Resistance in Candida albicans: exploring the cell wall barrier by proteomics

The incidence of candidiasis has dramatically increased and bloodstream infections due to different species of Candida are becoming a prime cause of morbidity and mortality in different types of immunocompromised patients. Azole and echinocandin drug resistance accounts for the dramatic increase in incidence of nosocomial bloodstream candidiasis found in recent years. Cell wall constitutes the barrier between the yeast and the host and resistant strains change the proteome of this compartment. In the last decade different proteomic platforms have been applied to study cell wall and markers of resistance to drugs have been pointed out. Modulation of these proteins seem to suggest that although resistance is based on a specific mutation able to counteract the toxicity of the antifungal drug, a set of other molecular modifications takes place contemporary or subsequently the establishment of the resistance and seems to support the viability of the resistant yeast. Profiled proteins by proteomics may be valuable in design therapy using classical antifungal along with complementary drugs able to abolish pathways that strengthen the resistance and attenuate virulence of the mutated cell

Hyperexpression of CDRs and HWP1 genes negatively impacts on Candida albicans virulence

C. albicans is a commensal organism present in the human microbiome of more than 60% of the healthy population. Transition from commensalism to invasive candidiasis may occur after a local or a general failure of host's immune system. This transition to a more virulent phenotype may reside either on the capacity to form hyphae or on an acquired resistance to antifungal drugs. Indeed, overexpression of genes coding drug efflux pumps or adhesins, cell wall proteins facilitating the contact between the fungus and the host, usually marks the virulence profile of invasive Candida spp. In this paper, we compare virulence of two clinical isolates of C. albicans with that of laboratory-induced resistant strains by challenging G. mellonella larvae with these pathogens along with monitoring transcriptional profiles of drug efflux pumps genes CDR1, CDR2, MDR1 and the adhesin genes ALS1 and HWP1. Although both clinical isolates were found resistant to both fluconazole and micafungin they were found less virulent than laboratory-induced resistant strains. An unexpected behavior emerged for the former clinical isolate in which three genes, CDR1, CDR2 and HWP1, usually correlated with virulence, although hyperexpressed, conferred a less aggressive phenotype. On the contrary, in the other isolate, we observed a decreased expression of CDR1, CDR2 and HWP1as well as of MDR1 and ALS1 that may be consistent with the less aggressive performance observed in this strain. These altered gene expressions might directly influence Candida virulence or they might be an epiphenomenon of a vaster rearrangement occurred in these strains during the challenge with the host's environment. An in-deepth comprehension of this scenario could be crucial for developing interventions able to counteract C. albicans invasiveness and lethality.C. albicans is a commensal organism present in the human microbiome of more than 60% of the healthy population. Transition from commensalism to invasive candidiasis may occur after a local or a general failure of host's immune system. This transition to a more virulent phenotype may reside either on the capacity to form hyphae or on an acquired resistance to antifungal drugs. Indeed, overexpression of genes coding drug efflux pumps or adhesins, cell wall proteins facilitating the contact between the fungus and the host, usually marks the virulence profile of invasive Candida spp. In this paper, we compare virulence of two clinical isolates of C. albicans with that of laboratory-induced resistant strains by challenging G. mellonella larvae with these pathogens along with monitoring transcriptional profiles of drug efflux pumps genes CDR1, CDR2, MDR1 and the adhesin genes ALS1 and HWP1. Although both clinical isolates were found resistant to both fluconazole and micafungin they were found less virulent than laboratory-induced resistant strains. An unexpected behavior emerged for the former clinical isolate in which three genes, CDR1, CDR2 and HWP1, usually correlated with virulence, although hyperexpressed, conferred a less aggressive phenotype. On the contrary, in the other isolate, we observed a decreased expression of CDR1, CDR2 and HWP1as well as of MDR1 and ALS1 that may be consistent with the less aggressive performance observed in this strain. These altered gene expressions might directly influence Candida virulence or they might be an epiphenomenon of a vaster rearrangement occurred in these strains during the challenge with the host's environment. An in-deepth comprehension of this scenario could be crucial for developing interventions able to counteract C. albicans invasiveness and lethality

Membrane protein remodeling in microglia exposed to amyloid peptides

Infection, neurodegeneration, and other conditions associated with loss of brain homeostasis, induce changes in microglial morphology, gene expression and function, generally referred to as “activation”. Alzheimer’s disease (AD) is the most common dementia and is characterized by neuroinfammatory changes, including alterations in the morphology and distribution of microglia and astrocytes, and deposition of complement and other infammatory mediators. Our previous observations show that microglial cells challenged in vitro with amyloid peptides clustered and rounded up, dramatically changing their morphology. Besides, in these cells we observed the early acetylation and then the phosphorylation of STAT3 which is required for the expression of the epsilon isoform of 14-3-3, a marker of Abeta-activated microglia (1, 2). We applied afnity partitioning approach combined with high throughput mass spectrometric analysis in order to identify variation of proteins on plasma membrane of BV2 immortalized microglia upon treatment with amyloid peptides. By this method several proteins up- or down-regulated by amyloid treatment were identifed in microglial plasma membrane. Among them annexins (5 and 7), IFITM3 and MARK3. These data have been confrmed in primary microglial cultures. In microglia, plasma membrane plays a relevant role in the cross-talking with the external neuronal environment and in the resulting trophic or infammatory response of these sentinel cells. As such, knowledge of the microglia responsiveness to beta amyloids in term of changes in its plasma membrane proteome is imperative for unveiling the molecular landscape in which AD occurs

Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed

<p>Abstract</p> <p>Background</p> <p>Several mutations have been described as responsible for rifampicin resistance in <it>Neisseria meningitidis</it>. However, the intriguing question on why these strains are so rare remains open. The aim of this study was to investigate the protein content and to identify differential expression in specific proteins in two rifampicin resistant and one susceptible meningococci using two-dimensional electrophoresis (2-DE) combined with mass spectrometry.</p> <p>Results</p> <p>In our experimental conditions, able to resolve soluble proteins with an isoelectric point between 4 and 7, twenty-three proteins have been found differentially expressed in the two resistant strains compared to the susceptible. Some of them, involved in the main metabolic pathways, showed an increased expression, mainly in the catabolism of pyruvate and in the tricarboxylic acid cycle. A decreased expression of proteins belonging to gene regulation and to those involved in the folding of polypeptides has also been observed. 2-DE analysis showed the presence of four proteins displaying a shift in their isoelectric point in both resistant strains, confirmed by the presence of amino acid changes in the sequence analysis, absent in the susceptible.</p> <p>Conclusions</p> <p>The analysis of differentially expressed proteins suggests that an intricate series of events occurs in <it>N. meningitidis </it>rifampicin resistant strains and the results here reported may be considered a starting point in understanding their decreased invasion capacity. In fact, they support the hypothesis that the presence of more than one protein differentially expressed, having a role in the metabolism of the meningococcus, influences its ability to infect and to spread in the population. Different reports have described and discussed how a drug resistant pathogen shows a high biological cost for survival and that may also explain why, for some pathogens, the rate of resistant organisms is relatively low considering the widespread use of a particular drug. This seems the case of rifampicin resistant meningococci.</p

Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting

In this study we report the effect of AZT on the whole protein expression profile both in the control and the AZT-treated K562 cells, evidenced by two-dimensional gel electrophoresis and peptide mass fingerprinting analysis. Two-dimensional gels computer digital image analysis showed two spots that appeared up-regulated in AZT-treated cells and one spot present only in the drug exposed samples. Upon extraction and analysis by peptide mass fingerprinting, the first two spots were identified as PDI-A3 and stathmin, while the third one was proved to be NDPK-A. Conversely, two protein spots were present only in the untreated K562 cells, and were identified as SOD1 and HSP-60, respectively

Effects of Mentha suaveolens

Candidosis is the most important cause of fungal infections in humans. The yeast Candida albicans can form biofilms, and it is known that microbial biofilms play an important role in human diseases and are very difficult to treat. The prolonged treatment with drugs has often resulted in failure and resistance. Due to the emergence of multidrug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to analyse the effects induced by essential oil of Mentha suaveolens Ehrh (EOMS) on Candida albicans and its potential synergism when used in combination with conventional drugs. Morphological differences between control and EOMS treated yeast cells or biofilms were observed by scanning electron microscopy and transmission electron microscopy (SEM and TEM resp.,). In order to reveal the presence of cell cycle alterations, flow cytometry analysis was carried out as well. The synergic action of EOMS was studied with the checkerboard method, and the cellular damage induced by different treatments was analysed by TEM. The results obtained have demonstrated both the effects of EOMS on C. albicans yeast cells and biofilms and the synergism of EOMS when used in combination with conventional antifungal drugs as fluconazole (FLC) and micafungin (MCFG), and therefore we can hypothesize on its potential use in therapy. Further studies are necessary to know its mechanism of action

Clinical validation of 13-Gene DNA methylation analysis from oral brushing: a non invasive sampling procedure for early detection of oral squamous cell carcinoma. A multicentric study

1. Introduction In a recent study our research group described a non-invasive sampling procedure based on DNA methylation analysis of a set of 13 genes with a high level of accuracy (sensitivity 96.6%, specificity 100%) in the detection of squamous cell carcinoma of the oral cavity (OSCC) [1]. The purpose of the present study was to test the diagnostic performance of this non invasive sampling procedure in an italian multicentric study. 2. Materials and Methods Oral brushing specimens were collected in ten different italian units of oral medicine. Each oral medicine unit collected blindly 10 brushing specimens from patients affected by OSCC and an equal number of age and sex-matched healthy controls. 13-gene DNA methylation analysis was performed and each sample was considered positive or negative in relation to a predefined cut-off value. 3. Results 181 out of 200 planned specimens were analyzed. DNA could not be amplified in 4 cases (2.2%). 86/93 (92.5%) specimens derived from OSCC patients were detected as positive and 70/84 (83.3%) specimens derived from healthy donors showed a negative score. 4. Conclusions Data from multicentric study confirmed a high level of sensitivity of our procedure whereas level of specificity is slightly lower if compared to our previous study. These data suggest that our procedure may be proposed as a first level diagnostic test with the aim to avoid a diagnostic delay in Oral Squamous Cell Carcinoma. Conflicts of Interest: As a possible conflict of interest, L. Morandi and D.B.G. submitted a patent (the applicant is the University of Bologna) in November 2016 to the National Institute of 398 Industrial Property; however, we believe that this is a natural step of translational research (bench-to-bedside) 399 and guarantee that the scientific results are true. The remaining authors declare that they have no competing 400 interest

Anxiety and depression in keratotic oral lichen planus: a multicentric study from the SIPMO

Objectives: Oral lichen planus with exclusive keratotic reticular, papular, and/or plaque-like lesions (K-OLP) is a clinical pattern of OLP that may be associated with a complex symptomatology and psychological alteration. The aim of the study was to evaluate the prevalence of anxiety (A) and depression (D) in patients with K-OLP, analyzing the potential predictors which can affect mental health status. Methods: Three hundred K-OLP patients versus 300 healthy controls (HC) were recruited in 15 Italian universities. The Numeric Rating Scale (NRS), Total Pain Rating Index (T-PRI), and Hamilton Rating Scales for Depression and for Anxiety (HAM-D and HAM-A) were administered. Results: The K-OLP patients showed statistically higher scores in the NRS, T-PRI, HAM-D, and HAM-A compared with the HC (p-value < 0.001**). A and D were found in 158 (52.7%) and 148 (49.3%) K-OLP patients. Strong linear correlations were identified between HAM-A, HAM-D, NRS, T-PRI, and employment status and between HAM-D, HAM-A, NRS, T-PRI, employment status, and female gender. Multivariate logistic regression revealed that HAM-D and HAM-A showed the greatest increase in the R2 value for A and D in the K-OLP patients, respectively (DR2 = 55.5% p-value < 0.001**; DR2 = 56.5% p-value < 0.001**). Conclusions: The prevalence of A and D is higher in the K-OLP patients compared with the HC, also found in K-OLP subjects without pain, suggesting that the processing of pain may be in a certain way independent of the processing of mood. Clinical relevance: Mood disorders and pain assessment should be carefully performed in relation to K-OLP to obtain a complete analysis of the patients

Anti-proliferative effect of Rosmarinus officinalis L. extract on human melanoma A375 cells

Rosemary (Rosmarinus officinalis L.) has been used since ancient times in traditional medicine, while nowadays various rosemary formulations are increasingly exploited by alternative medicine to cure or prevent a wide range of health disorders. Rosemary's bioproperties have prompted scientific investigation, which allowed us to ascertain antioxidant, anti-inflammatory, cytostatic, and cytotoxic activities of crude extracts or of pure components. Although there is a growing body of experimental work, information about rosemary's anticancer properties, such as chemoprotective or anti-proliferative effects on cancer cells, is very poor, especially concerning the mechanism of action. Melanoma is a skin tumor whose diffusion is rapidly increasing in the world and whose malignancy is reinforced by its high resistance to cytotoxic agents; hence the availability of new cytotoxic drugs would be very helpful to improve melanoma prognosis. Here we report on the effect of a rosemary hydroalcoholic extract on the viability of the human melanoma A375 cell line. Main components of rosemary extract were identified by liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) and the effect of the crude extract or of pure components on the proliferation of cancer cells was tested by MTT and Trypan blue assays. The effect on cell cycle was investigated by using flow cytometry, and the alteration of the cellular redox state was evaluated by intracellular ROS levels and protein carbonylation analysis. Furthermore, in order to get information about the molecular mechanisms of cytotoxicity, a comparative proteomic investigation was performed

Protection by Anti-β-Glucan Antibodies Is Associated with Restricted β-1,3 Glucan Binding Specificity and Inhibition of Fungal Growth and Adherence

Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model