pH-dependent effect of Congo Red on the growth of Aspergillus nidulans and Aspergillus niger.

The azo dye Congo Red (CR) is frequently used as an agent to elicit cell wall integrity stress in fungi. This highly toxic aromatic, heterocyclic compound contains two azo bonds as chromophore, which are responsible for protonation under acidic conditions, leading to changes in the molecular structure of the dye and the color of the solution. The investigation of how CR affects the growth of Aspergillus nidulans and Aspergillus niger on surface cultures provided us with evidence about its pH-dependent toxicity. Reducing the starting pH of the media from 7 to 3 decreased both the toxicity of CR and the dose-dependence of its toxicity substantially. These changes can be explained by the pH-dependent structural changes of CR and its precipitation at low pH. The pH also depended on the fungi; they could induce a decrease or even an increase, which could be important in the loss of dose-dependence. Our experiments led to the conclusion that in studies to evaluate the antifungal effect of CR, properly buffered solutions with pH values adjusted to above 5 are highly recommended to achieve a well-detectable and dose-dependent antifungal effect. However, for decolorization of CR solutions, lower pH is suggested where the decreased toxicity and solubility of CR could help this process.

Origanum vulgare manganese ferrite nanocomposite: An advanced multifunctional hybrid material for dye remediation.

The purpose of the study was to fabricate sustainable and cost-effective material for the thorough cleansing of polluted water. In this context, an economical, phytogenic and multifunctional Origanum vulgare plant-based nanocomposite material, MnFe2O4/OV, was prepared via one-pot synthetic technique. The synthesized nanocomposite with a band gap of 2.02 eV behaved as an efficient nano-photocatalyst for the degradation of both cationic (crystal violet) and anionic (congo red) dyes under direct sunlight irradiation. The material also inhibited the growth of E. coli and S. aureus bacteria and simultaneously adsorbed both cationic and anionic dyes from water through adsorption. A variety of techniques have been used to characterize the nanocomposite, including X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and transmission electron microscopy (TEM). Additionally, the kinetics of photodegradation of the aforementioned organic dyes has also been investigated. The MnFe2O4/OV exhibited excellent photocatalytic performance, leading to 43% and 72% degradation within 3 h at rate constants of 2.0 × 10-3 min-1 and 6.0 × 10-3 min-1 for crystal violet and congo red, respectively. The crystal violet and congo red were used to testify to the composite's potential for adsorption under the influence of several process variables, including initial solution pH, contact time, temperature, initial dye concentration, and amount of MnFe2O4/OV. The Langmuir maximum adsorption capacity Qmax as in the range 14.06-14.59 mgg-1 for crystal violet and 34.45-23.93 mgg-1 for congo red at pH 7 within 90 min contact time in the temperature range of 30-50 °C. The phenomenon of adsorption was found feasible and endothermic at all the investigated temperatures. Also, E. coli and S. Aureus bacteria have shown growth suppression activity when exposed to MnFe2O4/OV.As a result, the synthesized nanocomposite, MnFe2O4/OV, proved to be an antimicrobial, multifunctional novel nanocomposite, which is in high demand, and could serve as an affordable, and sustainable material for comprehensive water filtration.

The effect of minocycline on beta-amyloid-induced memory and learning deficit in male rats: A behavioral, biochemical, and histological study.

OBJECTIVES: Minocycline hydrochloride is a semi-synthetic, second-generation tetracycline with neuroprotective, neurorestorative, anti-amyloidogenic, anti-inflammatory, antioxidant, and anti-apoptotic properties. The present study was designed to investigate the potential protective effects of minocycline against beta-amyloid (Aß)-induced Alzheimer's disease (AD), recognition memory decline, and the possible involved anti-apoptotic mechanisms. METHODS: The rats were treated with minocycline (50 and 100 mg/kg/day; P.O.) after AD induction for 30 days. Behavioral functions were assessed by employing standard behavioral tests, including novel object recognition (NOR) and passive avoidance learning (PAL) tasks. Then, total antioxidant capacity (TAC) and total oxidant status (TOS) were measured in blood serum using ELISA kits. Apoptosis and the number of Aß plaques were examined by the TUNEL and Congo red staining, respectively. RESULTS: Treatment of Aß rats with minocycline improved memory deficit in the PAL task and a decline in recognition memory in the NOR test. Minocycline at 50 and 100 mg/kg significantly reduced the TOS levels and increased the TAC levels (P < 0.0001). Also, minocycline at 50 and 100 mg/kg reduced the apoptotic index in the hippocampus of Aß rats. After Congo red staining, the minocycline group showed improved cell morphology and markedly fewer Aß plaques. CONCLUSIONS: Minocycline reduced memory and learning deficit in behavioral experiments after Aß injection, which may be due to its anti-inflammatory and anti-apoptotic effects.

Synthesis of HAp/CS-SA composite for effective removal of highly toxic dyes in aqueous solution.

Textile industry is the major backbone of economy of the developing countries. The major problems associated in the textile factories are release of undesired dye effluents, which is a potential pollution risk for human health as well as the environmental aquatic system. The objective of this study was fabrication of a novel composite to treat textile industry effluents in an ecofriendly manner. In this context, hydroxyapatite (HAp) was derived from the mussel shell biowaste and fabricated with chitosan-sodium alginate through the in-situ method. The prepared HAp/CS-SA composite was physicochemically characterized by using Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (FESEM-EDX) and High Resolution-Transmission Electron Microscope (HR-TEM). The photocatalytic activity of the HAp/CS-SA composite was evaluated by using Congo red (CR), Malachite green (MG) and Methylene blue (MB) as model dyes for degradation in aqueous solutions under solar irradiation. The degradation rate was recorded as CR (88%), MG (81%) and MB (93%) respectively within 75 min of irradiation. The degraded end products were subjected to toxicity assessment by evaluating the phytotoxicity on seed germination of Vigna radiata plant in pot study. The degraded end products were also tested for toxic inhibition of E. coli and P. aeruginosa by agar well diffusion method. The prepared HAp/CS-SA composite had an enhanced photocatalytic activity due to the presence of natural biopolymers and their biological properties. HAp/CS-SA composite showed potential dye degradation properties and it could be effective for dye removal from industrial wastewaters.

Congo Red as a Supramolecular Carrier System for Doxorubicin: An Approach to Understanding the Mechanism of Action.

The uptake and distribution of doxorubicin in the MCF7 line of breast-cancer cells were monitored by Raman measurements. It was demonstrated that bioavailability of doxorubicin can be significantly enhanced by applying Congo red. To understand the mechanism of doxorubicin delivery by Congo red supramolecular carriers, additional monolayer measurements and molecular dynamics simulations on model membranes were undertaken. Acting as molecular scissors, Congo red particles cut doxorubicin aggregates and incorporated them into small-sized Congo red clusters. The mixed doxorubicin/Congo red clusters were adsorbed to the hydrophilic part of the model membrane. Such behavior promoted transfer through the membrane.

Islet amyloid polypeptide aggregation exerts cytotoxic and proinflammatory effects on the islet vasculature in mice.

AIMS/HYPOTHESIS: The islet vasculature, including its constituent islet endothelial cells, is a key contributor to the microenvironment necessary for normal beta cell health and function. In type 2 diabetes, islet amyloid polypeptide (IAPP) aggregates, forming amyloid deposits that accumulate between beta cells and islet capillaries. This process is known to be toxic to beta cells but its impact on the islet vasculature has not previously been studied. Here, we report the first characterisation of the effects of IAPP aggregation on islet endothelial cells/capillaries using cell-based and animal models. METHODS: Primary and immortalised islet endothelial cells were treated with amyloidogenic human IAPP (hIAPP) alone or in the presence of the amyloid blocker Congo Red or the Toll-like receptor (TLR) 2/4 antagonist OxPAPc. Cell viability was determined0 along with mRNA and protein levels of inflammatory markers. Islet capillary abundance, morphology and pericyte coverage were determined in pancreases from transgenic mice with beta cell expression of hIAPP using conventional and confocal microscopy. RESULTS: Aggregated hIAPP decreased endothelial cell viability in immortalised and primary islet endothelial cells (by 78% and 60%, respectively) and significantly increased expression of inflammatory markers Il6, Vcam1 and Edn1 mRNA relative to vehicle treatment in both cell types (p<0.05; n=4). Both cytotoxicity and the proinflammatory response were ameliorated by Congo Red (p<0.05; n=4); whereas TLR2/4-inhibition blocked inflammatory gene expression (p<0.05; n=6) without improving viability. Islets from high-fat-diet-fed amyloid-laden hIAPP transgenic mice also exhibited significantly increased expression of most markers of endothelial inflammation (p<0.05; n=5) along with decreased capillary density compared with non-transgenic littermates fed the same diet (p<0.01). Moreover, a 16% increase in capillary diameter was observed in amyloid-adjacent capillaries (p<0.01), accompanied by a doubling in pericyte structures positive for neuron-glial antigen 2 (p<0.001). CONCLUSIONS/INTERPRETATION: Islet endothelial cells are susceptible to hIAPP-induced cytotoxicity and exhibit a TLR2/4-dependent proinflammatory response to aggregated hIAPP. Additionally, we observed amyloid-selective effects that decreased islet capillary density, accompanied by increased capillary diameter and increased pericyte number. Together, these data demonstrate that the islet vasculature is a target of the cytotoxic and proinflammatory effects of aggregated hIAPP that likely contribute to the detrimental effects of hIAPP aggregation on beta cell function and survival in type 2 diabetes.

Aspergillus nidulans Septa Are Indispensable for Surviving Cell Wall Stress.

Septation in filamentous fungi is a normal part of development, which involves the formation of cross-hyphal bulkheads, typically containing pores, allowing cytoplasmic streaming between compartments. Based on previous findings regarding septa and cell wall stress, we hypothesized that septa are critical for survival during cell wall stress. To test this hypothesis, we used known Aspergillus nidulans septation-deficient mutants (ΔsepH, Δbud3, Δbud4, and Δrho4) and six antifungal compounds. Three of these compounds (micafungin, Congo red, and calcofluor white) are known cell wall stressors which activate the cell wall integrity signaling pathway (CWIS), while the three others (cycloheximide, miconazole, and 2,3-butanedione monoxime) perturb specific cellular processes not explicitly related to the cell wall. Our results show that deficiencies in septation lead to fungi which are more susceptible to cell wall-perturbing compounds but are no more susceptible to other antifungal compounds than a control. This implies that septa play a critical role in surviving cell wall stress. IMPORTANCE The ability to compartmentalize potentially lethal damage via septation appears to provide filamentous fungi with a facile means to tolerate diverse forms of stress. However, it remains unknown whether this mechanism is deployed in response to all forms of stress or is limited to specific perturbations. Our results support the latter possibility by showing that presence of septa promotes survival in response to cell wall damage but plays no apparent role in coping with other unrelated forms of stress. Given that cell wall damage is a primary effect caused by exposure to the echinocandin class of antifungal agents, our results emphasize the important role that septa might play in enabling resistance to these drugs. Accordingly, the inhibition of septum formation could conceivably represent an attractive approach to potentiating the effects of echinocandins and mitigating resistance in human fungal pathogens.

Fungal tolerance to Congo red, a cell wall integrity stress, as a promising indicator of ecological niche.

Differential sensitivities to the cell wall stress caused by Congo red (CR) have been observed in many fungal species. In this study, the tolerances and sensitivities to CR was studied with an assorted collection of fungal species from three phylogenetic classes: Sordariomycetes, Dothideomycetes, and Eurotiomycetes, three orders, and eight families. These grouped into different ecological niches, such as insect pathogens, plant pathogens, saprotrophs, and mycoparasitics. The saprotroph Aspergillus niger and the mycoparasite Trichoderma atroviride stood out as the most resistant species to cell wall stress caused by CR, followed by the plant pathogenic fungi, a mycoparasite, and other saprotrophs. The insect pathogens had low tolerance to CR. The insect pathogens Metarhizium acridum and Cordyceps fumosorosea were the most sensitive to CR. In conclusion, Congo red tolerance may reflect ecological niche, accordingly, the tolerances of the fungal species to Congo red were closely aligned with their ecology.

Functional Genomic and Biochemical Analysis Reveals Pleiotropic Effect of Congo Red on Aspergillus fumigatus.

Inhibition of fungal growth by Congo red (CR) has been putatively associated with specific binding to ß-1,3-glucans, which blocks cell wall polysaccharide synthesis. In this study, we searched for transcription factors (TFs) that regulate the response to CR and interrogated their regulon. During the investigation of the susceptibility to CR of the TF mutant library, several CR-resistant and -hypersensitive mutants were discovered and further studied. Abnormal distorted swollen conidia called Quasimodo cells were seen in the presence of CR. Quasimodo cells in the resistant mutants were larger than the ones in the sensitive and parental strains; consequently, the conidia of the resistant mutants absorbed more CR than the germinating conidia of the sensitive or parental strains. Accordingly, this higher absorption rate by Quasimodo cells resulted in the removal of CR from the culture medium, allowing a subset of conidia to germinate and grow. In contrast, all resting conidia of the sensitive mutants and the parental strain were killed. This result indicated that the heterogeneity of the conidial population is essential to promote the survival of Aspergillus fumigatus in the presence of CR. Moreover, amorphous surface cell wall polysaccharides such as galactosaminogalactan control the influx of CR inside the cells and, accordingly, resistance to the drug. Finally, long-term incubation with CR led to the discovery of a new CR-induced growth effect, called drug-induced growth stimulation (DIGS), since the growth of one of them could be stimulated after recovery from CR stress.IMPORTANCE The compound Congo red (CR) has been historically used for coloring treatment and histological examination as well to inhibit the growth of yeast and filamentous fungi. It has been thought that CR binds to ß-1,3-glucans in the fungal cell wall, disrupting the organization of the cell wall structure. However, other processes have been implicated in affecting CR sensitivity. Here, we explore CR susceptibility through screening a library of genetic null mutants. We find several previously uncharacterized genetic regulators important for CR susceptibility. Through biochemical and molecular characterization, we find cell membrane permeability to be important. Additionally, we characterize a novel cell type, Quasimodo cells, that occurs upon CR exposure. These cells take up CR, allowing the growth of the remaining fungi. Finally, we find that priming with CR can enhance long-term growth in one mutant.

Inhibition of the Vesicular Glutamate Transporter (VGLUT) with Congo Red Analogs: New Binding Insights.

The vesicular glutamate transporter (VGLUT) facilitates the uptake of glutamate (Glu) into neuronal vesicles. VGLUT has not yet been fully characterized pharmacologically but a body of work established that certain azo-dyes bearing two Glu isosteres via a linker were potent inhibitors. However, the distance between the isostere groups that convey potent inhibition has not been delineated. This report describes the synthesis and pharmacologic assessment of Congo Red analogs that contain one or two glutamate isostere or mimic groups; the latter varied in the interatomic distance and spacer properties to probe strategic binding interactions within VGLUT. The more potent inhibitors had two glutamate isosteres symmetrically linked to a central aromatic group and showed IC50 values ~ 0.3-2.0 µM at VGLUT. These compounds contained phenyl, diphenyl ether (PhOPh) or 1,2-diphenylethane as the linker connecting 4-aminonaphthalene sulfonic acid groups. A homology model for VGLUT2 using D-galactonate transporter (DgoT) to dock and identify R88, H199 and F219 as key protein interactions with Trypan Blue, Congo Red and selected potent analogs prepared and tested in this report.

Antibacterial Therapy by Ag+ Ions Complexed with Titan Yellow/Congo Red and Albumin during Anticancer Therapy of Urinary Bladder Cancer.

According to the World Health Organization report, the increasing antibiotic resistance of microorganisms is one of the biggest global health problems. The percentage of bacterial strains showing multidrug resistance (MDR) to commonly used antibiotics is growing rapidly. Therefore, the search for alternative solutions to antibiotic therapy has become critical to combat this phenomenon. It is especially important as frequent and recurring infections can cause cancer. One example of this phenomenon is urinary tract infections that can contribute to the development of human urinary bladder carcinoma. This tumor is one of the most common malignant neoplasms in humans. It occurs almost three times more often in men than in women, and in terms of the number of cases, it is the fifth malignant neoplasm after prostate, lung, colon, and stomach cancer. The risk of developing the disease increases with age. Despite the improvement of its treatment methods, the current outcome in the advanced stages of this tumor is not satisfactory. Hence, there is an urgent need to introduce innovative solutions that will prove effective even in the advanced stage of the disease. In our study, a nanosystem based on ionic silver (Ag+) bound to a carrier-Titan yellow (TY) was analyzed. The possibility of binding the thus formed TY-Ag system to Congo red (CR) and albumin (BSA) was determined. TY-Ag binding to CR provides for better nanosystem solubility and enables its targeted intracellular transport and binding to immune complexes. The binding of TY-Ag or CR-TY-Ag to albumin also protects the system against the uncontrolled release of silver ions. It will also allow the delivery of silver in a targeted manner directly to the desired site in the case of intravenous administration of such a system. In this study, the MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values of the TY-Ag or BSA-TY-Ag systems were determined in two reference strains (Escherichia coli and Staphylococcus aureus). The paper presents nanosystems with a size of about 40-50 nm, with an intense antibacterial effect obtained at concentrations of 0.019 mM. We have also discovered that TY-Ag free or complexed with BSA (with a minimal Ag+ dose of 15-20 µM) inhibited cancer cells proliferation. TY-Ag complex diminished migration and effectively inhibited the T24 cell viability and induced apoptosis. On the basis of the obtained results, it has been shown that the presented systems may have anti-inflammatory and antitumor properties at the same time. TY-Ag or BSA-TY-Ag are new potential drugs and may become in future important therapeutic compounds in human urinary bladder carcinoma treatment and/or potent antimicrobial factors as an alternative to antibiotics.

Compromised chitin synthesis in lager yeast affects its Congo red resistance and release of mannoproteins from the cells.

A mutant lager strain resistant to the cell wall-perturbing agent Congo red (CR) was isolated and the genetic alterations underlying CR resistance were investigated by whole genome sequencing. The parental lager strain was found to contain three distinct Saccharomyces cerevisiae (Sc)-type CHS6 (CHitin Synthase-related 6) alleles, two of which have one or two nonsense mutations in the open reading frame, leaving only one functional allele, whereas the functional allele was missing in the isolated CR-resistant strain. On the other hand, the Saccharomyces eubayanus-type CHS6 alleles shared by both the parental and mutant strains appeared to contribute poorly to chitin synthase-activating function. Therefore, the CR resistance of the mutant strain was attributable to the overall compromised activity of CHS6 gene products. The CR-resistant mutant cells exhibited less chitin production on the cell surface and smaller amounts of mannoprotein release into the medium. All these traits, in addition to the CR resistance, were complemented by the functional ScCHS6 gene. It is of great interest whether the frequent nonsense mutations found in ScCHS6 open reading frame in lager yeast strains are a consequence of the domestication process of lager yeast.

Characterization of gfdB, putatively encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans.

The genome of Aspergillus nidulans accommodates two glycerol 3-phosphate dehydrogenase genes, gfdA and gfdB. Previous studies confirmed that GfdA is involved in the osmotic stress defence of the fungus. In this work, the physiological role of GfdB was characterized via the construction and functional characterization of the gene deletion mutant ΔgfdB. Unexpectedly, ΔgfdB strains showed oxidative stress sensitivity in the presence of a series of well-known oxidants including tert-butyl-hydroperoxide (tBOOH), diamide as well as hydrogen peroxide. Moderate sensitivity of the mutant towards the cell wall stress inducing agent CongoRed was also observed. Hence, both Gfd isoenzymes contributed to the environmental stress defence of the fungus but their functions were stress-type-specific. Furthermore, the specific activities of certain antioxidant enzymes, like catalase and glutathione peroxidase, were lower in ΔgfdB hyphae than those recorded in the control strain. As a consequence, mycelia from ΔgfdB cultures accumulated reactive species at higher levels than the control. On the other hand, the specific glutathione reductase activity was higher in the mutant, most likely to compensate for the elevated intracellular oxidative species concentrations. Nevertheless, the efficient control of reactive species failed in ΔgfdB cultures, which resulted in reduced viability and, concomitantly, early onset of programmed cell death in mutant hyphae. Inactivation of gfdB brought about higher mannitol accumulation in mycelia meanwhile the erythritol production was not disturbed in unstressed cultures. After oxidative stress treatment with tBOOH, only mannitol was detected in both mutant and control mycelia and the accumulation of mannitol even intensified in the ΔgfdB strain.

Coding the α-subunit of SNF1 kinase, Snf1 is required for the conidiogenesis and pathogenicity of the Alternaria alternata tangerine pathotype.

To well cope with various external carbon sources, fungi have evolved an adaptive mechanism to overcome the adversity of carbon source deficiency. The sucrose non-fermenting (SNF1) protein kinase mainly mediates the utilization of non-fermentable carbon sources. In this study, we determined the function of Snf1, coding the α-subunit of SNF1 kinase, in the phytopathogenic fungus Alternaria alternata via analyzing the Snf1 deletion mutants (ΔAasnf1). Aasnf1 is required for growth, development of aerial mycelium, and conidiation. Results of pathogenicity test showed that ΔAasnf1 induced smaller lesions on detached citrus leaves. Moreover, in the carbon utilization assay, ΔAasnf1 showed growth inhibition on the minimal medium supplemented with polygalacturonic acid, sucrose or alcohol as the only carbon source. Compared to the wild type, ΔAasnf1 also exhibited stronger resistance to cell wall stressors of sodium dodecyl sulfate and congo red. In conclusion, Aasnf1 played important roles in the carbon utilization, vegetative growth, conidiation, cell wall functions and pathogenicity of A. alternata. This study is the first report on the functions of Aasnf1 and our results suggest that Snf1 is critical for the conidiogenesis and pathogenesis of the A. alternata tangerine pathotype.

Functional comparison of the group III hybrid histidine kinases TcsC of Aspergillus fumigatus and NikA of Aspergillus nidulans.

In filamentous fungi, group III hybrid histidine kinases (HHKs) are major and nonredundant sensing proteins of the high osmolarity glycerol pathway. In this study, we have compared the biological functions of the two homologous group III HHKs TcsC of Aspergillus fumigatus and NikA of A. nidulans. As expected from previous studies, the corresponding mutants are severely impaired in their ability to adapt to hyperosmotic stress and are both resistant to the antifungal agent fludioxonil. However, our data also reveal novel phenotypes and differences between these mutants. Both TcsC and NikA are required for wild-type-like growth on Czapek-Dox medium and a normal resistance to certain oxidative stressors, whereas an increased resistance to the cell wall disturbing agents Congo red and Calcofluor white was found for the ΔtcsC but not for the ΔnikA mutant. With respect to the cell wall reorganizations that are triggered by fludioxonil in a TcsC/NikA-dependent manner, we observed similarities but also striking differences. Strains from seven Aspergillus species, including A. fumigatus and A. nidulans incorporated more chitin into their cell walls in response to fludioxonil. In contrast, fludioxonil treatment resulted in a shedding of surface accessible galactomannan and ß-1,3-glucan in all Aspergillus strains tested except A. nidulans. Hence, the fludioxonil-induced activation of NikA results in a distinct and apparently A. nidulans-specific pattern of cell wall reorganizations that is not due to NikA itself, but its integration into the A. nidulans signaling network.

Tet38 of Staphylococcus aureus Binds to Host Cell Receptor Complex CD36-Toll-Like Receptor 2 and Protects from Teichoic Acid Synthesis Inhibitors Tunicamycin and Congo Red.

Using an affinity column retention assay, we showed that the purified Tet38 membrane transporter of Staphylococcus aureus bound specifically to host cell CD36 and to the complex CD36-Toll-like receptor 2 (TLR-2), but not to TLR-2 alone or TLR-2 and S. aureus lipoteichoic acid (LTA). We tested the effect of LTA on the internalization of S. aureustet38 mutant QT7 versus RN6390 by A549 epithelial cells. Addition of anti-LTA antibody to the bacteria prior to adding to A549 cells reduced internalization of QT7 2-fold compared to that with nonspecific antibody treatment. QT7 internalized 4- to 6-fold less than RN6390 with or without anti-LTA antibody. These data suggested that Tet38 and LTA were independently involved in the invasion process. The wall teichoic acid (WTA) inhibitor tunicamycin had an 8-fold decrease in activity with overexpression of tet38 and a 2-fold increase in activity in QT7 (tet38). Reserpine (an inhibitor of efflux pumps) reduced the effect of tet38 overexpression on tunicamycin resistance 4-fold. In addition, tet38 affected growth in the presence of LTA inhibitor Congo red, with overexpression increasing growth and deletion of tet38 reducing growth. In conclusion, Tet38 contributes to S. aureus invasion of A549 via direct binding to CD36 of the complex CD36-TLR-2, and LTA independently bound to TLR-2. The reduction of tunicamycin resistance in the presence of reserpine and the survival ability of the tet38 overexpressor in the presence of Congo red suggest that Tet38 can also protect the synthesis of LTA and WTA in S. aureus against their inhibitors, possibly functioning as an efflux pump.

Genotypic and phenotypic characterization of Candida albicans Lebanese hospital isolates resistant and sensitive to caspofungin.

The fungus Candida albicans is both a commensal and an opportunistic human pathogen, present as part of the normal human microflora causing serious mucosal, and systemic life threatening infections. The antifungal drug caspofungin of the echinocandin family is the latest generation of antifungal drugs to be developed. It functions by inhibiting glucan synthase thus weakening the fungal cell wall leading to death. Recently reports of resistance to caspofungin have been reported mainly through mutations in the FKS encoded subunits of glucan synthase at hot spot 1 (amino acids 641 to 649, FSTLSLRDP) and hot spot 2 (amino acids 1357 to 1364, DWIRRYTL). Our study aimed at sequencing both hot spots from 16 C. albicans Lebanese hospital isolates resistant and sensitive to caspofungin to determine whether mutations in these hot spots are present, and whether such mutations also impart resistance to our isolates. In addition, we wanted to determine any relationship between resistance and pathogenicity related attributes such as virulence, resistance to cell wall disrupting agents, biofilm formation, and cell wall chitin deposition. Five isolates were found to contain mutations with the mutations restricted to resistant strains. Within hot spot 1 substitution at positions S642, T643, L644, R647, and D648 were found, while within hot spot 2 substitutions at positions L1364, T1363, and R1360, W1358 and R1361 were identified with some of the mutations not previously documented. Strains that were resistant to caspofungin also showed increased resistance to Congo red but decreased biofilm formation and attenuated virulence in a mouse model of infection. Caspofungin sensitive strains showed decreased resistance to Congo red yet increased virulence and biofilm formation. Chitin content analysis showed that caspofungin resistant strains had elevated levels of chitin resulting in cell wall thickening that counters the effect of caspofungin, while sensitive strains showed decreased chitin content. Our results demonstrate an inverse correlation between resistance and virulence whereby resistance is due to thickening of the cell wall preventing the cell from gaining virulence attributes, while a more susceptible cell wall increases susceptibility to drugs but allows increased virulence.

The puzzling construction of the conidial outer layer of Aspergillus fumigatus.

If the mycelium of Aspergillus fumigatus is very short-lived in the laboratory, conidia can survive for years. This survival capacity and extreme resistance to environmental insults is a major biological characteristic of this fungal species. Moreover, conidia, which easily reach the host alveola, are the infective propagules. Earlier studies have shown the role of some molecules of the outer conidial layer in protecting the fungus against the host defense. The outer layer of the conidial cell wall, directly in contact with the host cells, consists of α-(1,3)-glucan, melanin, and proteinaceous rodlets. This study is focused on the global importance of this outer layer. Single and multiple mutants without one to three major components of the outer layer were constructed and studied. The results showed that the absence of the target molecules resulting from multiple gene deletions led to unexpected phenotypes without any logical additivity. Unexpected compensatory cell wall surface modifications were indeed observed, such as the synthesis of the mycelial virulence factor galactosaminogalactan, the increase in chitin and glycoprotein concentration or particular changes in permeability. However, sensitivity of the multiple mutants to killing by phagocytic host cells confirmed the major importance of melanin in protecting conidia.

Congo Red affects the growth, morphology and activity of glucosamine-6-phosphate synthase in the human pathogenic fungus Sporothrix schenckii.

Sporothrix schenckii is the etiological agent of sporotrichosis, a mycosis of humans and other mammals. Little is known about the responses of this thermodimorphic pathogen to perturbations in the cell wall (CW) by different stress conditions. Here we describe the effect of Congo Red (CR) on the fungal growth, morphogenesis and activity of glucosamine-6-phosphate (GlcN-6-P) synthase. Under conditions of yeast development, 15 µM CR abolished conidia (CN) germination, but when yeast cells were first obtained in the absence of the dye and then post-incubated in its presence, yeasts rapidly differentiated into mycelial cells. On the other hand, under conditions of mycelium development, 150 µM CR did not affect CN germination, but filamentous cells underwent structural changes characterized by a distorted CW contour, the loss of polarity and the formation of red-pigmented, hyphal globose structures. Under these conditions, CR also induced a significant and transient increase in the activity of GlcN-6-P synthase, an essential enzyme in CW biogenesis.

Evaluation of the chitin-binding dye Congo red as a selection agent for the isolation, classification, and enumeration of ascomycete yeasts.

Thirty-nine strains of ascomycete yeasts representing 35 species and 33 genera were tested for their ability to grow on solid agar medium containing increasing concentrations of the chitin-binding dye Congo red. Six strains were classified as hypersensitive (weak or no growth at 10 mg/l Congo red), five were moderately sensitive (weak or no growth at 50 mg/l), three were moderately tolerant (weak or no growth at 100 mg/l), while the remaining 25 strains were classified as resistant (robust growth at ≥ 100 mg/l) with 20 of these strains classified as hyper-resistant (robust growth at 200 mg/l). Congo red growth phenotypes were consistent within some families but not others. The frequency of Congo red resistance among ascomycete yeasts was deemed too high for the practical use of Congo red as a selection agent for targeted isolation, but can be useful for identification and enumeration of yeasts.