Ribosomal protein S6 kinase beta-1 gene variants cause hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic heart muscle disease with preserved or increased ejection fraction in the absence of secondary causes. Mutations in the sarcomeric protein-encoding genes predominantly cause HCM. However, relatively little is known about the genetic impact of signalling proteins on HCM. METHODS AND RESULTS: Here, using exome and targeted sequencing methods, we analysed two independent cohorts comprising 401 Indian patients with HCM and 3521 Indian controls. We identified novel variants in ribosomal protein S6 kinase beta-1 (RPS6KB1 or S6K1) gene in two unrelated Indian families as a potential candidate gene for HCM. The two unrelated HCM families had the same heterozygous missense S6K1 variant (p.G47W). In a replication association study, we identified two S6K1 heterozygotes variants (p.Q49K and p.Y62H) in the UK Biobank cardiomyopathy cohort (n=190) compared with matched controls (n=16 479). These variants are neither detected in region-specific controls nor in the human population genome data. Additionally, we observed an S6K1 variant (p.P445S) in an Arab patient with HCM. Functional consequences were evaluated using representative S6K1 mutated proteins compared with wild type in cellular models. The mutated proteins activated the S6K1 and hyperphosphorylated the rpS6 and ERK1/2 signalling cascades, suggesting a gain-of-function effect. CONCLUSIONS: Our study demonstrates for the first time that the variants in the S6K1 gene are associated with HCM, and early detection of the S6K1 variant carriers can help to identify family members at risk and subsequent preventive measures. Further screening in patients with HCM with different ethnic populations will establish the specificity and frequency of S6K1 gene variants.

Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation.

The main characteristic of biofilm formation is extracellular matrix (ECM) production. The cells within the biofilm are surrounded by ECM which provides structural integrity and protection. During an infection, this protection is mainly against cells of the immune system and antifungal drugs. A. fumigatus forms biofilms during static growth on a solid substratum and in chronic aspergillosis infections. It is important to understand how, and which, A. fumigatus signal transduction pathways are important for the adhesion and biofilm formation in a host during infection. Here we investigated the role of MAP kinases and protein phosphatases in biofilm formation. The loss of the MAP kinases MpkA, MpkC and SakA had an impact on the cell surface and the ECM during biofilm formation and reduced the adherence of A. fumigatus to polystyrene and fibronectin-coated plates. The phosphatase null mutants ΔsitA and ΔptcB, involved in regulation of MpkA and SakA phosphorylation, influenced cell wall carbohydrate exposure. Moreover, we characterized the A. fumigatus protein phosphatase PphA. The ΔpphA strain was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and reduced chitin, decreased conidia phagocytosis by Dictyostelium discoideum and reduced adhesion and biofilm formation. Finally, ΔpphA strain was avirulent in a murine model of invasive pulmonary aspergillosis and increased the released of tumor necrosis factor alpha (TNF-α) from bone marrow derived macrophages (BMDMs). These results show that MAP kinases and phosphatases play an important role in signaling pathways that regulate the composition of the cell wall, extracellular matrix production as well as adhesion and biofilm formation in A. fumigatus.

The Anti-Adhesive Effect of Curcumin on Candida albicans Biofilms on Denture Materials.

The use of natural compounds as an alternative source of antimicrobials has become a necessity given the growing concern over global antimicrobial resistance. Polyphenols, found in various edible plants, offers one potential solution to this. We aimed to investigate the possibility of using curcumin within the context of oral health as a way of inhibiting and preventing the harmful development of Candida albicans biofilms. We undertook a series of adsorption experiments with varying concentrations of curcumin, showing that 50 µg/ml could prevent adhesion. This effect could be further synergized by the curcumin pre-treatment of yeast cells to obtain significantly greater inhibition (>90%, p < 0.001). Investigation of the biological impact of curcumin showed that it preferentially affected immature morphological forms (yeast and germlings), and actively promoted aggregation of the cells. Transcriptional analyses showed that key adhesins were down-regulated (ALS1 and ALS3), whereas aggregation related genes (ALS5 and AAF1) were up-regulated. Collectively, these data demonstrated that curcumin elicits anti-adhesive effects and that induces transcription of genes integrally involved in the processes related to biofilm formation. Curcumin and associated polyphenols therefore have the capacity to be developed for use in oral healthcare to augment existing preventative strategies for candidal biofilms on the denture surface.

New strategic insights into managing fungal biofilms.

Fungal infections have dramatically increased in the last decades in parallel with an increase of populations with impaired immunity, resulting from medical conditions such as cancer, transplantation, or other chronic diseases. Such opportunistic infections result from a complex relationship between fungi and host, and can range from self-limiting to chronic or life-threatening infections. Modern medicine, characterized by a wide use of biomedical devices, offers new niches for fungi to colonize and form biofilm communities. The capability of fungi to form biofilms is well documented and associated with increased drug tolerance and resistance. In addition, biofilm formation facilitates persistence in the host promoting a persistent inflammatory condition. With a limited availability of antifungals within our arsenal, new therapeutic approaches able to address both host and pathogenic factors that promote fungal disease progression, i.e., chronic inflammation and biofilm formation, could represent an advantage in the clinical setting. In this paper we discuss the antifungal properties of myriocin, fulvic acid, and acetylcholine in light of their already known anti-inflammatory activity and as candidate dual action therapeutics to treat opportunistic fungal infections.

Aspergillus fumigatus enhances elastase production in Pseudomonas aeruginosa co-cultures.

In the cystic fibrosis (CF) lung the presence of bacteria and fungi in the airways promotes an inflammatory response causing progressive lung damage, ultimately leading to high rates of morbidity and mortality. We hypothesized that polymicrobial interactions play an important role in promoting airway pathogenesis. We therefore examined the interplay between the most commonly isolated bacterial CF pathogen, Pseudomonas aeruginosa, and the most prevalent filamentous fungi, Aspergillus fumigatus, to test this. Co-culture experiments showed that in the presence of A. fumigatus the production of P. aeruginosa elastase was enhanced. This was confirmed by the presence of zones of clearance on Elastin-Congo Red (ECR) agar, which was identified as elastase by mass spectrometry. When P. aeruginosa were grown in a co-culture model with mature A. fumigatus biofilms, 60% of isolates produced significantly more elastase in the presence of the filamentous fungi than in its absence (P < .05). The expression of lasB also increased when P. aeruginosa isolates PA01 and PA14 were grown in co-culture with A. fumigatus. Supernatants from co-culture experiments were also significantly toxic to a human lung epithelial cell line (19-38% cell cytotoxicity) in comparison to supernatants from P. aeruginosa only cultures (P < .0001). Here we report that P. aeruginosa cytotoxic elastase is enhanced in the presence of the filamentous fungi A. fumigatus, suggesting that this may have a role to play in the damaging pathology associated with the lung tissue in this disease. This indicates that patients who have a co-colonisation with these two organisms may have a poorer prognosis.

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence.

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

Utilising polyphenols for the clinical management of Candida albicans biofilms.

Polyphenols (PPs) are secondary metabolites abundant in plant-derived foods. They are reported to exhibit antimicrobial activity that may offer an alternative to existing antimicrobials. The aim of this study was to evaluate the antifungal potential of PPs against Candida albicans biofilms that are commonly recalcitrant to antifungal therapy. The antifungal activity of 14 PPs was assessed in terms of planktonic and sessile minimum inhibitory concentrations (PMICs and SMICs, respectively) against various C. albicans clinical isolates. The most active PPs were further tested for their effect on C. albicans adhesion and biofilm growth using standard biomass assays, microscopy and quantitative gene expression. Of the 14 PPs tested, 7 were effective inhibitors of planktonic growth, of which pyrogallol (PYG) was the most effective (PMIC50=78 µg/mL), followed by curcumin (CUR) (PMIC50=100 µg/mL) and pyrocatechol (PMIC50=625 µg/mL). Both PYG and CUR displayed activity against C. albicans biofilms (SMIC50=40 µg/mL and 50 µg/mL, respectively), although they did not disrupt the biofilm or directly affect the cellular structure. Overall, CUR displayed superior biofilm activity, significantly inhibiting initial cell adhesion following pre-coating (P<0.01), biofilm growth (P<0.05) and gene expression (P<0.05). This inhibitory effect diminished with prolonged CUR exposure, although it still inhibited by 50% after 4h adhesion. Overall, CUR exhibited positive antibiofilm properties that could be used at the basis for development of similar molecules, although further cellular and in vivo studies are required to explore its precise mechanism of action.

A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.

A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.

Activity of pyocin S2 against Pseudomonas aeruginosa biofilms.

In cystic fibrosis patients, chronic lung infection with Pseudomonas aeruginosa and the associated decline in lung function are the major cause of mortality. In this report, we show that pyocin S2 displays potent activity against P. aeruginosa biofilms, thus representing a potentially improved therapeutic option. Using an invertebrate model of P. aeruginosa infection, we also show that pyocin S2 is highly active in vivo.

Antifungal treatment affects the laboratory diagnosis of invasive aspergillosis.

AIMS: The purpose of this study was to investigate the performance of non-invasive diagnostic tests such as galactomannan enzyme immunoassay and quantitative PCR in the early diagnosis of invasive aspergillosis (IA), and how these tests are impacted upon by the use of different classes of antifungal agents in an in-vivo model of IA. METHODS: A standardised rat inhalation model of IA was used to examine the effects of an azole, posaconazole, a polyene, amphotericin B and an echinocandin caspofungin. Daily blood samples were collected for subsequent analysis using a commercially available galactomannan assay and an inhouse qPCR assay. RESULTS: No significant differences were observed in the CE/g of Aspergillus fumigatus in the lungs of each group. qPCR was statistically more sensitive than galactomannan for both the early detection of infected controls (p=0.045) and for overall detection (p=0.018). However, antifungal treatment significantly reduced the overall sensitivity of qPCR (p=0.020); these effects were due to posaconazole and caspofungin. In the latter stages of infection (days 4 and 5) there were no significant differences in the numbers of infections detected by galactomannan and qPCR; however, the antifungal class used caused significant qualitative differences (p=0.041). Galactomannan showed improved detection in posaconazole-treated animals. CONCLUSIONS: Previous exposure to antifungal therapy must be considered when interpreting either qPCR or galactomannan-based IA diagnostics as this study has shown that individual classes of antifungal agents impact upon the dynamics of antigen and DNA release into the circulation.

Commercial mouthwashes are more effective than azole antifungals against Candida albicans biofilms in vitro.

OBJECTIVE: The aim of this study was to evaluate and compare the activity of prescription and over-the-counter antimicrobial compounds against planktonic and biofilm forms of Candida albicans isolated from cases of oral candidiasis in vitro. STUDY DESIGN: The efficacy of azoles, polyenes, an echinocandin, and 4 over-the-counter mouthwashes were tested against C. albicans-derived planktonic and biofilm cells. RESULTS: Planktonic cells were shown to be highly sensitive to all of the antifungal agents tested. Sessile cells were highly resistant to azoles (≥128 mg/L) but equally sensitive to caspofungin and short treatments with Corsodyl, Listerine, and Oraldene. CONCLUSIONS: Although C. albicans is sensitive to azole antifungal agents in planktonic form, it is highly resistant within the biofilm. The good efficacy of the over-the-counter mouthwashes against candidal biofilms in vitro suggests that clinical trials should now be designed to establish their role in the clinical management of oral candidal infections.

Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation.

Aspergillus fumigatus is often isolated from the lungs of cystic fibrosis (CF) patients, but unlike in severely immunocompromised individuals, the mortality rates are low. This suggests that competition from bacteria within the CF lung may be inhibitory. The purpose of this study was to investigate how Pseudomonas aeruginosa influences A. fumigatus conidial germination and biofilm formation. Aspergillus fumigatus biofilm formation was inhibited by direct contact with P. aeruginosa, but had no effect on preformed biofilm. A secreted heat-stable soluble factor was also shown to exhibit biofilm inhibition. Coculture of P. aeruginosa quorum-sensing mutants (PAO1:ΔLasI, PAO1:ΔLasR) did not significantly inhibit A. fumigatus biofilms (52.6-58.8%) to the same extent as that of the PA01 wild type (22.9-30.1%), both by direct and by indirect interaction (P<0.001). Planktonic and sessile inhibition assays with a series of short carbon chain molecules (decanol, decanoic acid and dodecanol) demonstrated that these molecules could both inhibit and disrupt biofilms in a concentration-dependent manner. Overall, this suggests that small diffusible and heat-stable molecules may be responsible for the competitive inhibition of filamentous fungal growth in polymicrobial environments such as the CF lung.