Human plasma-like medium (HPLM) induces Cryptococcus neoformans in vivo cell morphologies.

The fungal pathogen Cryptococcus neoformans (C. neoformans) forms yeast cells of different sizes and morphological characteristics during infection. These features are usually not seen in standard laboratory in vitro conditions. Here, we describe in vivo cell morphologies when C. neoformans is grown in human plasma-like medium at 37°C, 5% CO2. We observed mixed-size populations of cells less than 1 µm up to 16.8 µm in cell diameter, increased capsule size, high chitin, and DNA content in larger cells. Our findings show that serum is not required for human plasma-like medium (HPLM)-induced C. neoformans cellular heterogeneity. Thus, this new method offers an opportunity to investigate factors of C. neoformans that mediate pathogenesis or host-pathogen interactions in a physiologically relevant setting.IMPORTANCEWe provide a description of new in vitro culture condition using the human plasma-like medium that supports the formation of the full range of in vivo cell morphologies of C. neoformans.

In Vitro Titan Cell Generation in Cryptococcus neoformans and Automated Cell Size Measurements.

A special feature of the human fungal pathogen Cryptococcus neoformans is its morphological changes triggered by the interaction with the host. During infection, a specific increase in cell size is observed, particularly in lung tissue, from a typical cell size of 5-7 µm cells to cells larger than 10 µm, dubbed titan cells (TCs). However, the study of this specific cell subpopulation was, until now, only possible via recovery of TCs from lungs of mice during experimental infections where stable and reproducible generation of TCs occurs.The protocol described here generates TCs using in vitro conditions and measures cell size using a rapid, automated method. TC generation in vitro is robust and reproducible, generating yeast cells harboring the same characteristics of TCs generated in vivo.

C1q Confers Protection Against Cryptococcal Lung Infection by Alleviating Inflammation and Reducing Cryptococcal Virulence.

Background: To define the role of C1qa in host defense against Cryptococcus neoformans lung infection, we investigated its susceptibility to cryptococcal lung infection in mice deficient in complement factor C1qa (C1qa-/- ). Methods: We established a wild-type (WT) and C1qa-deficient murine inhalation model with C. neoformans. We compared the host survival rate, inflammatory responses, and pathogenicity of C. neoformans during the infection course between WT and C1qa-/- mice. Results: The mortality rate of C1qa-deficient mice was significantly higher than that of wild-type mice. The increased formation of Titan cells in the lungs was associated with augmented inflammation in C1qa-deficient mice. The capacity of lung homogenate supernatant from C1qa-deficient mice to induce Titan formation in vitro was greater compared with that of wild-type mice. The C. neoformans isolated from the lungs of infected C1qa-deficient mice was more resistant to macrophage killing in vitro and caused significantly higher mortality after administration to mice compared with that isolated from WT mice. Conclusions: These findings reveal a novel role of C1qa in host defense against C. neoformans infection by regulating host inflammation and pathogen virulence and provide new insight into the C1q-mediated lung environment underlying the transition from yeast to Titan cell.

Intraerythrocytic Mycoplasma-like organism diagnosed ultrastructurally as an agent of anaemia in laboratory-reared cyprinid hybrids.

A study targeting the etiology of severe anaemia that sporadically occurred in laboratory-bred cyprinid hybrids resulted in a diagnosis of a Mycoplasma-like organism selectively invading the cytoplasm of erythrocytes. Despite the fact that there was a concurrent yeast infection in moribund anaemic hybrids, the primary role in the development of anaemia was assigned to the Mycoplasma-like organism due to its regular occurrence in erythrocytes of both the moribund hybrids and hybrids that were free of yeast infection yet showed early to advanced symptoms of the disease. Novel data on the Mycoplasma-like organism's cytoskeleton were obtained from ultrathin sections of affected erythrocytes. An ultrastructural study of the concurrent yeast infection in moribund hybrids manifesting the most advanced anaemia revealed the presence of Titan cells in ascitic fluid. The original findings presented in this study underline the diagnostic relevance of transmission electron microscopy in the research of similar infections.

Establishing Minimal Conditions Sufficient for the Development of Titan-like Cells in Cryptococcus neoformans/gattii Species Complex.

Opportunistic pathogens of the anamorphic genus Cryptococcus are unique considering their virulence factors that in the context of pathogenesis allowed them to achieve evolutionary success. Morphological transformation into giant (Titan) cells is one of the factors contributing to cryptococcosis. Recently established in vitro protocols demonstrate that 5 or 10% fetal bovine serum (FBS) combined with 5% CO2, 37 °C, and sufficiently low cell density, triggers cellular enlargement (Serum protocols). However, the FBS components that promote this morphological transition remain incompletely characterized. In search of minimal conditions necessary for stimulating the formation of Titan cells, we performed a study where we eliminated serum from the protocol (Serum-free protocol) and instead systematically adjusted the amount of glucose, source of nitrogen (ammonium sulfate), and the pH. We found that exposing cells to PBS with adjusted pH to 7.3, and supplemented with 0.05% glucose, 0.025% ammonium sulfate, 0.004% K2HPO4, 0.0035% MgSO4, in the presence of 5% CO2 at 37 °C triggers Titan-like cell formation to the same degree as the previously established protocol that utilized 10% FBS as the sole nutrient source. Titan-like cells obtained according to this Serum-free protocol were characterized by cell body size over ten microns, a single enlarged vacuole, thick cell wall, extensive polysaccharide capsule, and changes in the level of cell ploidy, all currently known hallmarks of Titan cells found in vivo. Strikingly, we found that in both, Serum and Serum-free protocols, an optimal pH for Titan-like cell development is ~7.3 whereas relatively acidic pH (5.5) prevents this morphological transition and promotes robust proliferation, while alkaline pH (~8.0) has a profound growth inhibitory effect. Our study demonstrates a critical role of pH response to the formation of Titan cells and indicates that conditions that allow restricted proliferation in the presence of 5% CO2 are sufficient for this morphological transition to form enlarged cells in Cryptococcus neoformans and Cryptococcus gattii species complex.

Role of IL-17 in Morphogenesis and Dissemination of Cryptococcus neoformans during Murine Infection.

Cryptococcus neoformans is a pathogenic yeast that can form Titan cells in the lungs, which are fungal cells of abnormally large size. The factors that regulate Titan cell formation in vivo are still unknown, although an increased proportion of these fungal cells of infected mice correlates with induction of Th2-type responses. Here, we focused on the role played by the cytokine IL-17 in the formation of cryptococcal Titan cells using Il17a-/- knockout mice. We found that after 9 days of infection, there was a lower proportion of Titan cells in Il17a-/- mice compared to the fungal cells found in wild-type animals. Dissemination to the brain occurred earlier in Il17a-/- mice, which correlated with the lower proportion of Titan cells in the lungs. Furthermore, knockout-infected mice increased brain size more than WT mice. We also determined the profile of cytokines accumulated in the brain, and we found significant differences between both mouse strains. We found that in Il17a-/-, there was a modest increase in the concentrations of the Th1 cytokine TNF-α. To validate if the increase in this cytokine had any role in cryptococcal morphogenesis, we injected wild-type mice with TNF-α t and observed that fungal cell size was significantly reduced in mice treated with this cytokine. Our results suggest a compensatory production of cytokines in Il17a-/- mice that influences both cryptococcal morphology and dissemination.

Too much of a good thing: Overproduction of virulence factors impairs cryptococcal pathogenicity.

The regulation of virulence factor production and deployment is crucial for the establishment of microbial infection and subsequent pathogenesis. If these processes are not properly coordinated, the infecting pathogen is less likely to both survive the immune response and cause damage to the host. One key virulence factor of the opportunistic fungal pathogen Cryptococcus neoformans, which kills almost 200,000 people each year worldwide, is a polysaccharide capsule that surrounds the cell wall; this structure helps the fungal cells resist engulfment and elimination by host phagocytes. Another important virulence trait is the development of a giant (Titan) cell morphotype that increases fungal resistance to phagocytosis, oxidative stress, and antifungal treatment. We recently identified the transcription factor Pdr802 as essential for C. neoformans adaptation to and survival under host conditions both in vitro and in vivo (Reuwsaat et al., mBio, doi: 10.1128/mBio.03457-20). Cryptococci lacking Pdr802 display enlarged capsules and enhanced Titan cell production, along with dramatically reduced virulence in a mouse model of infection. These results demonstrate that more is not necessarily better when it comes to virulence factors. Instead, precise regulation of these traits, to avoid both under- and overexpression, is critical for the success of this pathogen as it faces the challenges imposed by the host environment.

The Transcription Factor Pdr802 Regulates Titan Cell Formation and Pathogenicity of Cryptococcus neoformans.

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills almost 200,000 people worldwide each year. It is acquired when mammalian hosts inhale the infectious propagules; these are deposited in the lung and, in the context of immunocompromise, may disseminate to the brain and cause lethal meningoencephalitis. Once inside the host, C. neoformans undergoes a variety of adaptive processes, including secretion of virulence factors, expansion of a polysaccharide capsule that impedes phagocytosis, and the production of giant (Titan) cells. The transcription factor Pdr802 is one regulator of these responses to the host environment. Expression of the corresponding gene is highly induced under host-like conditions in vitro and is critical for C. neoformans dissemination and virulence in a mouse model of infection. Direct targets of Pdr802 include the quorum sensing proteins Pqp1, Opt1, and Liv3; the transcription factors Stb4, Zfc3, and Bzp4, which regulate cryptococcal brain infectivity and capsule thickness; the calcineurin targets Had1 and Crz1, important for cell wall remodeling and C. neoformans virulence; and additional genes related to resistance to host temperature and oxidative stress, and to urease activity. Notably, cryptococci engineered to lack Pdr802 showed a dramatic increase in Titan cells, which are not phagocytosed and have diminished ability to directly cross biological barriers. This explains the limited dissemination of pdr802 mutant cells to the central nervous system and the consequently reduced virulence of this strain. The role of Pdr802 as a negative regulator of Titan cell formation is thus critical for cryptococcal pathogenicity.IMPORTANCE The pathogenic yeast Cryptococcus neoformans presents a worldwide threat to human health, especially in the context of immunocompromise, and current antifungal therapy is hindered by cost, limited availability, and inadequate efficacy. After the infectious particle is inhaled, C. neoformans initiates a complex transcriptional program that integrates cellular responses and enables adaptation to the host lung environment. Here, we describe the role of the transcription factor Pdr802 in the response to host conditions and its impact on C. neoformans virulence. We identified direct targets of Pdr802 and also discovered that it regulates cellular features that influence movement of this pathogen from the lung to the brain, where it causes fatal disease. These findings significantly advance our understanding of a serious disease.

Human IgM Inhibits the Formation of Titan-Like Cells in Cryptococcus neoformans.

Human studies have shown associations between cryptococcal meningitis and reduced IgM memory B cell levels, and studies in IgM- and/or B cell-deficient mice have demonstrated increased Cryptococcus neoformans dissemination from lungs to brain. Since immunoglobulins are part of the immune milieu that C. neoformans confronts in a human host, and its ability to form titan cells is an important virulence mechanism, we determined the effect of human immunoglobulins on C. neoformans titan cell formation in vitro (i) Fluorescence microscopy showed normal human IgG and IgM bind C. neoformans (ii) C. neoformans grown in titan cell-inducing medium with IgM, not IgG, inhibited titan-like cell formation. (iii) Absorption of IgM with laminarin or curdlan (branched and linear 1-3-beta-d-glucans, respectively) decreased this effect. (iv) Transmission electron microscopy revealed that cells grown with IgM had small capsules and unique features not seen with cells grown with IgG. (v) Comparative transcriptional analysis of cell wall, capsule, and stress response genes showed that C. neoformans grown with IgM, not IgG or phosphate-buffered saline (PBS), had decreased expression of chitin synthetase, CHS1, CHS2, and CHS8, and genes encoding cell wall carbohydrate synthetases α-1-3-glucan (AGS1) and ß-1,3-glucan (FKS1). IgM also decreased expression of RIM101 and HOG1, genes encoding central regulators of C. neoformans stress response pathways and cell morphogenesis. Our data show human IgM affects C. neoformans morphology in vitro and suggest that the hypothesis that human immunoglobulins may affect C. neoformans virulence in vivo warrants further investigation.

The cAMP/Protein Kinase a Pathway Regulates Virulence and Adaptation to Host Conditions in Cryptococcus neoformans.

Nutrient sensing is critical for adaptation of fungi to environmental and host conditions. The conserved cAMP/PKA signaling pathway contributes to adaptation by sensing the availability of key nutrients such as glucose and directing changes in gene expression and metabolism. Interestingly, the cAMP/PKA pathway in fungal pathogens also influences the expression of virulence determinants in response to nutritional and host signals. For instance, protein kinase A (PKA) in the human pathogen Cryptococcus neoformans plays a central role in orchestrating phenotypic changes, such as capsule elaboration and melanin production, that directly impact disease development. In this review, we focus first on insights into the role of the cAMP/PKA pathway in nutrient sensing for the model yeast Saccharomyces cerevisiae to provide a foundation for understanding the pathway in C. neoformans. We then discuss key features of cAMP/PKA signaling in C. neoformans including new insights emerging from the analysis of transcriptional and proteomic changes in strains with altered PKA activity and expression. Finally, we highlight recent studies that connect the cAMP/PKA pathway to cell surface remodeling and the formation of titan cells.

Titan cell production in Cryptococcus neoformans reshapes the cell wall and capsule composition during infection.

Cryptococcus neoformans is a human fungal pathogen that often causes infections in immunocompromised individuals. Upon inhalation into the lungs C. neoformans differentiates into cells with altered size and morphology, including production of large titan cells. Titan cells possess thickened cell wall and dense, cross-linked capsule when compared to in vitro grown cells. In addition, titan cells have increased cell wall chitin that is associated with a detrimental anti-inflammatory immune response. Here we examined the cell wall and capsule composition of in vitro, in vivo typical-sized and in vivo titan cells using High Performance Liquid Chromatography (HPLC). The monomer composition of cell wall polysaccharides showed that in vivo C. neoformans cells contained more glucosamine and less glucose than in vitro cells, suggesting alteration in abundance of both chitin and glucans, respectively. Low levels of galactosamine were also detected in carbohydrates from both in vivo and vitro cells. Within the in vivo cell population, differences in the proportions of cell wall and capsule monomers between typical and titan cells were also observed. Taken together, these results demonstrate that C. neoformans reshapes its cell wall and capsule composition during infection. These cell wall and capsule alterations likely help C. neoformans escape recognition by, and allow modulation of, the host immune system.

The formation of titan cells in Cryptococcus neoformans depends on the mouse strain and correlates with induction of Th2-type responses.

Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN-γ, TNF-α and IL17, while C57BL/BL mice had an increase in the anti-inflammatory cytokine IL-4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2-type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.

Clinicopathological features of pulmonary cryptococcosis with cryptococcal titan cells: a comparative analysis of 27 cases.

In addition to the typical size, Cryptococcus neoformans can enlarge its size to form titan cells during infection, and its diameter can reach up to 100 µm. Clinical reports about cryptococcal titan cells are rare. Most studies focus on aspects of animal models of infection with titan cells. Herein, we report the clinical and imaging characteristics and histopathologic features of 3 patients with titan cells and 27 patients with pathogens of typical size, and describe the morphological characteristics of titan cells in details. Histologically, 3 patients with titan cells show necrosis, fibrosis and macrophage accumulation. The titan cells appear in necrotic tissue and between macrophages, and have thick wall with unstained halo around them and diameters range from 20 to 80 µm with characteristic of narrow-necked single budding. There are also organisms with typical size. All 27 patients with normal pathogens show epithelioid granulomatous lesions. There is no significantly difference in clinical and imaging feature between the two groups. Cryptococcus neoformans exhibits a striking morphological change for the formation of titan cells during pulmonary infection, which will result in misdiagnosis and under diagnosis. The histopathological changes may be new manifestation, which need to be further confirmed by the study with animal models of infection and the observation of more clinical cases. Careful observation of the tissue sections is necessary.