Slicing through the challenge of maintaining Pneumocystis in the laboratory.

Pneumocystis jirovecii is a major fungal pathogen of humans that causes life-threatening lung infections in immunocompromised individuals. Despite its huge global impact upon human health, our understanding of the pathobiology of this deadly fungus remains extremely limited, largely because it is not yet possible to cultivate Pneumocystis in vitro, independently of the host. However, a recent paper by Munyonho et al. offers a major step forward (F. T. Munyonho, R. D. Clark, D. Lin, M. S. Khatun, et al., 2023, mBio 15:e01464-23, https://doi.org/10.1128/mbio.01464-23). They show that it is possible to maintain both the trophozoite and cyst forms of the mouse pathogen, Pneumocystis murina, in precision-cut lung slices for several weeks. Furthermore, they demonstrate that this offers the exciting opportunity to examine potential virulence factors such as possible biofilm formation as well as antifungal drug responses in the lung.

Alternative Oxidase - Aid or obstacle to combat the rise of fungal pathogens?

Fungal pathogens present a growing threat to both humans and global health security alike. Increasing evidence of antifungal resistance in fungal populations that infect both humans and plant species has increased reliance on combination therapies and shown the need for new antifungal therapeutic targets to be investigated. Here, we review the roles of mitochondria and fungal respiration in pathogenesis and discuss the role of the Alternative Oxidase enzyme (Aox) in both human fungal pathogens and phytopathogens. Increasing evidence exists for Aox within mechanisms that underpin fungal virulence. Aox also plays important roles in adaptability that may prove useful within dual targeted fungal-specific therapeutic approaches. As improved fungal specific mitochondrial and Aox inhibitors are under development we may see this as an emerging target for future approaches to tackling the growing challenge of fungal infection.

The pathogenesis of experimental Emergomycosis in mice.

Emergomyces africanus is a recently identified thermally-dimorphic fungal pathogen that causes disseminated infection in people living with advanced HIV disease. Known as emergomycosis, this disseminated disease is associated with very high case fatality rates. Over the last decade, improved diagnostics and fungal identification in South Africa resulted in a dramatic increase in the number of reported cases. Although the true burden of disease is still unknown, emergomycosis is among the most frequently diagnosed dimorphic fungal infections in Southern Africa; and additional species in the genus have been identified on four continents. Little is known about the pathogenesis and the host's immune response to this emerging pathogen. Therefore, we established a murine model of pulmonary infection using a clinical isolate, E. africanus (CBS 136260). Both conidia and yeast forms caused pulmonary and disseminated infection in mice with organisms isolated in culture from lung, spleen, liver, and kidney. Wild-type C57BL/6 mice demonstrated a drop in body weight at two weeks post-infection, corresponding to a peak in fungal burden in the lung, spleen, liver, and kidney. An increase in pro-inflammatory cytokine production was detected in homogenized lung supernatants including IFN-γ, IL-1ß, IL-6, IL12-p40 and IL-17 at three- and four-weeks post-infection. No significant differences in TNF, IL-12p70 and IL-10 were observed in wild-type mice between one and four-weeks post-infection. Rag-1-deficient mice, lacking mature T-and B-cells, had an increased fungal burden associated with reduced IFN-γ production. Together our data support a protective T-helper type-1 immune response to E. africanus infection. This may provide a possible explanation for the susceptibility of only a subset of people living with advanced HIV disease despite hypothesized widespread environmental exposure. In summary, we have established a novel murine model of E. africanus disease providing critical insights into the host immune components required for eliminating the infection.

Cbp1, a fungal virulence factor under positive selection, forms an effector complex that drives macrophage lysis.

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel "binocular" fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.

Key thermally dimorphic fungal pathogens: shaping host immunity.

Exposure to fungal pathogens from the environment is inevitable and with the number of at-risk populations increasing, the prevalence of invasive fungal infection is on the rise. An interesting group of fungal organisms known as thermally dimorphic fungi predominantly infects immunocompromised individuals. These potential pathogens are intriguing in that they survive in the environment in one form, mycelial phase, but when entering the host, they are triggered by the change in temperature to switch to a new pathogenic form. Considering the growing prevalence of infection and the need for improved diagnostic and treatment approaches, studies identifying key components of fungal recognition and the innate immune response to these pathogens will significantly contribute to our understanding of disease progression. This review focuses on key endemic dimorphic fungal pathogens that significantly contribute to disease, including Histoplasma, Coccidioides and Talaromyces species. We briefly describe their prevalence, route of infection and clinical presentation. Importantly, we have reviewed the major fungal cell wall components of these dimorphic fungi, the host pattern recognition receptors responsible for recognition and important innate immune responses supporting adaptive immunity and fungal clearance or the failure thereof.

The potential of respiration inhibition as a new approach to combat human fungal pathogens.

The respiratory chain has been proposed as an attractive target for the development of new therapies to tackle human fungal pathogens. This arises from the presence of fungal-specific electron transport chain components and links between respiration and the control of virulence traits in several pathogenic species. However, as the physiological roles of mitochondria remain largely undetermined with respect to pathogenesis, its value as a potential new drug target remains to be determined. The use of respiration inhibitors as fungicides is well developed but has been hampered by the emergence of rapid resistance to current inhibitors. In addition, recent data suggest that adaptation of the human fungal pathogen, Candida albicans, to respiration inhibitors can enhance virulence traits such as yeast-to-hypha transition and cell wall organisation. We conclude that although respiration holds promise as a target for the development of new therapies to treat human fungal infections, we require a more detailed understanding of the role that mitochondria play in stress adaption and virulence.

Inhibition of Classical and Alternative Modes of Respiration in Candida albicans Leads to Cell Wall Remodeling and Increased Macrophage Recognition.

The human fungal pathogen Candida albicans requires respiratory function for normal growth, morphogenesis, and virulence. Mitochondria therefore represent an enticing target for the development of new antifungal strategies. This possibility is bolstered by the presence of characteristics specific to fungi. However, respiration in C. albicans, as in many fungal organisms, is facilitated by redundant electron transport mechanisms, making direct inhibition a challenge. In addition, many chemicals known to target the electron transport chain are highly toxic. Here we made use of chemicals with low toxicity to efficiently inhibit respiration in C. albicans We found that use of the nitric oxide donor sodium nitroprusside (SNP) and of the alternative oxidase inhibitor salicylhydroxamic acid (SHAM) prevents respiration and leads to a loss of viability and to cell wall rearrangements that increase the rate of uptake by macrophages in vitro and in vivo We propose that treatment with SNP plus SHAM (SNP+SHAM) leads to transcriptional changes that drive cell wall rearrangement but which also prime cells to activate the transition to hyphal growth. In line with this, we found that pretreatment of C. albicans with SNP+SHAM led to an increase in virulence. Our data reveal strong links between respiration, cell wall remodeling, and activation of virulence factors. Our findings demonstrate that respiration in C. albicans can be efficiently inhibited with chemicals that are not damaging to the mammalian host but that we need to develop a deeper understanding of the roles of mitochondria in cellular signaling if they are to be developed successfully as a target for new antifungals.IMPORTANCE Current approaches to tackling fungal infections are limited, and new targets must be identified to protect against the emergence of resistant strains. We investigated the potential of targeting mitochondria, which are organelles required for energy production, growth, and virulence, in the human fungal pathogen Candida albicans Our findings suggest that mitochondria can be targeted using drugs that can be tolerated by humans and that this treatment enhances their recognition by immune cells. However, release of C. albicans cells from respiratory inhibition appears to activate a stress response that increases the levels of traits associated with virulence. Our results make it clear that mitochondria represent a valid target for the development of antifungal strategies but that we must determine the mechanisms by which they regulate stress signaling and virulence ahead of successful therapeutic advance.

High Resolution Respirometry in Candida albicans.

Many Candida species, such as the opportunistic human pathogen Candida albicans, are Crabtree-Negative yeasts and are therefore highly dependent on the energy generated through oxidative phosphorylation. Respiration control is linked to a range of aspects of C. albicans cell physiology that appear to be important for virulence, most notably its ability to switch from yeast to hyphal forms and the maintenance of the cell wall. The following protocol allows for the measurement and characterization of respiration in C. albicans using high resolution respirometry. We outline how addition of respiration inhibitors can be used to assay the "mode" of respiration, mitochondrial health and the level of electron transport that is coupled to ATP synthase activity in living cell cultures. These data provide useful insight into the effects of external factors, such as exposure to anti-fungal compounds, or internal changes such as genetic alterations on respiratory performance.

Expression in tobacco and purification of beak and feather disease virus capsid protein fused to elastin-like polypeptides.

Psittacine beak and feather disease, caused by beak and feather disease virus (BFDV), is a threat to endangered psittacine species. There is currently no vaccine against BFDV, which necessitates the development of safe and affordable vaccine candidates. A subunit vaccine based on BFDV capsid protein (CP), the major antigenic determinant, expressed in the inexpensive and highly scalable plant expression system could satisfy these requirements. Full-length CP and a truncated CP (ΔN40 CP) were transiently expressed in tobacco (Nicotiana benthamiana) as fusions to elastin-like polypeptide (ELP). These two proteins were fused to ELPs of different lengths in order to increase expression levels and to provide a simple means of purification. The ELP fusion proteins were purified by inverse transition cycling (ITC) and it was found that a membrane filtration-based ITC method improved the recovery of ΔN40 CP-ELP51 fusion protein relative to a centrifugation-based method.