Sillanpää, A. | Search bioRxiv

bioRxiv2024

The inflammasome adaptor pycard is essential for immunity against Mycobacterium marinum infection in adult zebrafish

Inflammasome regulates the host response to intracellular pathogens including mycobacteria. We have previously shown that the course of Mycobacterium marinum infection in adult zebrafish (Danio rerio) has similar features than the course of tuberculosis in human. In this study, we have investigated the role of the inflammasome adaptor pycard in M. marinum infection in zebrafish. We produced two zebrafish knock-out mutant lines for the pycard gene with CRISPR/Cas9 mutagenesis. While the zebrafish larvae devoid of pycard develop normally and have unaltered resistance against M. marinum, the loss of pycard led to impaired survival and increased bacterial burden in the adult zebrafish. Based on histological analysis, immune cell aggregates, granulomas, were larger in pycard deficient fish compared to wild type controls. Transcriptome analysis with RNA sequencing of a zebrafish haematopoietic tissue, kidney, suggests a role for pycard in neutrophil mediated defence as well as in haematopoiesis and myelopoiesis during infection. Transcriptome analysis of fluorescently labelled kidney neutrophils further supported the importance of pycard for neutrophil-mediated immunity against M. marinum. Genes associated with neutrophil degranulation, haematopoiesis and PI3K signalling were differentially expressed in the pycard deficient neutrophils when compared to wild type controls. All in all, our results indicate that pycard is essential for resistance against mycobacteria in adult zebrafish. Based on transcriptional profiling of pycard mutants, we postulate that pycard mutant phenotype is mediated in part via defects in neutrophil function including neutrophil degranulation. Author summaryInflammasome is a multiprotein complex which is a part of immune response especially against intracellular microbes. Tuberculosis, caused by Mycobacterium tuberculosis, is a global health problem causing 1.3 million deaths yearly. A natural fish pathogen, Mycobacterium marinum, causes a systemic infection in adult zebrafish resembling human tuberculosis including granuloma formation and an asymptomatic, latent state. Here, we have used zebrafish as a model to investigate the role of an inflammasome regulator pycard in resistance against M. marinum infection in zebrafish. We show that while pycard is dispensable for normal immunity against mycobacterial infection in larval stages, it has a central role in the immune defense against M. marinum in adult zebrafish. Based on our results, we suggest that this relates to blood cell formation, haematopoiesis and especially to the function of neutrophils. By analysing transcriptrional profile of pycard deficient neutrophils upon Mycobacterial challenge, it seems that pycard has a role in PI3K signalling and neutrophil degranulation. Our results indicate that pycard is essential for normal resistance against M. Marinum in adult zebrafish, and that it regulates neutrophil function during Mycobacterial infection.

immunology↗

bioRxiv2024

Aerobic Adaptation and Metabolic Dynamics of Propionibacterium freudenreichii DSM 20271: Insights from Comparative Transcriptomics and Surfaceome Analysis

Propionibacterium freudenreichii (PFR) DSM 20271 is a bacterium known for its ability to thrive in diverse environments and to produce vitamin B12. Despite its anaerobic preference, recent studies have elucidated its ability to prosper in the presence of oxygen, prompting a deeper exploration of its physiology under aerobic conditions. Here, we investigated the response of DSM 20271 to aerobic growth by employing comparative transcriptomic and surfaceome analyses alongside metabolite profiling. Cultivation under controlled partial pressure of oxygen (pO2) conditions revealed significant increases in biomass formation and altered metabolite production, notably of B12 vitamin, pseudovitamin-B12, propionate and acetate, under aerobic conditions. Transcriptomic analysis identified differential expression of genes involved in lactate metabolism, TCA cycle, and electron transport chain, suggesting metabolic adjustments to aerobic environments. Moreover, surfaceome analysis unveiled growth environment-dependent changes in surface protein abundance, with implications for sensing and adaptation to atmospheric conditions. Supplementation experiments with key compounds highlighted the potential for enhancing aerobic growth, emphasizing the importance of iron and -ketoglutarate availability. Furthermore, in liquid culture, FeSO4 supplementation led to increased heme production and reduced vitamin B12 production, highlighting the impact of oxygen and iron availability on the metabolic pathways. These findings deepen our understanding of PFRs physiological responses to oxygen availability and offer insights for optimizing its growth in industrial applications. ImportanceThe study of the response of Propionibacterium freudenreichii to aerobic growth is crucial for understanding how this bacterium adapts to different environments and produces essential compounds like vitamin B12. By investigating its physiological changes under aerobic conditions, we can gain insights into its metabolic adjustments and potential for enhanced growth. These findings not only deepen our understanding of P. freudenreichii responses to oxygen availability but also offer valuable information for optimizing its growth in industrial applications. This research sheds light on the adaptive mechanisms of this bacterium, providing a foundation for further exploration and potential applications in various fields.

microbiology↗