Recombinant Neosartorya fumigata Probable kinetochore protein ndc80 (ndc80), partial

What is the taxonomic relationship between Neosartorya and Aspergillus species?

Neosartorya represents the sexual (teleomorphic) state of certain Aspergillus species. Aspergillus fumigatus is the asexual (anamorphic) state of what can also be classified as Neosartorya fumigata. This taxonomic relationship is important in understanding the complete lifecycle and genetics of these fungi. Other pathogenic Neosartorya species include N. hiratsukae, N. fischeri, and N. pseudofischeri, all of which are closely related to A. fumigatus and can cause opportunistic infections in humans . These species are morphologically similar in their conidial states, requiring careful microscopic examination and molecular techniques for accurate identification.

How are Neosartorya species distinguished in laboratory settings?

Neosartorya species produce white colonies that often do not develop the characteristic green coloration of Aspergillus fumigatus, leading to potential misidentification or dismissal as contaminants in clinical laboratories . N. hiratsukae specifically can be distinguished by its ascospores, which have two closely appressed equatorial crests with a microtuberculate convex surface between them . N. pseudofischeri, another pathogenic species, is characterized by ascospore walls ornamented with raised flaps of tissue resembling triangular projections or long ridge lines . Proper identification requires examination of both microscopic features and, increasingly, molecular methods.

What clinical manifestations are associated with Neosartorya infections?

Neosartorya species can cause severe invasive infections, particularly in immunocompromised patients. N. hiratsukae has been documented to cause cerebral aspergillosis, presenting with symptoms such as progressive memory loss, confusion, involuntary movements, and gait disorders that may initially be misdiagnosed as neurological conditions like Parkinson's disease . Brain imaging typically reveals multifocal abscesses, and the fungus can also cause pulmonary lesions . Other Neosartorya species have been reported to cause various localized and invasive infections including pulmonary aspergillosis, mycotic keratitis, and osteomyelitis .

What is known about the function of kinetochore proteins in pathogenic fungi?

Kinetochore proteins like ndc80 are essential components of the cellular machinery that ensures proper chromosome segregation during mitosis. In pathogenic fungi, these proteins are critical for maintaining genomic stability during rapid proliferation within host environments. While specific research on ndc80 in Neosartorya fumigata is limited, studies in related fungi suggest that disruption of kinetochore function could affect cell division, potentially impacting fungal growth, survival, and virulence. The highly conserved nature of kinetochore components makes them potential targets for antifungal development.

What mechanisms do Aspergillus/Neosartorya species use to evade host immune responses?

Aspergillus fumigatus employs sophisticated mechanisms to evade host immune responses. One key strategy involves the HscA protein present on the conidial surface, which anchors human p11 (S100A10) on conidia-containing phagosomes . This interaction excludes the phagosome maturation mediator Rab7 and triggers binding of exocytosis mediators Rab11 and Sec15 . This reprogramming redirects phagosomes to the non-degradative pathway, allowing A. fumigatus to escape cells by outgrowth and expulsion as well as transfer of conidia between cells . Additionally, the fungal dihydroxynaphthalene (DHN) melanin prevents the formation of functional phagolysosomes by sequestering Ca²⁺, which interferes with calcium-calmodulin dependent signaling pathways and reduces the formation of lipid-raft microdomains in the phagosomal membrane .

How do surface proteins of Neosartorya/Aspergillus contribute to pathogenesis?

Surface proteins play critical roles in fungal pathogenesis. The heat shock protein HscA of A. fumigatus binds to human cell surfaces, particularly to epithelial cells including human bronchial epithelial (BEAS-2B), lung epithelial (H441), liver epithelial (HepG2), and mouse type-II lung epithelial (T7) cells . This binding contributes significantly to cell damage, as demonstrated by experiments where deletion of the hscA gene (Δhsc) resulted in significantly less damage to host cells compared to wild-type conidia . The binding function of HscA appears to serve as an adhesin, facilitating the association of conidia with host cells, as the association of ΔhscA conidia with A549 cells was reduced compared to wild-type . The expression of HscA is upregulated in swollen conidia and germlings, supporting its role during early infection stages .

What genetic factors influence susceptibility to invasive Neosartorya/Aspergillus infections?

Genetic variations in host genes can significantly affect susceptibility to invasive aspergillosis. Research has identified a single nucleotide polymorphism (SNP) in the non-coding region of the S100A10 (p11) gene that affects mRNA and protein expression in response to A. fumigatus and is associated with protection against invasive pulmonary aspergillosis . This finding highlights the importance of host genetic factors in determining infection outcomes. The p11 protein serves as a regulatory node of phagosome maturation that is targeted by A. fumigatus, illustrating how genetic variations can impact critical host-pathogen interactions . Understanding these genetic factors could help identify high-risk patients and develop targeted preventive strategies.

How might the ndc80 protein complex contribute to antifungal resistance mechanisms?

While direct evidence on ndc80's role in antifungal resistance is limited, its function in chromosome segregation may indirectly contribute to genomic adaptability. Proper kinetochore assembly and function are essential for accurate chromosome distribution during cell division. Disruptions or modifications in kinetochore proteins could potentially affect recombination frequencies or chromosome stability, which might contribute to genetic variations that confer resistance. Additionally, the regulation of ndc80 expression under stress conditions (including antifungal exposure) remains an important area for investigation to understand if alterations in chromosome segregation machinery play roles in adaptation to antifungal therapies.

What techniques are most effective for studying protein-protein interactions between fungal pathogens and host cells?

Several techniques have proven effective for studying protein-protein interactions between fungal pathogens and host cells. In the case of A. fumigatus interactions with human cells, researchers successfully employed a targeted crosslinking approach coupled with affinity purification . This approach allowed them to identify interactions between the fungal HscA protein and human proteins. Immunofluorescence imaging was used to visualize the binding of fungal proteins to host cell surfaces, and western blot analysis identified candidate interacting proteins . To provide additional evidence for specific interactions, researchers used recombinant tagged proteins (such as N-terminal Twin-Strep-tagged recombinant HscA) and demonstrated their binding to human cells . Generation of fungal strains expressing tagged proteins (such as hscA-myc and hscA-gfp strains) also provided tools for visualizing and studying these interactions .

What are recommended methods for isolating and identifying Neosartorya species in clinical samples?

Isolation and identification of Neosartorya species from clinical samples require a combination of conventional and molecular approaches. For isolation, standard mycological media can be used, but it's crucial to be aware that Neosartorya species may produce white colonies that don't turn green like typical A. fumigatus . For definitive identification, microscopic examination of both asexual and sexual structures is important. The presence and characteristics of ascospores are particularly valuable for distinguishing between Neosartorya species - for example, N. hiratsukae has ascospores with two closely appressed equatorial crests, while N. pseudofischeri has ascospore walls with raised flaps resembling triangular projections . Molecular identification through DNA sequencing of appropriate genetic markers (like ITS, β-tubulin, or calmodulin genes) is recommended for confirmation, especially for unusual or rare isolates from clinical specimens.

How can researchers test the antifungal susceptibility of Neosartorya isolates?

Antifungal susceptibility testing of Neosartorya isolates can be performed using microdilution methods adapted from the reference method for molds recommended by standardizing bodies like the National Committee for Clinical Laboratory Standards . A specific protocol that has been successfully used involves RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid, an inoculum of approximately 9.1 × 10⁵ CFU/mL, incubation at 30°C, a second-day reading (48 h), and an additive drug-dilution procedure . This approach allows determination of both Minimum Inhibitory Concentrations (MICs) and Minimum Fungicidal Concentrations (MFCs). For N. hiratsukae, testing against amphotericin B, flucytosine, itraconazole, voriconazole, and experimental triazoles has been documented, with results showing good activity of azole derivatives while amphotericin B and flucytosine showed limited effectiveness .

What experimental models are most suitable for studying recombinant Neosartorya proteins?

For studying recombinant Neosartorya proteins, several experimental models can be employed. Expression systems such as E. coli, Pichia pastoris, or baculovirus-insect cell systems are commonly used for producing recombinant fungal proteins. For functional studies of recombinant proteins, cell culture models using relevant human cell lines such as A549 (alveolar epithelial cells), BEAS-2B (bronchial epithelial cells), or immune cells like macrophages and neutrophils provide valuable insights . When studying protein-protein interactions, techniques such as pull-down assays, co-immunoprecipitation, surface plasmon resonance, or yeast two-hybrid systems can be employed. For in vivo studies, mouse models of pulmonary or disseminated aspergillosis provide physiologically relevant systems to evaluate protein function in the context of infection.

What recent discoveries have been made about Aspergillus fumigatus interactions with host cells?

Recent research has revealed that A. fumigatus employs sophisticated strategies to manipulate host cell functions and evade immune responses. A significant discovery is that the fungal heat shock protein HscA interacts with human p11 (S100A10) to reprogram phagosome maturation . This interaction excludes the phagosome maturation mediator Rab7 and triggers binding of exocytosis mediators Rab11 and Sec15, redirecting phagosomes to a non-degradative pathway . This mechanism allows A. fumigatus to escape cells by outgrowth and expulsion, as well as transfer between cells . Researchers have also identified that HscA functions as an adhesin, contributing to the association of conidia with host cells and subsequent cellular damage . These findings provide new insights into the molecular mechanisms underlying A. fumigatus pathogenesis and potential targets for therapeutic intervention.

What is the current understanding of antifungal susceptibility patterns in Neosartorya species?

Current research on antifungal susceptibility of Neosartorya species indicates variable patterns depending on the specific species and antifungal agents. For N. hiratsukae, studies have shown that azole derivatives (itraconazole, voriconazole, and experimental triazoles like UR-9825) demonstrate good activity with relatively low MICs, while amphotericin B and flucytosine show limited effectiveness with very high MFCs . Specifically, for a clinical isolate of N. hiratsukae, the MICs and MFCs were: amphotericin B (1 and >16 μg/mL), flucytosine (64 and >64 μg/mL), itraconazole (0.25 and 0.25 μg/mL), voriconazole (0.25 and 0.5 μg/mL), and UR-9825 (0.06 and 0.5 μg/mL) . These in vitro findings correlated with clinical outcomes, as treatment with amphotericin B was ineffective while the patient responded well to itraconazole at daily doses of 400 mg . This highlights the importance of accurate species identification and susceptibility testing to guide therapeutic decisions.

How do Neosartorya species cause cerebral infections, and what are the clinical implications?

Neosartorya species can cause severe cerebral infections, particularly in immunocompromised individuals. The pathogenesis involves the ability of these fungi to withstand higher temperatures, allowing them to invade the brain . Many Neosartorya species, including N. hiratsukae, are thermotolerant and can grow at temperatures above 37°C, demonstrating their inherent ability to survive in the human body . In a documented case of cerebral aspergillosis caused by N. hiratsukae, the infection presented with progressive memory loss, confusion, involuntary movements, and gait disorders that initially led to a misdiagnosis of Parkinson's disease . Brain imaging revealed multifocal abscesses in both subtentorial and supratentorial regions . The infection was resistant to treatment with amphotericin B but responded to itraconazole therapy . This case highlights the importance of considering fungal etiologies in patients with progressive neurological symptoms, particularly those who are immunocompromised or unresponsive to standard therapies.

Table: Comparative Analysis of Antifungal Susceptibility Patterns in Clinical Neosartorya Isolates

This table summarizes the in vitro susceptibility testing results for a clinical isolate of N. hiratsukae, showing the superior activity of azole derivatives compared to amphotericin B and flucytosine. The clinical efficacy column reflects observations from treatment outcomes in a documented case .

What are the potential applications of understanding kinetochore protein function in pathogenic fungi?

Understanding the function of kinetochore proteins like ndc80 in pathogenic fungi opens several avenues for future research and applications. Kinetochore proteins are essential for proper chromosome segregation and cell division, making them potential targets for novel antifungal development. Research could focus on identifying structural or functional differences between fungal and human kinetochore components that could be exploited for selective inhibition. Additionally, understanding how environmental stresses or antifungal exposure affects kinetochore function might provide insights into mechanisms of adaptation and resistance. The essential nature of these proteins also makes them potential targets for gene silencing or CRISPR-based approaches to controlling fungal growth and virulence.