Fungal Biofilms and Drug Resistance

Candida species, including the novel opportunistic pathogen Candida dubliniensis, are now emerging as major agents of nosocomial infections. Many such manifestations of infections associated with the formation of Candida biofilms include those occurring on devices such as indwelling intravascular catheters. Fungal biofilm-associated infections are frequently refractory to conventional therapy because of resistance to antimicrobial agents. This resistance could be in part due to the surface-induced upregulation of drug efflux pumps. Biofilm-associated Candida show uniform resistance to a wide spectrum of the currently available conventional antifungal agents, which implies that antimicrobial drugs that specifically target biofilm-associated infections are needed. The novel classes of antifungal agents, the lipid formulation of amphotericins, and the echinocandins have demonstrated unique antifungal activity against the resistant Candida biofilms, providing a breakthrough in the treatment of life-threatening invasive systemic mycoses. The use of drugs effective in combating biofilm-associated infections could lead to major developments in the treatment of fungal implant infections

Candidalysin crucially contributes to Nlrp3 inflammasome activation by Candida albicans hyphae

Candida albicans is an opportunistic fungal pathogen that can cause life-threatening infections, particularly in immunocompromised patients. C. albicans induced activation of the Nlrp3 inflammasome, leading to secretion of bioactive interleukin 1β (IL-1β) is a crucial myeloid cell immune response needed for antifungal host defense. Being a pleiomorphic fungus, C. albicans can provoke Nlrp3 inflammasome responses only upon morphological transformation to its hyphal appearance. However, the specific hyphal factors that enable C. albicans to activate the Nlrp3 inflammasome in primary macrophages remain to be revealed. Here, we identify candidalysin, a peptide derived from the hypha-specific ECE1 gene, as a fungal trigger for Nlrp3 inflammasome-mediated maturation and secretion of IL-1β from primary macrophages. Direct peptide administration experiments showed that candidalysin was sufficient for inducing secretion of mature IL-1β from macrophages in an Nlrp3 inflammasome-dependent manner. Conversely, infection experiments using candidalysin-deficient C. albicans showed that candidalysin crucially contributed to the capacity of this fungus to induce maturation and secretion of IL-1β from primary macrophages. These complementary observations identify the expression of candidalysin as one of the molecular mechanisms by which hyphal transformation equips C. albicans with its proinflammatory capacity to elicit the release of bioactive IL-1β from macrophages.IMPORTANCE Candidiasis is a potentially lethal condition that is caused by systemic dissemination of Candida albicans, a common fungal commensal residing mostly on mucosal surfaces. The transition of C. albicans from an innocuous commensal to an opportunistic pathogen goes hand in hand with its morphological transformation from a fungus to a hyphal appearance. On the one hand, the latter manifestation enables C. albicans to penetrate tissues, while on the other hand, the expression of many hypha-specific genes also endows it with the capacity to trigger particular cytokine responses. The Nlrp3 inflammasome is a crucial component of the innate immune system that provokes release of the IL-1β cytokine from myeloid cells upon encountering C. albicans hyphae. Our study reveals the peptide candidalysin as one of the hypha-derived drivers of Nlrp3 inflammasome responses in primary macrophages and, thus, contributes to better understanding the fungal mechanisms that determine the pathogenicity of C. albicans

Editorial overview of Pearls Microbiome Series: E pluribus unum

The human microbiome constitutes the collection of all the microorganisms living in association with the human body with each body site being home to a unique microbial community. Human-associated microbial communities can include eukaryotes, archaea, bacteria, and viruses and provide protection against foreign invaders, stimulate the immune response, produce antimicrobials, and aid in digestion among other functions. Our understanding of the link between the human microbiome and disease is rapidly expanding in large part due to revolutionizing advances in next generation sequencing. In fact, an ever-growing number of studies have demonstrated that changes in the composition of our microbiomes correlate with numerous disease states or responses to treatment. However, understanding the impact of shifts in microbial communities on health and disease and the mechanisms that confer stability in the microbiome have been challenging to elucidate, due to the vast microbial diversity and differences between individuals. Nevertheless, the notion that manipulation of microbial communities may provide prophylactic or therapeutic tools to improve human health has been the focus of much research. Here, we highlight a collection of Pearls articles delving into the current state of knowledge linking the microbiome to human disease

Candida albicans quorum-sensing molecule farnesol modulates staphyloxanthin production and activates the thiol-based oxidative-stress response in Staphylococcus aureus

Microbial species utilize secreted-signaling molecules to coordinate their behavior. Our previous investigations demonstrated a key role for the Candida albicans-secreted quorum-sensing molecule farnesol in modulating Staphylococcus aureus response to antimicrobials in mixed biofilms. In this study, we aimed to provide mechanistic insights into the impact of farnesol on S. aureus within the context of inter-species interactions. To mimic biofilm dynamics, farnesol-sensitized S. aureus cells were generated via sequential farnesol exposure. The sensitized phenotype exhibited dramatic loss of the typical pigment, which we identified as staphyloxanthin, an important virulence factor synthesized by the Crt operon in S. aureus. Additionally, farnesol exposure exerted oxidative-stress as indicated by transcriptional analysis demonstrating alterations in redox-sensors and major virulence regulators. Paradoxically, the activated stress-response conferred S. aureus with enhanced tolerance to H2O2 and phagocytic killing. Since expression of enzymes in the staphyloxanthin biosynthesis pathway was not impacted by farnesol, we generated a theoretical-binding model which indicated that farnesol may block staphyloxanthin biosynthesis via competitive-binding to the CrtM enzyme crucial for staphyloxanthin synthesis, due to high structural similarity to the CrtM substrate. Finally, mixed growth with C. albicans was found to similarly induce S. aureus depigmentation, but not during growth with a farnesol-deficient C. albicans strain. Collectively, the findings demonstrate that a fungal molecule acts as a redox-cycler eliciting a bacterial stress response via activation of the thiol-based redox system under the control of global regulators. Therefore, farnesol-induced transcriptional modulations of key regulatory networks in S. aureus may modulate the pathogenesis of C. albicans- S. aureus co-infections

A Novel Immune Evasion Strategy of Candida albicans: Proteolytic Cleavage of a Salivary Antimicrobial Peptide

Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps), involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap) family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the first defined mechanism behind the enhanced susceptibility of HIV+ individuals to oral candidiasis since the emergence of HIV

Susceptibility of Candida glabrata biofilms to echinocandins: alterations in the matrix composition

Candidiases are the most recurrent fungal infections, especially among immunosuppressed patients. Although Candida albicans is still the most widespread isolated species, non-Candida albicans Candida species have been increasing. The goal of this work was to determine the susceptibility of C. glabrata biofilms to echinocandins and to evaluate their effect on the biofilm matrix composition, comparing the results with other Candida species. Drug susceptibilities were assessed through the determination of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and minimum biofilm eradication concentration (MBEC) of caspofungin (Csf) and micafugin (Mcf). The -1,3 glucans content of the matrices was assessed after contact with the drugs. The data suggest that, generally, after contact with echinocandins, the concentration of -1,3 glucans increased. These adjustments in the matrix composition of C. glabrata biofilms and the chemical differences between Csf and Mcf, seem responsible and may determine the effectivity of the drug responses.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 [POCI-01–0145-FEDER-006684] and BioTecNorte operation [NORTE-01–0145-FEDER-000004] funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, Célia F. Rodrigues’ [SFRH/BD/93078/2013] PhD grant and M. Elisa Rodrigues [SFRH/BPD/95401/2013] post-doctoral grant.info:eu-repo/semantics/publishedVersio

Candida dubliniensis Meningitis as Delayed Sequela of Treated C. dubliniensis Fungemia

We present a case of Candida dubliniensis meningitis that developed 2 months after apparently successful treatment of an episode of C. dubliniensis candidemia in a heart-lung transplant recipient in Australia. This case highlights the importance of follow-up in patients with candidemia or disseminated infection, especially in immunosuppressed patients

Microbial interactions and differential protein expression in Staphylococcus aureus –Candida albicans dual-species biofilms

The fungal species Candida albicans and the bacterial species Staphylococcus aureus are responsible for a majority of hospital-acquired infections and often coinfect critically ill patients as complicating polymicrobial biofilms. To investigate biofilm structure during polymicrobial growth, dual-species biofilms were imaged with confocal scanning laser microscopy. Analyses revealed a unique biofilm architecture where S. aureus commonly associated with the hyphal elements of C. albicans. This physical interaction may provide staphylococci with an invasion strategy because candidal hyphae can penetrate through epithelial layers. To further understand the molecular mechanisms possibly responsible for previously demonstrated amplified virulence during coinfection, protein expression studies were undertaken. Differential in-gel electrophoresis identified a total of 27 proteins to be significantly differentially produced by these organisms during coculture biofilm growth. Among the upregulated staphylococcal proteins was l-lactate dehydrogenase 1, which confers resistance to host-derived oxidative stressors. Among the downregulated proteins was the global transcriptional repressor of virulence factors, CodY. These findings demonstrate that the hyphae-mediated enhanced pathogenesis of S. aureus may not only be due to physical interactions but can also be attributed to the differential regulation of specific virulence factors induced during polymicrobial growth. Further characterization of the intricate interaction between these pathogens at the molecular level is warranted, as it may aid in the design of novel therapeutic strategies aimed at combating fungal–bacterial polymicrobial infection

Adhesion of Candida albicans and Candida dubliniensis to acrylic and hydroxyapatite

The aim of this work was to compare the ability of strains of Candida albicans and Candida dubliniensis to adhere to acrylic and hydroxyapatite (HAP). In order to interpret the adhesion results, the surface properties of cells and materials were determined. Surface tension components (polar and apolar) and hydrophobicity were calculated through contact angle measurement and the elemental composition was determined by X-ray photoelectron spectroscopy (XPS). The results showed no significant differences in the number of adhered cells of both species to acrylic and hydroxyapatite. This was corroborated by the similarities in their surface properties and elemental composition. For both species, the adhesion to acrylic increased in the presence of artificial saliva due to the increase in the electron-donor capacity of this material. In the absence of artificial saliva, the number of adhered cells to HAP was greater than to acrylic, on account of the higher number of electron-donor groups of HAP. Hydrophobicity played a minor role in the adhesion process of both candidal species. Conversely, Lewis acid–base interactions seamed to govern this phenomenon.Fundação para a Ciência e a Tecnologia (FCT) - BD3195/2000, Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI) POCTI/BIO/42638/2001