Biology subjects

Ramage, G.

Publications and source records attributed to Ramage, G..

5 recordsLinked to original sources

bioRxiv2021

Recurrent Vulvovaginal Candidiasis; a dynamic interkingdom biofilm disease of Candida and Lactobacillus

Vulvovaginal Candidiasis (VVC) is the most prevalent Candida infection in humans affecting 75% of women at least once throughout their lifetime. In its debilitating recurrent form, RVVC is estimated to affect 140 million women annually. Despite this strikingly high prevalence, treatment options for RVVC remain limited with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown with few studies identifying potential mechanisms of failed treatment. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, vaginal microbiome and antagonism between Candida and Lactobacillus on disease pathology. 16S rRNA sequencing characterised disease by a reduction in specific health-associated Lactobacillus such as L. crispatus, coupled with an increase in L. iners. In vitro analysis showed Candida albicans clinical isolates are capable of heterogeneous biofilm formation and show the presence of hyphae and C. albicans aggregates in vaginal lavage. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to exploit a possible mechanism by which L. crispatus may aim to re-establish a healthy vaginal environment through amino-acid acquisition from C. albicans. This study suggests RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and understanding interactions between the yeast and vaginal Lactobacillus species may be more crucial to elucidating the cause of RVVC and developing appropriate therapies.

microbiology↗

bioRxiv2020

A novel Hsp90 phospho-switch modulates virulence in the major human fungal pathogen Candida albicans

The ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. Hsp90 also regulates cellular morphogenesis, drug resistance, and virulence in human pathogenic fungi, which kill more than 1.6 million patients each year worldwide. Invasive fungal infections are difficult to treat due to the lack of effective antifungal therapies, resulting in mortality rates of up to 95%. As a key regulator of fungal virulence, Hsp90 is an attractive therapeutic target. However, fungal and animal homologs are highly conserved, impeding fungal-specific targeting. Thus, understanding the factors that regulate Hsp90 could provide an alternative strategy aimed at exclusively targeting this regulator of fungal virulence. Here, we demonstrate how CK2-mediated phosphorylation of two Hsp90 residues modulates virulence in a major fungal pathogen of humans, Candida albicans. We combined proteomics, molecular evolution and structural modelling with molecular biology to identify and characterize two Hsp90 phosphorylation sites. Phosphorylation negatively affects thermal stress response, morphogenesis, drug susceptibility and fungal virulence. Our results provide the first record of specific Hsp90 phosphorylation sites acting as modulators of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitation as antifungal drug targets.

microbiology↗

bioRxiv2020

PEA polymer-coated nanotopography delivers solid-state BMP2, enhances mesenchymal stem cell adhesion, prevents bacterial biofilm formation and protects cells from quorum sensing virulence factors

Post-operative infection is a major complication in patients recovering from orthopaedic surgery. As such, there is a clinical need to develop biomaterials for use in regenerative surgery that can promote mesenchymal stem cell (MSC) osteospecific differentiation and that can prevent infection caused by biofilm-forming pathogens. Nanotopographical approaches to pathogen control are being identified, including in orthopaedic materials such as titanium and its alloys. These topographies use high aspect ratio nanospikes or nanowires to prevent bacterial adhesion but these features puncture adhering cells, thus also reducing MSC adhesion. Here, we use a poly(ethyl acrylate) (PEA) polymer coating on titanium nanowires to spontaneously organise fibronectin (FN) and to deliver bone morphogenetic protein 2 (BMP2) to enhance MSC adhesion and osteospecific signalling. This nanotopography when combined with the PEA coating enhanced osteogenesis and reduced adhesion of Pseudomonas aeruginosa in culture. Using a novel MSC-Pseudomonas aeruginosa co-culture, we also show that the coated nanotopographies protect MSCs from cytotoxic quorum sensing and signalling molecules. We conclude that the PEA polymer-coated nanotopography can both support MSCs and prevent pathogens from adhering to a biomaterial surface, thus protecting from biofilm formation and bacterial infection and supporting osteogenic repair.

bioengineering↗

bioRxiv2020

Curcumin-Sophorolipid nano-conjugate inhibits Candida albicans filamentation and biofilm development

Candida albicans is an opportunistic fungal pathogen that is highly resistant to contemporary antifungals, and a major reason for this appears to be their predominant, filamentation-mediated, biofilm lifestyle. Hence, agents that inhibit biofilms and filamentation of the yeast offer promise as next-generation antifungals. Curcumin is a natural polyphenol with several beneficial pharmacological attributes, yet limitations such as poor solubility, acid, and enzyme tolerance have impeded its practical utility. Sophorolipids are biologically-derived surfactants that serve as efficient carriers and delivery agents of hydrophobic molecules, such as curcumin, into biofilms. The aim of this study was to investigate the effects of a novel, curcumin-sophorolipid (CU-ASL) nano-conjugate on Candida albicans biofilms and filamentation. The effects of CU and ASL, in combination, and alone, were investigated on planktonic cells of the yeast. The effects of sub-inhibitory concentrations of the compounds were investigated on biofilm biomass and biofilm architecture. Their effects on filamentation was compared by scanning electron microscopic imaging, and gene expression analysis by qRT-PCR. Our results demonstrated that sub-inhibitory concentration of CU-ASL (9.37 {micro}g/mL) significantly inhibited candidal adhesion to substrates, and subsequent biofilm development, maturation, and filamentation. This effect was associated with significant downregulation of a select group of biofilm, adhesins, and hyphal regulatory genes. In conclusion, the curcumin-sophorolipid nano-conjugate is a potent inhibitor of the two major virulence attributes of C. albicans, biofilm formation and filamentation, thus highlighting its promise as a putative anti-candidal agent with low toxicity and biofilm penetrative potential.

microbiology↗

bioRxiv2020

Candida auris phenotypic heterogeneity determines pathogenicity in vitro

Candida auris is an enigmatic yeast that provides substantial global risk in healthcare facilities and intensive care units. A unique phenotype exhibited by certain isolates of C. auris is their ability to form small clusters of cells known as aggregates, which have been to a limited extent described in the context of pathogenic traits. In this study, we screened several non-aggregative and aggregative C. auris isolates for biofilm formation, where we observed a level of heterogeneity amongst the different phenotypes. Next, we utilised an RNA-sequencing approach to investigate the transcriptional responses during biofilm formation of a non-aggregative and aggregative isolate of the initial pool. Observations from these analyses indicate unique transcriptional profiles in the two isolates, with several genes identified relating to proteins involved in adhesion and invasion of the host in other fungal species. From these findings we investigated for the first time the fungal recognition and inflammatory responses of a three-dimensional skin epithelial model to these isolates. In these models, a wound was induced to mimic a portal of entry for C. auris. We show both phenotypes elicited minimal response in the model minus induction of the wound, yet in the wounded tissue both phenotypes induced a greater response, with the aggregative isolate more pro-inflammatory. This capacity of aggregative C. auris biofilms to generate such responses in the wounded skin highlights how this opportunistic yeast is a high risk within the intensive care environment where susceptible patients have multiple indwelling lines. ImportanceCandida auris has recently emerged as an important cause of concern within healthcare environments due to its ability to persist and tolerate commonly used antiseptics and disinfectants, particularly when surface attached (biofilms). This yeast is able to colonise and subsequently infect patients, particularly those that are critically ill or immunosuppressed, which may result in death. We have undertaken analysis on two different types of this yeast, using molecular and immunological tools to determine whether either of these has a greater ability to cause serious infections. We describe that both isolates exhibit largely different transcriptional profiles during biofilm development. Finally, we show that the inability to form small aggregates (or clusters) of cells has an adverse effect on the organisms immuno-stimulatory properties, suggestive the non-aggregative phenotype may exhibit a certain level of immune evasion.

microbiology↗