Intracellular Pathogens: Infection, Immunity, and Intervention.

Intracellular pathogens comprise a diverse group of pathogens that all share a required location in a host cell to infect, survive, and replicate. Intracellular location allows pathogens to hide from host immune responses, avoid competition with other pathogens, mediate host cellular functions, replicate safely, and cause infection that is difficult to target with therapeutics. All intracellular pathogens have varying routes of infiltration into host cells and different host cell preferences. For example, bacteria Mycobacterium tuberculosis chooses to invade antigen-presenting cells, which allows them to moderate host antigen presentation to memory cells, whereas rabies virus prefers to invade neurons because they have pre-existing innate immunity protection systems. Regardless of the pathway that each intracellular pathogen follows, all share the capacity to cause disease if they succeed in entering host cells. Here, we give an overview of selected intracellular pathogens and infections they cause, immune responses they induce, and intervention strategies used to treat and control them.

A food color-based colorimetric assay for Cryptococcus neoformans laccase activity.

Cryptococcus neoformans is a fungal pathogen that causes cryptococcosis primarily in immunocompromised patients, such as those with HIV/AIDS. One survival mechanism of C. neoformans during infection is melanin production, which catalyzed by laccase and protects fungal cells against immune attack. Hence, the comparative assessment of laccase activity is useful for characterizing cryptococcal strains. We serendipitously observed that culturing C. neoformans with food coloring resulted in degradation of some dyes with phenolic structures. Consequently, we investigated the color changes for the food dyes metabolized by C. neoformans laccase and by using this effect explored the development of a colorimetric assay to measure laccase activity. We developed several versions of a food dye-based colorimetric laccase assay that can be used to compare the relative laccase activities between different C. neoformans strains. We found that phenolic color degradation was glucose-dependent, which may reflect changes in the reduction properties of the media. Our food color-based colorimetric assay has several advantages, including lower cost, irreversibility, and not requiring constant monitoring , over the commonly used 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for determining laccase activity. This method has potential applications to bioremediation of water pollutants in addition to its use in determining laccase virulence factor expression.IMPORTANCECryptococcus neoformans is present in the environment, and while infection is common, disease occurs mostly in immunocompromised individuals. C. neoformans infection in the lungs results in symptoms like pneumonia, and consequently, cryptococcal meningitis occurs if the fungal infection spreads to the brain. The laccase enzyme catalyzes the melanization reaction that serves as a virulence factor for C. neoformans. Developing a simple and less costly assay to determine the laccase activity in C. neoformans strains can be useful for a variety of procedures ranging from studying the relative virulence of cryptococci to environmental pollution studies.

Phenotypic characterization of HAM1, a novel mating regulator of the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen responsible for >200,000 yearly cases with a mortality as high as 81%. This burden results, in part, from an incomplete understanding of its pathogenesis and ineffective antifungal treatments; hence, there is a pressing need to understand the biology and host interactions of this yeast to develop improved treatments. Protein palmitoylation is important for cryptococcal virulence, and we previously identified the substrates of its main palmitoyl transferase. One of them was encoded by the uncharacterized gene CNAG_02129. In the filamentous fungus Neurospora crassa, a homolog of this gene named hyphal anastomosis protein 13 plays a role in proper cellular communication and filament fusion. In Cryptococcus, cellular communication is essential during mating; therefore, we hypothesized that CNAG_02129, which we named hyphal anastomosis protein 1 (HAM1), may play a role in mating. We found that ham1Δ mutants produce more fusion products during mating, filament more robustly, and exhibit competitive fitness defects under mating and non-mating conditions. Additionally, we found several differences with the major virulence factor, the polysaccharide capsule, that may affect virulence, consistent with prior studies linking virulence to mating. We observed that ham1Δ mutants have decreased capsule attachment and transfer but exhibit higher amounts of exopolysaccharide shedding and biofilm production. Finally, HAM1 expression is significantly lower in mating media relative to non-mating conditions, consistent with it acting as a negative regulator of mating. Understanding the connection between mating and virulence in C. neoformans may open new avenues of investigation into ways to improve the treatment of this disease. IMPORTANCE: Fungal mating is a vital part of the lifecycle of the pathogenic yeast Cryptococcus neoformans. More than just ensuring the propagation of the species, mating allows for sexual reproduction to occur and generates genetic diversity as well as infectious propagules that can invade mammalian hosts. Despite its importance in the biology of this pathogen, we still do not know all of the major players regulating the mating process and if they are involved or impact its pathogenesis. Here, we identified a novel negative regulator of mating that also affects certain cellular characteristics known to be important for virulence. This gene, which we call HAM1, is widely conserved across the cryptococcal family as well as in many pathogenic fungal species. This study will open new avenues of exploration regarding the function of uncharacterized but conserved genes in a variety of pathogenic fungal species and specifically in serotype A of C. neoformans.

Host and fungal factors both contribute to cryptococcosis-associated hyperammonemia (cryptammonia).

Cryptococcus neoformans and Cryptococcus gattii are both known urease producers and have the potential to cause hyperammonemia. We hypothesized that the risk of hyperammonemia is increased by renal failure, burden of cryptococcal infection, and fungal strain characteristics. We performed a retrospective review of plasma ammonia levels in patients with cryptococcal infections. Risk factors for hyperammonemia were statistically compared between patients with and without hyperammonemia (>53 µmol/L). Cryptococcal cells from three patients included in the study were recovered from our biorepository. Strain characteristics including urease activity, ammonia production, growth curves, microscopy, melanin production, and M13 molecular typing were analyzed and compared with a wild-type (WT) C. neoformans strain. We included 29 patients, of whom 37.9% had hyperammonemia, 59% had disseminated cryptococcal infection (DCI), and 41% had isolated central nervous system infection. Thirty-eight percent of patients had renal failure and 28% had liver disease. Renal failure was associated with 4.4 times (95% confidence interval [CI] 1.5, 13.0) higher risk of hyperammonemia. This risk was higher in DCIs (RR 6.2, 95% CI 1.0, 40.2) versus isolated cryptococcal meningitis (RR 2.5, 95% CI, 0.40, 16.0). Liver disease and cryptococcal titers were not associated with hyperammonemia. C. neoformans from one patient with extreme hyperammonemia demonstrated a 4- to 5-fold increase in extracellular urease activity, slow growth, enlarged cell size phenotypes, and diminished virulence factors. Hyperammonemia was strongly associated with renal failure in individuals with DCI, surpassing associations with liver failure or cryptococcal titers. However, profound hyperammonemia in one patient was attributable to high levels of urease secretion unique to that cryptococcal strain. Prospective studies are crucial to exploring the significance of this association.IMPORTANCECryptococcus produces and secretes the urease enzyme to facilitate its colonization of the host. Urease breaks down urea into ammonia, overwhelming the liver's detoxification process and leading to hyperammonemia in some hosts. This underrecognized complication exacerbates organ dysfunction alongside the infection. Our study investigated this intricate relationship, uncovering a strong association between the development of hyperammonemia and renal failure in patients with cryptococcal infections, particularly those with disseminated infections. We also explore mechanisms underlying increased urease activity, specifically in strains associated with extreme hyperammonemia. Our discoveries provide a foundation for advancing research into cryptococcal metabolism and identifying therapeutic targets to enhance patient outcomes.

Cryptococcal Pleuritis in an Immunocompetent Patient: A Case Report and Literature Review.

Cryptococcosis, primarily an opportunistic infection, often occurs in immunocompromised patients but can also affect immunocompetent individuals. Cryptococcosis typically manifests in the lungs, but pleurisy is rare, particularly in immunocompetent patients. This report details a case of cryptococcal pleuritis in a 74-year-old immunocompetent male with a history of heart failure, presenting initially with pleural effusion. Diagnostic challenges arose due to the initial absence of intrapulmonary lesions. The diagnosis was eventually established through a surgical biopsy and tissue culture, revealing Cryptococcus neoformans. This case underscores the complexity of diagnosing cryptococcal infections, particularly in immunocompetent patients, and highlights the need for considering cryptococcosis in differential diagnoses of lymphocyte-predominant exudative pleural effusions.

Unraveling the mechanism of thermotolerance by Set302 in Cryptococcus neoformans.

The underlying mechanism of thermotolerance, which is a key virulence factor essential for pathogenic fungi such as Cryptococcus neoformans, is largely unexplored. In this study, our findings suggest that Set302, a homolog of Set3 and a subunit of histone deacetylase complex Set3C, contributes to thermotolerance in C. neoformans. Specifically, the deletion of the predicted Set3C core subunit, Set302, resulted in further reduction in the growth of C. neoformans at 39°C, and survival of transient incubation at 50°C. Transcriptomics analysis revealed that the expression levels of numerous heat stress-responsive genes altered at both 30°C and 39°C due to the lack of Set302. Notably, at 39°C, the absence of Set302 led to the downregulation of gene expression related to the ubiquitin-proteasome system (UPS). Based on the GFP-α-synuclein overexpression model to characterize misfolded proteins, we observed a pronounced accumulation of misfolded GFP-α-synuclein at 39°C, consequently inhibiting C. neoformans thermotolerance. Furthermore, the loss of Set302 exacerbated the accumulation of misfolded GFP-α-synuclein during heat stress. Interestingly, the set302∆ strain exhibited a similar phenotype under proteasome stress as it did at 39°C. Moreover, the absence of Set302 led to reduced production of capsule and melanin. set302∆ strain also displayed significantly reduced pathogenicity and colonization ability compared to the wild-type strain in the murine infection model. Collectively, our findings suggest that Set302 modulates thermotolerance by affecting the degradation of misfolded proteins and multiple virulence factors to mediate the pathogenicity of C. neoformans.IMPORTANCECryptococcus neoformans is a pathogenic fungus that poses a potential and significant threat to public health. Thermotolerance plays a crucial role in the wide distribution in natural environments and host colonization of this fungus. Herein, Set302, a critical core subunit for the integrity of histone deacetylase complex Set3C and widely distributed in various fungi and mammals, governs thermotolerance and affects survival at extreme temperatures as well as the formation of capsule and melanin in C. neoformans. Additionally, Set302 participates in regulating the expression of multiple genes associated with the ubiquitin-proteasome system (UPS). By eliminating misfolded proteins under heat stress, Set302 significantly contributes to the thermotolerance of C. neoformans. Moreover, Set302 regulates the pathogenicity and colonization ability of C. neoformans in a murine model. Overall, this study provides new insight into the mechanism of thermotolerance in C. neoformans.

Inbred Mouse Models in Cryptococcus neoformans Research.

Animal models are frequently used as surrogates to understand human disease. In the fungal pathogen Cryptococcus species complex, several variations of a mouse model of disease were developed that recapitulate different aspects of human disease. These mouse models have been implemented using various inbred and outbred mouse backgrounds, many of which have genetic differences that can influence host response and disease outcome. In this review, we will discuss the most commonly used inbred mouse backgrounds in C. neoformans infection models.

Card9 Broadly Regulates Host Immunity against Experimental Pulmonary Cryptococcus neoformans 52D Infection.

The ubiquitous soil-associated fungus Cryptococcus neoformans causes pneumonia that may progress to fatal meningitis. Recognition of fungal cell walls by C-type lectin receptors (CLRs) has been shown to trigger the host immune response. Caspase recruitment domain-containing protein 9 (Card9) is an intracellular adaptor that is downstream of several CLRs. Experimental studies have implicated Card9 in host resistance against C. neoformans; however, the mechanisms that are associated with susceptibility to progressive infection are not well defined. To further characterize the role of Card9 in cryptococcal infection, Card9em1Sq mutant mice that lack exon 2 of the Card9 gene on the Balb/c genetic background were created using CRISPR-Cas9 genome editing technology and intratracheally infected with C. neoformans 52D. Card9em1Sq mice had significantly higher lung and brain fungal burdens and shorter survival after C. neoformans 52D infection. Susceptibility of Card9em1Sq mice was associated with lower pulmonary cytokine and chemokine production, as well as reduced numbers of CD4+ lymphocytes, neutrophils, monocytes, and dendritic cells in the lungs. Histological analysis and intracellular cytokine staining of CD4+ T cells demonstrated a Th2 pattern of immunity in Card9em1Sq mice. These findings demonstrate that Card9 broadly regulates the host inflammatory and immune response to experimental pulmonary infection with a moderately virulent strain of C. neoformans.

Orthopedic Manifestations of Disseminated Cryptococcal neoformans Infection in an Immunocompromised Patient: a Case Report.

Introduction: Cryptococccus neoformans is a fungus which typically presents in immunocompromised hosts, commonly presenting as meningoencephalitis. There have been very few documented incidents of intramuscular manifestations of this pathogen. Case Report: We report on a case of a 45-year-old caucasian male with disseminated Cryptococcus neoformans who developed cryptococcal intramuscular abscesses of all extremities and osteomyelitis of the left upper limb. Clinical treatment and surgical debridement of the forearm was performed. Persistent infection resulted in a left humeral amputation and ultimately the patient's death. This is one of the few documented intramuscular abscesses of Cryptococcus neoformans. Conclusion: Orthopedic manifestations of cryptococcal infections are rare; however, awareness and prompt diagnosis may improve outcomes.

Brilacidin, a novel antifungal agent against Cryptococcus neoformans.

Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

Exploring a Rare Pulmonary Coinfection: Cryptococcal Pneumonia and Exophiala dermatitidis in an Immunocompetent Host.

Pulmonary cryptococcosis is becoming increasingly common in immunocompetent hosts, manifesting with variable clinical presentations ranging from asymptomatic colonization to severe pneumonia. Radiological findings are non-specific, such as nodular infiltrates, mass-like lesions, and mediastinal lymphadenopathy. We present a case of a 61-year-old woman with Cryptococcus neoformans pneumonia coinfected with Exophiala dermatitidis, an unusual occurrence in an immunocompetent host and the first of its kind. This coinfection posed significant diagnostic challenges due to the rare occurrence of each individual organism in immunocompetent patients as well as the difficulty of their laboratory diagnosis. Treatment regimens, particularly in coinfections, warrant careful consideration to mitigate mortality risk. This case underscores the importance of comprehensive diagnostic strategies and optimized treatment regimens for rare fungal coinfections in immunocompetent hosts.

Sirtulin-Ypk1 regulation axis governs the TOR signaling pathway and fungal pathogenicity in Cryptococcus neoformans.

Cryptococcus neoformans is a life-threatening fungal pathogen that is a causative agent for pulmonary infection and meningoencephalitis in both immunocompetent and immunodeficient individuals. Recent studies have elucidated the important function of the target of rapamycin (TOR) signaling pathway in the modulation of C. neoformans virulence factor production and pathogenicity in animal infection models. Herein, we discovered that Ypk1, a critical component of the TOR signaling pathway, acts as a critical modulator in fungal pathogenicity through post-translational modifications (PTMs). Mass spectrometry analysis revealed that Ypk1 is subject to protein acetylation at lysines 315 and 502, and both sites are located within kinase functional domains. Inhibition of the C. neoformans TOR pathway by rapamycin activates the deacetylation process for Ypk1. The YPK1Q strain, a hyper-acetylation of Ypk1, exhibited increased sensitivity to rapamycin, decreased capsule formation ability, reduced starvation tolerance, and diminished fungal pathogenicity, indicating that deacetylation of Ypk1 is crucial for responding to stress. Deacetylase inhibition assays have shown that sirtuin family proteins are critical to the Ypk1 deacetylation mechanism. After screening deacetylase mutants, we found that Dac1 and Dac7 directly interact with Ypk1 to facilitate the deacetylation modification process via a protein-protein interaction. These findings provide new insights into the molecular basis for regulating the TORC-Ypk1 axis and demonstrate an important function of protein acetylation in modulating fungal pathogenicity. IMPORTANCE: Cryptococcus neoformans is an important opportunistic fungal pathogen in humans. While there are currently few effective antifungal treatments, the absence of novel molecular targets in fungal pathogenicity hinders the development of new drugs. There is increasing evidence that protein post-translational modifications (PTMs) can modulate the pathogenicity of fungi. In this study, we discovered that the pathogenicity of C. neoformans was significantly impacted by the dynamic acetylation changes of Ypk1, the immediate downstream target of the TOR complex. We discovered that Ypk1 is acetylated at lysines 315 and 502, both of which are within kinase functional domains. Deacetylation of Ypk1 is necessary for formation of the capsule structure, the response to the TOR pathway inhibitor rapamycin, nutrient utilization, and host infection. We also demonstrate that the sirtuin protein family is involved in the Ypk1 deacetylation mechanism. We anticipate that the sirtuin-Ypk1 regulation axis could be used as a potential target for the development of antifungal medications.

Identification of a putative α-galactoside ß-(1 → 3)-galactosyltransferase involved in the biosynthesis of galactomannan side chain of glucuronoxylomannogalactan in Cryptococcus neoformans.

The cell surface of Cryptococcus neoformans is covered by a thick capsular polysaccharide. The capsule is the most important virulence factor of C. neoformans; however, the complete mechanism of its biosynthesis is unknown. The capsule is composed of glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). As GXM is the most abundant component of the capsule, many studies have focused on GXM biosynthesis. However, although GXMGal has an important role in virulence, studies on its biosynthesis are scarce. Herein, we have identified a GT31 family ß-(1 → 3)-galactosyltransferase Ggt2, which is involved in the biosynthesis of the galactomannan side chain of GXMGal. Comparative analysis of GXMGal produced by a ggt2 disruption strain revealed that Ggt2 is a glycosyltransferase that catalyzes the initial reaction in the synthesis of the galactomannan side chain of GXMGal. The ggt2 disruption strain showed a temperature-sensitive phenotype at 37°C, indicating that the galactomannan side chain of GXMGal is important for high-temperature stress tolerance in C. neoformans. Our findings provide insights into complex capsule biosynthesis in C. neoformans.

Blap-6, a Novel Antifungal Peptide from the Chinese Medicinal Beetle Blaps rhynchopetera against Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans) is a pathogenic fungus that can cause life-threatening meningitis, particularly in individuals with compromised immune systems. The current standard treatment involves the combination of amphotericin B and azole drugs, but this regimen often leads to inevitable toxicity in patients. Therefore, there is an urgent need to develop new antifungal drugs with improved safety profiles. We screened antimicrobial peptides from the hemolymph transcriptome of Blaps rhynchopetera (B. rhynchopetera), a folk Chinese medicine. We found an antimicrobial peptide named blap-6 that exhibited potent activity against bacteria and fungi. Blap-6 is composed of 17 amino acids (KRCRFRIYRWGFPRRRF), and it has excellent antifungal activity against C. neoformans, with a minimum inhibitory concentration (MIC) of 0.81 µM. Blap-6 exhibits strong antifungal kinetic characteristics. Mechanistic studies revealed that blap-6 exerts its antifungal activity by penetrating and disrupting the integrity of the fungal cell membrane. In addition to its direct antifungal effect, blap-6 showed strong biofilm inhibition and scavenging activity. Notably, the peptide exhibited low hemolytic and cytotoxicity to human cells and may be a potential candidate antimicrobial drug for fungal infection caused by C. neoformans.

In Vivo Modeling of Cryptococcus neoformans Infection and Collection of Murine Samples.

In vivo models provide advantages to study the progression of disease and to identify potential biomarkers to detect and monitor infections. For the human fungal pathogen Cryptococcus neoformans, murine intranasal models aim to recapitulate natural infection from inhalation of desiccated fungal cells from the environment and permit monitoring of disease over time. In this chapter, we describe the establishment of a murine model for cryptococcosis and the subsequent collection of organs, tissues, and fluids for sampling. These samples may support novel diagnostic strategies and opportunities to monitor dissemination of the fungal cells throughout the host and propose new treatment options to combat disease.

Biolistic Transformation of Cryptococcus neoformans.

Biolistic transformation of Cryptococcus neoformans is used as a molecular tool to genetically alter or delete targeted genes. The DNA is introduced into the yeast on DNA-coated gold beads by a helium shock wave produced using a biolistic particle system. The procedure often involves insertion of a dominant selectable marker into the desired site by homologous recombination. To increase the likelihood of homologous recombination, large fragments of overlapping DNA are used. The two most used dominant selectable markers are nourseothricin and Geneticin. With the need to generate multiple gene deletions in the same strain, there are recyclable marker systems, such as the bacteriophage P1 Cre-loxP system or CRISPR that provide additional useful molecular tools. While newer strategies exist to generate deletions and introduce markers and other gene modifications, biolistic transformation has remained a viable tool to facilitate the construction of genetically modified yeast strains. This chapter provides a working protocol on how to delete and restore a gene in C. neoformans.

Models for Inducing Experimental Cryptococcosis in Mice.

Cryptococcus neoformans and Cryptococcus gattii are the predominant etiological agents of cryptococcosis, a particularly problematic disease in immunocompromised individuals. The increased clinical use of immunosuppressive drugs, the inherent ability of Cryptococcus species to suppress and evade host immune responses, and the emergence of drug-resistant yeast support the need for model systems that facilitate the design of novel immunotherapies and antifungals to combat disease progression. The mouse model of cryptococcosis is a widely used system to study Cryptococcus pathogenesis and the efficacy of antifungal drugs in vivo. In this chapter, we describe three commonly used strategies to establish cryptococcosis in mice: intranasal, intratracheal, and intravenous inoculations. Also, we discuss the methodology for delivering drugs to mice via intraperitoneal injection.

Electron Microscopy of Cryptococcus neoformans: Processing Challenges to Avoid Artifacts.

This chapter describes methodological details for preparing specimens of Cryptococcus neoformans (although it can be applied to any species of the genus) and their subsequent analysis by scanning and transmission electron microscopy. Adaptations to conventional protocols for better preservation of the sample, as well as to avoid artifacts, are presented. The protocols may be used to examine both the surface ultrastructure and the interior of this pathogenic fungus in detail.

Assessing Phagocytosis of Cryptococcus neoformans Cells in Human Monocytes or the J774 Murine Macrophage Cell Line.

Monocyte/macrophage cells play a central role in innate immunity against C. neoformans and C. gattii, species known to cause human disease. Cryptococcus is the only fungal genus known to possess such a large extracellular polysaccharide capsule, which impacts interactions of innate cells with the yeast. This interaction results in different fates, such as phagocytosis and intracellular proliferation and, as the interaction progresses, vomocytosis, cell-to-cell transfer, lysis of macrophages, or yeast killing. Differentiating internalized versus external Cryptococcus cells is thus essential to evaluate monocyte-macrophage phagocytosis. We describe here a protocol that allows quantification of Cryptococcus spp. phagocytosis using quantitative flow cytometry in human monocytes and a murine macrophage cell line (J774).