Project description:Control of intracellular heme levels by extracellular scavenger proteins and intracellular heme oxygenases are essential functions during disease states with enhanced extracellular heme release. During severe hemolysis or rhabdomyolysis uncontrolled heme exposure can cause acute kidney injury and endothelial damage. The cytotoxic activity of heme has been primarily attributed to its pro-oxidative potential. However, the mechanisms of heme toxicity have never been systematically explored. Besides its redox reactivity, heme could also adversely alter cellular functions through its broad binding affinity to multiple non-hemoproteins. Such interactions may impair protein functions and support heme toxicity. In this study we mapped the gene expression profile of Hb triggered acute kidney injury in old blood transfused guinea pigs by serial analysis of gene expression (SAGE). Additionally, the toxic heme response of mouse embryo fibroblasts was systematically characterized on the gene and protein expression levels by gene array experiments and quantitative mass-spectrometry of stable isotope labeled cells. In all these studies, in addition to oxidative stress signals, the most significant signals were reproducibly found for biologic networks related to altered protein degradation, which ultimately triggers the response to unfolded proteins and apoptosis. These screening data could be mechanistically explained by heme-proteasome interactions and a proteasome inhibitor activity of heme. Proteasome inhibition drastically reduced the threshold of cellular toxicity during heme exposure. We therefore propose a novel model of heme toxicity whereby proteasome inhibition by the porphyrin fuels a vicious cycle of oxidative protein modification, accumulation of damaged proteins, cell damage and apoptosis. A two color common reference design was chosen with 2-4 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled). To compensate for dye bias control arrays with competitively hybridized Cy3- and Cy5-labeled non-treated reference samples were used. The latter allowed for a very robust statistical analysis with pair-wise comparison of treatment array replicates versus the corresponding control array replicates.

Project description:Control of intracellular heme levels by extracellular scavenger proteins and intracellular heme oxygenases are essential functions during disease states with enhanced extracellular heme release. During severe hemolysis or rhabdomyolysis uncontrolled heme exposure can cause acute kidney injury and endothelial damage. The cytotoxic activity of heme has been primarily attributed to its pro-oxidative potential. However, the mechanisms of heme toxicity have never been systematically explored. Besides its redox reactivity, heme could also adversely alter cellular functions through its broad binding affinity to multiple non-hemoproteins. Such interactions may impair protein functions and support heme toxicity. In this study we mapped the gene expression profile of Hb triggered acute kidney injury in old blood transfused guinea pigs by serial analysis of gene expression (SAGE). Additionally, the toxic heme response of mouse embryo fibroblasts was systematically characterized on the gene and protein expression levels by gene array experiments and quantitative mass-spectrometry of stable isotope labeled cells. In all these studies, in addition to oxidative stress signals, the most significant signals were reproducibly found for biologic networks related to altered protein degradation, which ultimately triggers the response to unfolded proteins and apoptosis. These screening data could be mechanistically explained by heme-proteasome interactions and a proteasome inhibitor activity of heme. Proteasome inhibition drastically reduced the threshold of cellular toxicity during heme exposure. We therefore propose a novel model of heme toxicity whereby proteasome inhibition by the porphyrin fuels a vicious cycle of oxidative protein modification, accumulation of damaged proteins, cell damage and apoptosis. A two color common reference design was chosen with 2-8 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled). To compensate for dye bias control arrays with competitively hybridized Cy3- and Cy5-labeled non-treated reference samples were used. The latter allowed for a very robust statistical analysis with pair-wise comparison of treatment array replicates versus the corresponding control array replicates.

Project description:Mosquitoes of the genus Aedes are primary vectors of arboviruses such as dengue, Zika, and chikungunya1. Their ability to transmit pathogens is intrinsically linked to their hematophagous behavior, which presents metabolic challenges, particularly during blood digestion2,3. One major challenge is the oxidative stress generated by heme, the prosthetic group of hemoglobin, which catalyzes the formation of reactive oxygen species (ROS)4. Excess heme can lead to cellular damage through lipid peroxidation and protein oxidation, necessitating specialized detoxification mechanisms in mosquitoes5,6.7. During blood digestion, heme concentrations in the mosquito midgut can exceed 10 mM, creating a highly oxidative environment that selects for resistant microbial species8. The midgut of A. aegypti hosts a diverse microbiota that plays a vital role in digestion, nutrient acquisition, and immune regulation9,10. Among these microbial inhabitants, bacteria of the genus Serratia are of particular interest due to their dominance in this oxidative environment11. As a Gram-negative, facultatively anaerobic bacterium, S. plymuthica exhibits resistance to oxidative stress, suggesting that it may play a role in heme homeostasis and redox balance within the mosquito midgut. Genomic analyses reveal that S. plymuthica encodes robust oxidative stress-response systems, including superoxide dismutase (sodA), catalases (katA/G), and glutathione S-transferases, which neutralize ROS and mitigate heme toxicity12. While mosquitoes have evolved antioxidant mechanisms, such as catalase induction, to mitigate heme toxicity, midgut bacteria like S. plymuthica may synergistically contribute to redox homeostasis. For instance, heme-peroxidases in mosquitoes regulate bacterial populations by modulating ROS levels and mucin crosslinking, creating a low-immunity zone that supports microbiota proliferation13. Conversely, Serratia species can internalize heme or produce antioxidant molecules, indirectly protecting the mosquito from oxidative damage while enhancing their own survival12,14,15. Such interactions are critical for maintaining microbial homeostasis and influencing vector competence, as dysbiosis in the midgut microbiota alters Plasmodium and arbovirus infections. Understanding the physiological and molecular adaptations of S. plymuthica to heme-induced oxidative stress is crucial for unraveling its survival strategies and potential role in mosquito-microbiota interactions. The midgut of A. aegypti presents a highly dynamic and oxidative environment, particularly following a blood meal, where bacterial symbionts must withstand heme toxicity while maintaining their metabolic functions12,13. We hypothesize that S. plymuthica plays an active role in mitigating oxidative stress in the mosquito gut, which may impact microbial homeostasis and vector competence. In this study, we assess the growth and oxidative stress tolerance of S. plymuthica in culture media supplemented with varying concentrations of iron and heme, coupled with a comparative proteomic analysis to identify differentially expressed proteins and metabolic pathways. By highlighting stress-response proteins, such as universal stress proteins and ABC transporters, we elucidate the adaptive mechanisms that enable S. plymuthica to persist in the mosquito midgut. These findings not only expand our understanding of mosquito-microbiota interactions but also offer new perspectives on vector competence and oxidative homeostasis, with potential implications for novel vector control strategies and biotechnological applications.

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