The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect

This is the final version. Available from American Society for Microbiology via the DOI in this record. Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis (IA), a life-threatening disease in immunocompromised humans. The echinocandin caspofungin, adopted as a second-line therapy in combating IA, is a -1,3-glucan synthase inhibitor, which, when used in high concentrations, reverts the anticipated A. fumigatus growth inhibition, a phenomenon called the “caspofungin paradoxical effect” (CPE). The CPE has been widely associated with increased chitin content in the cell wall due to a compensatory upregulation of chitin synthaseencoding genes. Here, we demonstrate that the CPE is dependent on the cell wall integrity (CWI) mitogen-activated protein kinase MpkAMPK1 and its associated transcription factor (TF) RlmARLM1, which regulate chitin synthase gene expression in response to different concentrations of caspofungin. Furthermore, the calcium- and calcineurin-dependent TF CrzA binds to and regulates the expression of specific chitin synthase genes during the CPE. These results suggest that the regulation of cell wall biosynthetic genes occurs by several cellular signaling pathways. In addition, CrzA is also involved in cell wall organization in the absence of caspofungin. Differences in the CPE were also observed between two A. fumigatus clinical isolates, which led to the identification of a novel basic leucine zipper TF, termed ZipD. This TF functions in the calcium-calcineurin pathway and is involved in the regulation of cell wall biosynthesis genes. This study therefore unraveled additional mechanisms and novel factors governing the CPE response, which ultimately could aid in developing more effective antifungal therapies.CNPqFAPES

The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence

This is the final version (corrected proof). The final published version is available from Public Library of Science via the DOI in this recordData Availability: Short reads were submitted to the NCBI’s Sequence Read Archive under accession number SRP154617 (https://www.ncbi.nlm.nih.gov/sra/?term=SRP154617). The ChIPseq data are available from NCBI SRA (sequence read archive) database under accession number PRJNA574873 (https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA574873&o=acc_s%3Aa).Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES)Wellcome TrustUniversity of MacauNational Science Foundation (NSF)Vanderbilt UniversityHoward Hughes Medical Institut

Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: Modulation of Innate Immune System and Variations in Carbohydrate Structure

The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2−/− and TLR4−/−) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-β, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages

Alloxan-Induced Diabetes Triggers the Development of Periodontal Disease in Rats

BACKGROUND: Periodontal disease in diabetic patients presents higher severity and prevalence; and increased severity of ligature-induced periodontal disease has been verified in diabetic rats. However, in absence of aggressive stimuli such as ligatures, the influence of diabetes on rat periodontal tissues is incompletely explored. The aim of this study was to evaluate the establishment and progression of periodontal diseases in rats only with diabetes induction. METHODOLOGY/PRINCIPAL FINDINGS: Diabetes was induced in Wistar rats (n = 25) by intravenous administration of alloxan (42 mg/kg) and were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The hemimandibles were removed and submitted to radiographical and histopathological procedures. A significant reduction was observed in height of bone crest in diabetic animals at 3, 6, 9 and 12 months, which was associated with increased numbers of osteoclasts and inflammatory cells. The histopathological analyses of diabetic rats also showed a reduction in density of collagen fibers, fibroblasts and blood vessels. Severe caries were also detected in the diabetic group. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that diabetes induction triggers, or even co-induces the onset of alterations which are typical of periodontal diseases even in the absence of aggressive factors such as ligatures. Therefore, diabetes induction renders a previously resistant host into a susceptible phenotype, and hence diabetes can be considered a very important risk factor to the development of periodontal disease