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.

SummaryCells 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 antiapoptotic 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.

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.The obligate intracellular protozoan Toxoplasma gondii has evolved an intimate relationship with its host that extends to the cellular and molecular levels (82, 83). The absolute requirement for invasion of an appropriate host cell in the replication of the parasite suggests that modification of host functions is central to pathogenesis. Intuitively, this subversion of the cell must be a complex process, since host cells are not inherently programmed to provide an environment conducive to pathogens. Host cells have evolved primary lines of defense as countermeasures to pathogen invasion, establishment, and replication. These include elaborate systems, such as the phagolysosomal fusion, reactive oxygen and nitrogen intermediates, the sequestration of nutrients, and even cell suicide (apoptosis), as defenses to limit pathogen growth. The implementation of these systems and the ability of successful pathogens to mitigate their effects are ultimately mediated by changes in both the levels and activities of key proteins. While some of these changes may be transcriptionally regulated and thus potentially revealed by microarray analysis, the true effectors are proteins,...

SummaryMedical implants are sometimes colonized by biofilm-forming bacteria that are very difficult to treat effectively. The combination of gentamicin and ultrasonic exposure for 24 h was previously shown to reduce the viability of E. coli biofilms in vivo. This article shows that such treatment for 48 h reduced viable E. coli bacteria to nearly undetectable levels. However, when P. aeruginosa biofilms were implanted and treated for 24 and 48 h, no significant ultrasonic-enhanced reduction of viable bacteria was observed. The difference in response of these two organisms is attributed to greater impermeability and stability of the outer membrane of P. aeruginosa.

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.