacterial Infections/surgery , Gram-Positive Bacterial Infections/microbiology , Gram-Positive Bacterial Infections/surgery , Humans , Lung Abscess/drug therapy , Lung Abscess/microbiology , Lung Abscess/surgery , Male , Microbial Sensitivity Tests , Middle Aged , Polymerase Chain Reaction , Suction , Young AdultABSTRACT
Cutaneous and mucocutaneous Candida infections are considered to be important targets for antimicrobial photodynamic therapy (PDT). Clinical application of antimicrobial PDT will require strategies that enhance microbial killing while minimizing damage to host tissue. Increasing the sensitivity of infectious agents to PDT will help achieve this goal. Our previous studies demonstrated that raising the level of oxidative stress in Candida by interfering with fungal respiration increased the efficiency of PDT. Therefore, we sought to identify compounds in clinical use that would augment the oxidative stress caused by PDT by contributing to reactive oxygen species (ROS) formation themselves. Based on the ability of the antifungal miconazole to induce ROS in Candida, we tested several azole antifungals for their ability to augment PDT in vitro. Although miconazole and ketoconazole both stimulated ROS production in Candida albicans, only miconazole enhanced the killing of C. albicans and induced prolonged fungistasis in organisms that survived PDT using the porphyrin TMP-1363 and the phenothiazine methylene blue as photosensitizers. The data suggest that miconazole could be used to increase the efficacy of PDT against C. albicans, and its mechanism of action is likely to be multifactorial.
Subject(s)
Antifungal Agents/pharmacology , Candida albicans/drug effects , Miconazole/pharmacology , Photochemotherapy , Candida albicans/metabolism , Fluconazole/pharmacology , Microbial Sensitivity Tests , Oxidative Stress , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Reactive Oxygen Species/metabolismABSTRACT
BACKGROUND AND OBJECTIVE: The fungus Candida albicans commonly causes mucosal and cutaneous infections in patients with impaired immunity. We investigated the effectiveness of the photosensitizer meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363) in the photodynamic treatment (PDT) of C. albicans infection in vitro and its selectivity in an animal model. MATERIALS AND METHODS: The efficacy of TMP-1363 in PDT of C. albicans in vitro was compared to that of methylene blue (MB) using a colony forming unit (CFU) assay. In vivo infection in the mouse was established by inoculation of C. albicans yeast in the intradermal space of the ear pinna. Two days post-infection, 0.3 mg ml(-1) TMP-1363 was administered topically. Thirty minutes after TMP-1363 application, the ears were irradiated at 514 nm using a fluence of 90 J cm(-2) delivered at an irradiance of 50 mW cm(-2) . The ears were excised 2 hours post-irradiation, homogenized, and the organism burden was determined by a CFU assay. In vivo wide field and confocal fluorescence imaging assessed the localization of the photosensitizer in relationship to C. albicans. RESULTS: Photosensitization with TMP-1363 resulted in a greater than three-log increase in killing of C. albicans in vitro compared to MB. In vivo fluorescence imaging demonstrated a high degree of selective labeling of C. albicans by TMP-1363. PDT of infection using TMP-1363 resulted in a significant reduction in CFU/ear relative to untreated controls. Infected ears subjected to PDT displayed complete healing over time with no observable damage to the pinna. CONCLUSION: Our in vitro and in vivo findings support TMP-1363-mediated PDT as a viable therapeutic approach for the PDT of candidiasis.
Subject(s)
Candida albicans/drug effects , Candidiasis/drug therapy , Photochemotherapy/methods , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Administration, Topical , Animals , Candida albicans/metabolism , Colony Count, Microbial , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Fluoroscopy , In Vitro Techniques , Methylene Blue/administration & dosage , Methylene Blue/pharmacology , Mice , Mice, Inbred BALB C , Photosensitivity Disorders , Random Allocation , Sensitivity and SpecificityABSTRACT
Respiratory deficiency increases the sensitivity of the pathogenic fungi Candida albicans and Candida glabrata to oxidative stress induced by photodynamic therapy (PDT) sensitized by the cationic porphyrin meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363). Since disruption of electron transport chain (ETC) function increases intracellular levels of reactive oxygen species in yeast, we determined whether interference with ETC assembly or function increased sensitivity to TMP-1363-PDT in C. albicans, C. glabrata and the non-pathogenic yeast Saccharomyces cerevisiae. Metabolic inhibitor antimycin A and defined genetic mutants were used to identify ETC components that contribute to the sensitivity to PDT. Inhibition of cytochrome bc(1) (Complex III) with antimycin A increases mitochondrial levels of reactive oxygen species. PDT performed following pre-treatment with antimycin A reduced colony forming units (CFU) of C. albicans and C. glabrata by approximately two orders of magnitude relative to PDT alone. A S. cerevisiae mitochondrial glutaredoxin grx5 mutant, defective in assembly of Fe-S clusters critical for Complex III function, displayed increased sensitivity to PDT. Furthermore, C. glabrata and S.cerevisiae mutants in cytochrome c oxidase (Complex IV) synthesis and assembly were also significantly more sensitive to PDT. These included suv3, encoding an ATP-dependent RNA helicase critical for maturation of cytochrome c oxidase subunit transcripts, and pet117, encoding an essential cytochrome c oxidase assembly factor. Following PDT, the reduction in CFU of these mutants was one to two orders of magnitude greater than in their respective parental strains. The data demonstrate that selective inhibition of ETC Complexes III and IV significantly increases the sensitivity of C. albicans, C. glabrata and S. cerevisiae to PDT sensitized with TMP-1363.
Subject(s)
Candida albicans/radiation effects , Candida glabrata/radiation effects , Electron Transport Complex III/antagonists & inhibitors , Electron Transport Complex IV/antagonists & inhibitors , Antimycin A/pharmacology , Candida albicans/drug effects , Candida glabrata/drug effects , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Electron Transport Complex III/metabolism , Electron Transport Complex IV/metabolism , Mitochondria/metabolism , Oxidative Stress , Photochemotherapy , Photosensitizing Agents/toxicity , Porphyrins/toxicity , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant DeltanarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase.
Subject(s)
Nitrate Reductase/metabolism , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/growth & development , Anaerobiosis , Base Sequence , Catalytic Domain/genetics , Cell Membrane/enzymology , DNA, Bacterial/genetics , Energy Metabolism , Gene Expression , Genes, Bacterial , Mutagenesis, Site-Directed , Mutation , Nitrate Reductase/genetics , Nitrites/metabolism , Operon , Periplasm/enzymology , Pseudomonas aeruginosa/geneticsABSTRACT
The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (Delta narGH) and nitrite reductase (Delta nirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to Delta nirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14.
Subject(s)
Bacterial Proteins/physiology , Membrane Transport Proteins/biosynthesis , Monocytes/microbiology , Nitrite Reductases/physiology , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/pathogenicity , Virulence Factors/physiology , Animals , Bacterial Proteins/genetics , Cell Line , Cell Survival , Colony Count, Microbial , Gene Deletion , Gene Knockout Techniques , Humans , Microbial Viability , Mutation , Nitrate Reductase/genetics , Nitrate Reductase/physiology , Nitrite Reductases/genetics , Pseudomonas aeruginosa/genetics , Virulence , Virulence Factors/geneticsABSTRACT
Mucosal infections caused by the pathogenic fungus Candida are a significant infectious disease problem and are often difficult to eradicate because of the high frequency of resistance to conventional antifungal agents. Photodynamic treatment (PDT) offers an attractive therapeutic alternative. Previous studies demonstrated that filamentous forms and biofilms of Candida albicans were sensitive to PDT using Photofrin as a photosensitizer. However, early stationary phase yeast forms of C. albicans and Candida glabrata were not adversely affected by treatment. We report that the cationic porphyrin photosensitizer meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363) is effective in PDT against yeast forms of C. albicans and C. glabrata. Respiratory-deficient (RD) strains of C. albicans and C. glabrata display a pleiotropic resistance pattern, including resistance to members of the azole family of antifungals, the salivary antimicrobial peptides histatins and other types of toxic stresses. In contrast to this pattern, RD mutants of both C. albicans and C. glabrata were significantly more sensitive to PDT compared to parental strains. These data suggest that intact mitochondrial function may provide a basal level of anti-oxidant defense against PDT-induced phototoxicity in Candida, and reveals pathways of resistance to oxidative stress that can potentially be targeted to increase the efficacy of PDT against this pathogenic fungus.
Subject(s)
Candida albicans/drug effects , Candida albicans/metabolism , Candida glabrata/drug effects , Candida glabrata/metabolism , Oxygen Consumption , Photosensitizing Agents/pharmacology , Porphyrins/pharmacology , Antifungal Agents/pharmacology , Biofilms/drug effects , Candida albicans/genetics , Candida albicans/pathogenicity , Candida glabrata/genetics , Candida glabrata/pathogenicity , Dihematoporphyrin Ether/pharmacology , Hyphae/drug effects , Microbial Sensitivity Tests , Mitochondria/drug effects , Mitochondria/metabolism , Mutation , Oxidative Stress , PhotochemotherapyABSTRACT
Although most individuals are colonized with Candida albicans, only patients with insufficient or nonfunctional phagocytes develop life-threatening C. albicans disease. Because recognition of bacterial pathogens through phagocyte receptors for IgG (FcgammaR) is known to augment phagocyte responses, we postulated that antibody opsonization would enhance monocyte damage to C. albicans and subsequent tumor necrosis factor-alpha (TNF-alpha) production. After exposure to the human monocytic cell line THP-1, opsonized yeast showed an 89% decrease in metabolic activity, compared with 40% for unopsonized yeast (P<0.05). Culture supernatants contained 1316 pg mL(-1) of TNF-alpha after monocytes were exposed to opsonized yeast vs. 341 pg mL(-1) for unopsonized yeast (P=0.003). Similar results were obtained using peripheral blood mononuclear cells. Antibody opsonization of C. albicans germ tubes enhanced TNF-alpha production but did not affect organism damage. Antibody-dependent and antibody-independent factors were found to act synergistically to increase TNF-alpha production. ERK activation was important for both antibody-dependent and antibody-independent stimulation of TNF-alpha production, but not for monocyte-mediated organism damage. These data suggest that FcgammaR cooperates positively with antibody-independent recognition mechanisms in what may be a novel link between innate and adaptive immunity to C. albicans.
Subject(s)
Antibodies, Fungal/immunology , Candida albicans/immunology , Monocytes/immunology , Cell Line , Extracellular Signal-Regulated MAP Kinases/physiology , Humans , MAP Kinase Signaling System , Phagocytosis , Phosphorylation , Toll-Like Receptors/physiology , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a biofilm, requiring motility for biofilm formation and dispersal and an ability to grow in nutrient- and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through the denitrification enzyme pathway that catalyzes the sequential reduction of nitrate to nitrogen gas. Mutants mutated in the two-component nitrate sensor-response regulator and in membrane nitrate reductase displayed altered motility and biofilm formation compared to wild-type P. aeruginosa PAO1. Analysis of additional nitrate dissimilation mutants demonstrated a second level of regulation in P. aeruginosa motility that is independent of nitrate sensor-response regulator function and is associated with nitric oxide production. Because motility and biofilm formation are important for P. aeruginosa pathogenicity, we examined the virulence of selected regulatory and structural gene mutants in the surrogate model host Caenorhabditis elegans. Interestingly, the membrane nitrate reductase mutant was avirulent in C. elegans, while nitrate sensor-response regulator mutants were fully virulent. The data demonstrate that nitrate sensing, response regulation, and metabolism are linked directly to factors important in P. aeruginosa pathogenesis.