John C. Carmen

The Differential Effect of Toxoplasma Gondii Infection on the Stability of BCL2-Family Members Involves Multiple Activities

The regulation of mitochondrial permeability, a key event in the initiation of apoptosis is governed by the opposing actions of the pro- and anti-apoptotic members of the BCL2-family of proteins. The BCL2-family can be classified further based on the number of BCL-homology (BH) domains they encode. Pathogen mediated modulation of BCL2-family members play a significant role in their ability to affect the apoptotic pathways in the infected host cell. The protozoan parasite Toxoplasma gondii establishes a profound blockade of apoptosis noted by a requirement for host NFκB activity and correlating with the selective degradation of pro-apoptotic BCL2-family members. In this study, we explore the potential activities associated with the inherent stability of the anti-apoptotic BCL2 as well as the selective degradation of the pro-apoptotic proteins BAX, BAD, and BID. We find that multiple activities govern the relative stability of BCL2-family members suggesting a complex and balanced network of stability-enhancing and–destabilizing activities are perturbed by parasite infection. The data leave open the possibility for both parasite induced host activities as well as the direct consequence of parasite effectors in governing the relative levels of BCL2-proteins in the course of infection.

Modulation of the Host Cell Proteome by the Intracellular Apicomplexan Parasite Toxoplasma gondii

ABSTRACT To investigate how intracellular parasites manipulate their host cell environment at the molecular level, we undertook a quantitative proteomic study of cells following infection with the apicomplexan parasite Toxoplasma gondii. Using conventional two-dimensional electrophoresis, difference gel electrophoresis (DIGE), and mass spectrometry, we identified host proteins that were consistently modulated in expression following infection. We detected modification of protein expression in key metabolic pathways, including glycolysis, lipid and sterol metabolism, mitosis, apoptosis, and structural-protein expression, suggestive of global reprogramming of cell metabolism by the parasite. Many of the differentially expressed proteins had not been previously implicated in the response to the parasite, while others provide important corroborative protein evidence for previously proposed hypotheses of pathogen-cell interactions. Significantly, over one-third of all modulated proteins were mitochondrial, and this was further investigated by DIGE analysis of a mitochondrion-enriched preparation from infected cells. Comparison of our proteomic data with previous transcriptional studies suggested that a complex relationship exits between transcription and protein expression that may be partly explained by posttranslational modifications of proteins and revealed the importance of investigating protein changes when interpreting transcriptional data. To investigate this further, we used phosphatase treatment and DIGE to demonstrate changes in the phosphorylation states of several key proteins following infection. Overall, our findings indicate that the host cell proteome responds in a dramatic way to T. gondii invasion, in terms of both protein expression changes and protein modifications, and reveal a complex and intimate molecular relationship between host and parasite.

Suicide prevention: disruption of apoptotic pathways by protozoan parasites

The modulation of apoptosis has emerged as an important weapon in the pathogenic arsenal of multiple intracellular protozoan parasites. Cryptosporidium parvum, Leishmania spp., Trypanosoma cruzi, Theileria spp., Toxoplasma gondii and Plasmodium spp. have all been shown to inhibit the apoptotic response of their host cell. While the pathogen mediators responsible for this modulation are unknown, the parasites are interacting with multiple apoptotic regulatory systems to render their host cell refractory to apoptosis during critical phases of intracellular infection, including parasite invasion, establishment and replication. Additionally, emerging evidence suggests that the parasite life cycle stage impacts the modulation of apoptosis and possibly parasite differentiation. Dissection of the host–pathogen interactions involved in modulating apoptosis reveals a dynamic and complex interaction that recent studies are beginning to unravel.

Toxoplasma gondii inhibits ultraviolet light-induced apoptosis through multiple interactions with the mitochondrion-dependent programmed cell death pathway.

Cells infected with the protozoan parasite Toxoplasma gondii are resistant to diverse apoptotic stimuli. In this study, we perform a detailed analysis of the manipulation of the mitochondrial arm of the apoptotic cascade by the parasite. Apoptosis was induced using irradiation with ultraviolet light (UV), and the kinetics of caspase activation, cytochrome c release and activation of the upstream signalling pathways were examined. The evidence clearly points to T. gondii targeting multiple steps in the transmission [inhibition of c‐Jun N‐terminal kinase (JNK) activation in response to UV], triggering (inhibition of cytochrome c release by affecting the balance of pro‐ and anti‐apoptotic BCL‐2 family members) and execution (inhibition of caspase 9 and caspase 3) phases of the apoptotic cascade. Interestingly, the multilevel pattern of inhibition that emerges suggests that the global inhibition of the mitochondrial arm of apoptosis is not likely to be contributed to by the small subset of mitochondria recruited to the T. gondii parasitophorous vacuole membrane.

Trypanosoma cruzi Infection and Nuclear Factor Kappa B Activation Prevent Apoptosis in Cardiac Cells

ABSTRACT Studies of cardiac pathology and heart failure have implicated cardiomyocyte apoptosis as a critical determinant of disease. Recent evidence indicates that the intracellular protozoan parasite Trypanosoma cruzi, which causes heart disease in chronically infected individuals, impinges on host apoptotic pathways in a cell type-dependent manner. T. cruzi infection of isolated neuronal cells and cardiomyocytes protects against apoptotic cell death, whereas apoptosis is triggered in T cells in T. cruzi-infected animals. In this study, we demonstrate that the ability of T. cruzi to protect cardiac cells in vitro from apoptosis triggered by a combination of tumor necrosis factor alpha and serum reduction correlates with the presence of intracellular parasites and involves activation of host cell NF-κB. We further demonstrate that the apoptotic block diminishes activation of caspase 3. The ability of T. cruzi to prevent apoptosis of infected cardiomyocytes is likely to play an important role in establishment of persistent infection in the heart while minimizing potential damage and remodeling that is associated with cardiomyocyte apoptosis in cardiovascular disease.

The dynamic genome and transcriptome of the human fungal pathogen Blastomyces and close relative Emmonsia

Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, Blastomyces, and two species of the related genus Emmonsia, typically pathogens of small mammals. Compared to related species, Blastomyces genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for gypsy elements, are variable in total size between isolates, and are least expanded in the avirulent B. dermatitidis strain ER-3 as compared with the virulent B. gilchristii strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the Blastomyces lineage. While gene content is highly conserved between Blastomyces and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of B. dermatitidis during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in Blastomyces, and upregulation of dioxygenases in vivo suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively in vivo, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in in vitro studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of Blastomyces to its host environment. These results reveal the dynamics of genome evolution and of factors contributing to virulence in Blastomyces.

Blastomyces dermatitidis yeast cells inhibit nitric oxide production by alveolar macrophage inducible nitric oxide synthase.

ABSTRACT The ability of pathogens to evade host antimicrobial mechanisms is crucial to their virulence. The dimorphic fungal pathogen Blastomyces dermatitidis can infect immunocompetent patients, producing a primary pulmonary infection that can later disseminate to other organs. B. dermatitidis possesses a remarkable ability to resist killing by alveolar macrophages. To date, no mechanism to explain this resistance has been described. Here, we focus on macrophage production of the toxic molecule nitric oxide as a potential target of subversion by B. dermatitidis yeast cells. We report that B. dermatitidis yeast cells reduce nitric oxide levels in the supernatants of activated alveolar macrophages. This reduction is not due to detoxification of nitric oxide, but rather to suppression of macrophage nitric oxide production. We show that B. dermatitidis yeast cells do not block upregulation of macrophage inducible nitric oxide synthase (iNOS) expression or limit iNOS access to its arginine substrate. Instead, B. dermatitidis yeast cells appear to inhibit iNOS enzymatic activity. Further investigation into the genetic basis of this potential virulence mechanism could lead to the identification of novel antifungal drug targets.

Isolation of Blastomyces dermatitidis yeast from lung tissue during murine infection for in vivo transcriptional profiling

Blastomyces dermatitidis belongs to a group of thermally dimorphic fungi that grow as sporulating mold in the soil and convert to pathogenic yeast in the lung following inhalation of spores. Knowledge about the molecular events important for fungal adaptation and survival in the host remains limited. The development of high-throughput analytic tools such as RNA sequencing (RNA-Seq) has potential to provide novel insight on fungal pathogenesis especially if applied in vivo during infection. However, in vivo transcriptional profiling is hindered by the low abundance of fungal cells relative to mammalian tissue and difficulty in isolating fungal cells from the tissues they infect. For the purpose of obtaining B. dermatitidis RNA for in vivo transcriptional analysis by RNA-Seq, we developed a simple technique for isolating yeast from murine lung tissue. Using a two-step approach of filtration and centrifugation following lysis of murine lung cells, 91% of yeast cells causing infection were isolated from lung tissue. B. dermatitidis recovered from the lung yielded high-quality RNA with minimal murine contamination and was suitable for RNA-Seq. Approximately 87% of the sequencing reads obtained from the recovered yeast aligned with the B. dermatitidis genome. This was similar to 93% alignment for yeast grown in vitro. The use of near-freezing temperature along with short ex vivo time minimized transcriptional changes that would have otherwise occurred with higher temperature or longer processing time. In conclusion, we have developed a technique that recovers the majority of yeast causing pulmonary infection and yields high-quality fungal RNA with minimal contamination by mammalian RNA.

The complexity of signaling in host–pathogen interactions revealed by the Toxoplasma gondii-dependent modulation of JNK phosphorylation

The inhibition of apoptosis by Toxoplasma gondii is governed by its modulation of several signaling cascades including the NFkappaappaB and JNK pathways. This is evident in the dysregulation of JNK activation following treatment with UV and TNFalpha, both apoptogenic stimuli. Infection-mediated interference with the JNK cascade was found to be highly reproducible in HeLa cells. In light of emerging evidence regarding cross talk between the JNK and NFkappaB cascades, we examined the impact of infection in wild type and RelA/p65-/- mouse embryonic fibroblasts (MEF). Remarkably, parasite infection failed to significantly impact both UV and TNFalpha-mediated JNK phosphorylation in both cell lines suggesting a cell type specific effect. Furthermore siRNA-mediated knockdown of RelA/p65 failed to impact the parasite mediated effects on stimulus dependent activation of JNK in HeLa cells. Finally, the infection mediated suppression of JNK phosphorylation in HeLa cells did not result in decreased JNK kinase activity. Rather, the reduced levels of phospho-JNK in infected cells correlated with increased phosphatase activity noted by the partial rescue of the phenotype following treatment with okadaic acid. Taken together the results indicate that manipulation of the JNK pathway does not involve NFkappaB and is furthermore not a central component of the parasite enforced block of apoptosis. It further highlights the complexity of these systems and the danger of extrapolating results both within and across pathogen-host cell systems based on limited studies.