Critical Role of Bcr1-Dependent Adhesins in C. albicans Biofilm Formation In Vitro and In Vivo

The fungal pathogen Candida albicans is frequently associated with catheter-based infections because of its ability to form resilient biofilms. Prior studies have shown that the transcription factor Bcr1 governs biofilm formation in an in vitro catheter model. However, the mechanistic role of the Bcr1 pathway and its relationship to biofilm formation in vivo are unknown. Our studies of biofilm formation in vitro indicate that the surface protein Als3, a known adhesin, is a key target under Bcr1 control. We show that an als3/als3 mutant is biofilm-defective in vitro, and that ALS3 overexpression rescues the biofilm defect of the bcr1/bcr1 mutant. We extend these findings with an in vivo venous catheter model. The bcr1/bcr1 mutant is unable to populate the catheter surface, though its virulence suggests that it has no growth defect in vivo. ALS3 overexpression rescues the bcr1/bcr1 biofilm defect in vivo, thus arguing that Als3 is a pivotal Bcr1 target in this setting. Surprisingly, the als3/als3 mutant forms a biofilm in vivo, and we suggest that additional Bcr1 targets compensate for the Als3 defect in vivo. Indeed, overexpression of Bcr1 targets ALS1, ECE1, and HWP1 partially restores biofilm formation in a bcr1/bcr1 mutant background in vitro, though these genes are not required for biofilm formation in vitro. Our findings demonstrate that the Bcr1 pathway functions in vivo to promote biofilm formation, and that Als3-mediated adherence is a fundamental property under Bcr1 control. Known adhesins Als1 and Hwp1 also contribute to biofilm formation, as does the novel protein Ece1

Hsp90 governs dispersion and drug resistance of fungal biofilms

Fungal biofilms are a major cause of human mortality and are recalcitrant to most treatments due to intrinsic drug resistance. These complex communities of multiple cell types form on indwelling medical devices and their eradication often requires surgical removal of infected devices. Here we implicate the molecular chaperone Hsp90 as a key regulator of biofilm dispersion and drug resistance. We previously established that in the leading human fungal pathogen, Candida albicans, Hsp90 enables the emergence and maintenance of drug resistance in planktonic conditions by stabilizing the protein phosphatase calcineurin and MAPK Mkc1. Hsp90 also regulates temperature-dependent C. albicans morphogenesis through repression of cAMP-PKA signalling. Here we demonstrate that genetic depletion of Hsp90 reduced C. albicans biofilm growth and maturation in vitro and impaired dispersal of biofilm cells. Further, compromising Hsp90 function in vitro abrogated resistance of C. albicans biofilms to the most widely deployed class of antifungal drugs, the azoles. Depletion of Hsp90 led to reduction of calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Reduction of Hsp90 levels led to a marked decrease in matrix glucan levels, providing a compelling mechanism through which Hsp90 might regulate biofilm azole resistance. Impairment of Hsp90 function genetically or pharmacologically transformed fluconazole from ineffectual to highly effective in eradicating biofilms in a rat venous catheter infection model. Finally, inhibition of Hsp90 reduced resistance of biofilms of the most lethal mould, Aspergillus fumigatus, to the newest class of antifungals to reach the clinic, the echinocandins. Thus, we establish a novel mechanism regulating biofilm drug resistance and dispersion and that targeting Hsp90 provides a much-needed strategy for improving clinical outcome in the treatment of biofilm infections

Neutropenia induced in outbred mice by a simplified low-dose cyclophosphamide regimen: characterization and applicability to diverse experimental models of infectious diseases

BACKGROUND: For its low cost and ease of handling, the mouse remains the preferred experimental animal for preclinical tests. To avoid the interaction of the animal immune system, in vivo antibiotic pharmacodynamic studies often employ cyclophosphamide (CPM) to induce neutropenia. Although high doses (350–450 mg/kg) are still used and their effects on mouse leukocytes have been described, a lower dose (250 mg/kg) is widely preferred today, but the characteristics and applicability of this approach in outbred mice have not been determined. METHODS: Fifteen female ICR mice were injected intraperitoneally with 150 and 100 mg/kg of CPM on days 1 and 4, respectively. Blood samples (~160 μL) were drawn from the retro-orbital sinus of each mouse on days 1, 4, 5, 6, 7 and 11. Leukocytes were counted manually and the number of granulocytes was based on microscopic examination of Wright-stained smears. The impact of neutropenia induced by this method was then determined with a variety of pathogens in three different murine models of human infections: pneumonia (Klebsiella pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus), meningoencephalitis (S. pneumoniae), and the thigh model (S. aureus, Escherichia coli, Bacteroides fragilis). RESULTS: The basal count of leukocytes was within the normal range for outbred mice. On day 4, there was an 84% reduction in total white blood cells, and by day 5 the leukopenia reached its nadir (370 ± 84 cells/mm(3)). Profound neutropenia (≤10 neutrophils/mm(3)) was demonstrated at day 4 and persisted through days 5 and 6. Lymphocytes and monocytes had a 92% and 96% decline between days 1 and 5, respectively. Leukocytes recovered completely by day 11. Mice immunosupressed under this protocol displayed clinical and microbiological patterns of progressive and lethal infectious diseases after inoculation in different organs with diverse human pathogens. CONCLUSION: A CPM total dose of 250 mg/kg is sufficient to induce profound and sustained neutropenia (<10 neutrophils/mm(3)) at least during 3 days in outbred mice, is simpler than previously described methods, and allows successful induction of infection in a variety of experimental models

Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium.

BACKGROUND: Invasive fungal diseases (IFDs) remain important causes of morbidity and mortality. The consensus definitions of the Infectious Diseases Group of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group have been of immense value to researchers who conduct clinical trials of antifungals, assess diagnostic tests, and undertake epidemiologic studies. However, their utility has not extended beyond patients with cancer or recipients of stem cell or solid organ transplants. With newer diagnostic techniques available, it was clear that an update of these definitions was essential. METHODS: To achieve this, 10 working groups looked closely at imaging, laboratory diagnosis, and special populations at risk of IFD. A final version of the manuscript was agreed upon after the groups' findings were presented at a scientific symposium and after a 3-month period for public comment. There were several rounds of discussion before a final version of the manuscript was approved. RESULTS: There is no change in the classifications of "proven," "probable," and "possible" IFD, although the definition of "probable" has been expanded and the scope of the category "possible" has been diminished. The category of proven IFD can apply to any patient, regardless of whether the patient is immunocompromised. The probable and possible categories are proposed for immunocompromised patients only, except for endemic mycoses. CONCLUSIONS: These updated definitions of IFDs should prove applicable in clinical, diagnostic, and epidemiologic research of a broader range of patients at high-risk

Murine model for Fusarium oxysporum invasive fusariosis reveals organ-specific structures for dissemination and long-term persistence

Peer reviewedPublisher PD

Genome expansion by allopolyploidization in the fungal strain <i>Coniochaeta </i>2T2.1 and its exceptional lignocellulolytic machinery

Background Particular species of the genus Coniochaeta (Sordariomycetes) exhibit great potential for bioabatement of furanic compounds and have been identified as an underexplored source of novel lignocellulolytic enzymes, especially Coniochaeta ligniaria. However, there is a lack of information about their genomic features and metabolic capabilities. Here, we report the first in-depth genome/transcriptome survey of a Coniochaeta species (strain 2T2.1). Results The genome of Coniochaeta sp. strain 2T2.1 has a size of 74.53 Mbp and contains 24,735 protein-encoding genes. Interestingly, we detected a genome expansion event, resulting similar to 98% of the assembly being duplicated with 91.9% average nucleotide identity between the duplicated regions. The lack of gene loss, as well as the high divergence and strong genome-wide signatures of purifying selection between copies indicates that this is likely a recent duplication, which arose through hybridization between two related Coniochaeta-like species (allopolyploidization). Phylogenomic analysis revealed that 2T2.1 is related Coniochaeta sp. PMI546 and Lecythophora sp. AK0013, which both occur endophytically. Based on carbohydrate-active enzyme (CAZy) annotation, we observed that even after in silico removal of its duplicated content, the 2T2.1 genome contains exceptional lignocellulolytic machinery. Moreover, transcriptomic data reveal the overexpression of proteins affiliated to CAZy families GH11, GH10 (endoxylanases), CE5, CE1 (xylan esterases), GH62, GH51 (alpha-l-arabinofuranosidases), GH12, GH7 (cellulases), and AA9 (lytic polysaccharide monoxygenases) when the fungus was grown on wheat straw compared with glucose as the sole carbon source. Conclusions We provide data that suggest that a recent hybridization between the genomes of related species may have given rise to Coniochaeta sp. 2T2.1. Moreover, our results reveal that the degradation of arabinoxylan, xyloglucan and cellulose are key metabolic processes in strain 2T2.1 growing on wheat straw. Different genes for key lignocellulolytic enzymes were identified, which can be starting points for production, characterization and/or supplementation of enzyme cocktails used in saccharification of agricultural residues. Our findings represent first steps that enable a better understanding of the reticulate evolution and "eco-enzymology" of lignocellulolytic Coniochaeta species

Clinical Practice Guidelines for the Management Candidiasis: 2009 Update by the Infectious Diseases Society of America

Guidelines for the management of patients with invasive candidiasis and mucosal candidiasis were prepared by an Expert Panel of the Infectious Diseases Society of America. These updated guidelines replace the previous guidelines published in the 15 January 2004 issue of Clinical Infectious Diseases and are intended for use by health care providers who care for patients who either have or are at risk of these infections. Since 2004, several new antifungal agents have become available, and several new studies have been published relating to the treatment of candidemia, other forms of invasive candidiasis, and mucosal disease, including oropharyngeal and esophageal candidiasis. There are also recent prospective data on the prevention of invasive candidiasis in high-risk neonates and adults and on the empiric treatment of suspected invasive candidiasis in adults. This new information is incorporated into this revised documen

Punk is just a state of mind: Exploring what punk means to older punk women

What does punk mean to older punk women? And how are such understandings interwoven with experiences of ageing and gender? The complexity in defining punk has been noted and it has been suggested that this complexity in part results from punk’s dislike of being labelled/categorised. Drawing upon interviews with 22 self-identifying older punk women, this article considers how they conceived punk as ‘a state of mind’, exploring the four shared punk values seen to comprise this: DIY, subversion, political consciousness and community. An unpacking of these values in terms of what they might ‘look like’ and how they are put into action by the women highlights the considerable roles ageing and gender play

Portrait of Candida albicans Adherence Regulators

Cell-substrate adherence is a fundamental property of microorganisms that enables them to exist in biofilms. Our study focuses on adherence of the fungal pathogen Candida albicans to one substrate, silicone, that is relevant to device-associated infection. We conducted a mutant screen with a quantitative flow-cell assay to identify thirty transcription factors that are required for adherence. We then combined nanoString gene expression profiling with functional analysis to elucidate relationships among these transcription factors, with two major goals: to extend our understanding of transcription factors previously known to govern adherence or biofilm formation, and to gain insight into the many transcription factors we identified that were relatively uncharacterized, particularly in the context of adherence or cell surface biogenesis. With regard to the first goal, we have discovered a role for biofilm regulator Bcr1 in adherence, and found that biofilm regulator Ace2 is a major functional target of chromatin remodeling factor Snf5. In addition, Bcr1 and Ace2 share several target genes, pointing to a new connection between them. With regard to the second goal, our findings reveal existence of a large regulatory network that connects eleven adherence regulators, the zinc-response regulator Zap1, and approximately one quarter of the predicted cell surface protein genes in this organism. This limited yet sensitive glimpse of mutant gene expression changes had thus defined one of the broadest cell surface regulatory networks in C. albicans

Antimicrobial therapy for acute cholangitis: Tokyo Guidelines

Antimicrobial agents should be administered to all patients with suspected acute cholangitis as a priority as soon as possible. Bile cultures should be performed at the earliest opportunity. The important factors which should be considered in selecting antimicrobial therapy include the agent’s activity against potentially infecting bacteria, the severity of the cholangitis, the presence or absence of renal and hepatic diseases, the patient’s recent history of antimicrobial therapy, and any recent culture results, if available. Biliary penetration of the microbial agents should also be considered in the selection of antimicrobials, but activity against the infecting isolates is of greatest importance. If the causative organisms are identified, empirically chosen antimicrobial drugs should be replaced by narrower-spectrum antimicrobial agents, the most appropriate for the species and the site of the infection