STAPHYLOMICS · Identifying host factors involved in staphylococcal infection

Identifying host factors involved in staphylococcal infection

SUMMARY DESCRIPTION OF THE PROJECT OBJECTIVES The overall aim of the project “Identifying host factors involved in staphylococcal infection” was to determine host factors involved in staphylococcal infection to increase understanding of host genes involved in susceptibility and resistance to staphylococcal infection. As suggested in previous research of the Fellow and work of others the parasitism of phagocytic host immune cells by Staphylococcus aureus is a key phase of the disease. Therefore, such infected phagocytes can act as reservoirs of disseminated infection. This has become particularly important as the recent rapid spread of antibiotic resistance reinforces the necessity to find alternative therapeutic strategies including host-targeted approaches to modulate the immune system and fight the infection. Within this fellowship project, we decided to combine the expertise of the Fellow on modelling S. aureus infection using embryonic/larval zebrafish that he has previously developed, with that of the host institution in application of transcriptomics and bioinformatics to zebrafish infectious disease models. The specific objectives of this study included defining the host transcriptional response to S. aureus infection in vivo by RNA-sequencing analysis of the two major types of phagocytes, neutrophils and macrophages, sorted from infected zebrafish larvae. Based on the sequencing results, a subset of candidate genes was selected for further functional analysis of their specific role in S. aureus pathogenesis using larval zebrafish. DESCRIPTION OF THE WORK PERFORMED The S. aureus-infected zebrafish larvae were enzymatically dissociated and resulting cell suspensions were subjected to fluorescence-activated cell sorting (FACS) to isolate pools of infected macrophages and neutrophils separately. Subsequently, the RNA was isolated from infected phagocytes, and RNA-sequencing using Illumina HiSeq was performed. The reads were then mapped and annotated against the zebrafish genome assembly. Statistical analysis of differentially expressed genes in infected phagocytes compared to their uninfected counterparts was performed using a number of available statistical packages. Following the statistical analysis, the lists of genes differentially expressed in infected macrophages and neutrophils have been compiled. Subsequently, a subset of candidate genes from the list was selected for further functional analysis of their specific role in S. aureus pathogenesis using gain and loss of function approaches. In addition, the role of the autophagic response to S. aureus was studied using larval zebrafish. DESCRIPTION OF THE MAIN RESULTS The results of RNA-sequencing of S. aureus-infected phagocytes indicated that S. aureus suppresses host inflammatory signaling. Therefore, the role of differentially expressed genes involved in balancing inflammation was further studied by manipulating their expression by either morpholino-mediated knockdown or overexpression. In addition, the top upregulated gene found was knocked out using CRISPR/Cas9 technology to validate the findings obtained by transient knockdown and also to provide a novel tool to the zebrafish scientific community. Two of the inflammation-associated genes identified in RNA-sequencing were found to play a role in S. aureus infection as manipulation of their expression resulted in altered survival and bacterial burden during infection. Additionally, it was shown that both phagocyte types mount the autophagic response upon internalisation of staphylococci and we identified genes important in this process. In future work, we aim to extend the analyses onto the role of other genes identified in the RNA-sequencing in the S. aureus infection process to gain better understanding of staphylococcal host-pathogen interaction. EXPECTED FINAL RESULTS AND THEIR POTENTIAL IMPACT AND USE In this project, supported by a European Marie-Curie postdoctoral fellowship, we successfully employed the larval zebrafish model of S. aureus infection to identify host genes involved in controlling staphylococcal growth during infection. During this fellowship, only a few most promising genes identified in the phagocyte-specific RNA-sequencing were tested for their role in S. aureus infection. However, the rest of the genes found in this study will be studied later, after the conclusion of this project, as the Fellow has already secured a new employment at the University of Sheffield. Development of new staphylococcal treatment strategies is especially urgent because virulent multi-drug resistant strains of S. aureus are rapidly emerging, and the spread of resistant staphylococci has significantly contributed to the importance of this organism, with significant associated morbidity and mortality, and rising public concern. Host-targeted treatments are now being considered for development of innovative therapeutic approaches that may overcome current limitations of antibiotic treatment. This underscores the importance to understand the key regulating host factors involved in staphylococcal infection and to identify potential targets for future drug development. Therefore, in the longer run a better understanding of the innate immune response to staphylococci could provide immunomodulatory strategies to enhance the host's immune response against this versatile pathogen. Web site links: https://www.universiteitleiden.nl/en/staffmembers/tomasz-prajsnar#tab-1 https://www.universiteitleiden.nl/en/research/research-projects/science/ibl-staphylomics-identifying-host-factors-involved-in-staphylococcal-infection

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