Dataset Information

Mildly pathogenic Rickettsia-macrophage interactions

ABSTRACT: The molecular details underlying differences in pathogenicity between Rickettsia species remain to be fully understood. Evidence points to macrophage permissiveness as a key mechanism in rickettsial virulence. Different studies have shown that several rickettsial species responsible for mild forms of rickettsioses can also escape macrophage-mediated killing mechanisms and establish a replicative niche within these cells. However, their manipulative capacity with respect to host cellular processes is far from being understood. A deeper understanding of the interplay between mildly pathogenic rickettsiae and macrophages and the commonalities and specificities of host responses to infection would illuminate differences in immune evasion mechanisms and pathogenicity. We have used quantitative proteomics by SWATH-MS/MS to profile alterations resulting from infection of THP-1 macrophages with three mildly pathogenic rickettsiae: R. parkeri, R. africae, and R. massiliae, all successfully proliferating in these cells. We show that all three species trigger different proteome signatures. Our results reveal a significant impact of infection in proteins categorized as type-I interferon responses, here included several components of the RIG-1-like signaling pathway, mRNA splicing, and protein translation. Moreover, significant differences in protein content between infection conditions anticipate species-specific induced alterations. Indeed, we confirm a distinct impact on host inflammatory responses between species during infection, demonstrating that these species trigger different levels of INF-β, differences in the bioavailability of the pro-inflammatory cytokine IL1-β, and in triggering pyroptotic events. This work reveals novel aspects and exciting nuances of macrophage-Rickettsia interactions, adding additional layers of complexity between Rickettsia and host cells constant arms race for survival.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Homo Sapiens (human) Rickettsia

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Cátia Santa

LAB HEAD: Bruno Manadas

PROVIDER: PXD026982 | Pride | 2022-01-11

REPOSITORIES: Pride

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Project description:We have previously reported that Rickettsia conorii and Rickettsia montanensis have distinct intracellular fates within THP-1 macrophages, suggesting that the ability to proliferate within macrophages may be a distinguishable factor between pathogenic and non-pathogenic Spotted fever group (SFG) members. To start unraveling the molecular mechanisms underlying the capacity (or not) of SFG Rickettsia to establish their replicative niche in macrophages, we have herein profiled the host proteomic alterations resulted by the infection of THP-1 macrophages with R. conorii and R. montanensis using a high throughput quantitative proteomics approach (SWATH-MS). Our results revealed that these two members of SFG Rickettsia with distinct pathogenicity attributes for humans, trigger differential proteomic signatures in macrophage-like cells. Although infection by both rickettsial species resulted in a lower abundance of enzymes of glycolysis and pentose phosphate pathway, the pathogenic R. conorii specifically induced the accumulation of several enzymes of the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid -oxidation and glutaminolysis, as well as of several inner and outer membrane mitochondrial transporters. These results suggest a profound metabolic rewriting of macrophages by R. conorii towards a metabolic signature of an M2-like (anti-inflammatory) activation program. Moreover, our results revealed that several subunits forming the proteasome and immunoproteasome are found in lower abundance upon infection with both rickettsial species, which may help bacteria to escape immune surveillance. Remarkably, R. conorii-infection specifically induced the accumulation of several host proteins implicated in protein processing and quality control in ER, suggesting that this pathogenic Rickettsia may be able to compensate the accumulation of misfolded proteins by increasing the ER protein folding capacity and subsequently restore host cell homeostasis. This work reveals novel aspects of macrophage-Rickettsia interactions, expanding our knowledge of how pathogenic rickettsiae explore host cells to their advantage.

Project description:Aspergillus fumigatus, a common airborne fungus, has been reported as one of the most frequent agents of human fungal infections. However, A. fumigatus conidia atmospheric resistance and longevity, and their aging mechanism are unknown. Therefore, the main purpose of this work is to clarify the main processes associated with conidial adaptation to nutrient limitation, similar to those found in their atmospheric transport. To that a time course evaluation of the changes in the proteome and in the ultrastructure of A. fumigatus’ conidia subjected to limiting nutrient conditions were performed, allowing a comprehensive characterization of conidia cells behavior and, revealing possible targets for the development of new antifungal medicines and improve infection control strategies. In the proposed work, a total of 712 proteins were identified and compared in the A. fumigatus conidia, with 387 proteins presenting significant differences among the considered conditions. These 387 proteins were then grouped into clusters according to similarities in their dynamic profile (relative levels) among the three points in time analyzed, and four completely distinct profiles can be observed. According with the proteomics changes, it was observed that the conidia go from a highly active metabolic state to a dormant state, and culminate in a cell autolysis state revealed by increased levels of hydrolytic enzymes. The proteomics alterations observed were further corroborate with the structural results, where it was observed changes in mitochondria, nucleus, and plasma membrane ultrastructure, consistent with an apoptotic process. Altogether the results indicate that the changes in protein levels anticipated those in cell morphology, clearly highlighting the need for further studies that may lead to significant improvement in infection control strategies. Additionally, a total of 28 proteins were identified with one acetylated peptide. Among them, it was possible to quantify the acetylation levels in 21 proteins with 11 presenting a statistical meaningful difference between the tested time points. Furthermore, from the proteins evaluated, all except the protein Histone H3, presented N-terminal acetylation. In the case of the Histone H3, the modification was observed at the lysine 56. In general, the proteins regulated by acetylation quantified in our study could be grouped into four distinct groups and although only some of the proteins have a statistical meaningful variation between the experimental time points, those proteins are spread along the different groups. Moreover, statistical variation if observed for all the proteins belonging to the group of proteins that have higher acetylation levels at time 15 minutes. In conclusion, this work presents a comprehensive characterization of the proteomics changes associated with the adaptive mechanism of Aspergillus fumigatus’ conidia to nutrient restriction, raveling some on the main pathways associated with such capacity. Furthermore, this proteomic characterization is associated with the conidia’s ultrastructural evaluation allowing to link the changes observed at the proteome level with the respective phenotype. Finally, it was also presented an evaluation of the levels of the protein acetylation, one of the principal regulatory PTMs in eukaryotes. Therefore, the present study may be an important dataset to be used as a tool to understand and identified potential targets associated with conidia resistance, and therefore improving infection control strategies.

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Mildly pathogenic Rickettsia-macrophage interactions

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