Identification of novel targets for host-directed therapeutics against intracellular Staphylococcus aureus

Natalia Bravo-Santano,Pablo Capilla-Lasheras,Yolanda Calle,Michal Letek,Volker Behrends,Luis M Mateos

doi:10.1038/s41598-019-51894-3

Natalia Bravo-Santano, Pablo Capilla-Lasheras + Show 4 more

Open Access

https://doi.org/10.1038/s41598-019-51894-3

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Journal: Scientific Reports	Publication Date: Oct 28, 2019
Citations: 9	License type: open-access

Affiliation: University of Exeter, University of Roehampton

Abstract

During patient colonization, Staphylococcus aureus is able to invade and proliferate within human cells to evade the immune system and last resort drugs such as vancomycin. Hijacking specific host molecular factors and/or pathways is necessary for pathogens to successfully establish an intracellular infection. In this study, we employed an unbiased shRNA screening coupled with ultra-fast sequencing to screen 16,000 human genes during S. aureus infection and we identified several host genes important for this intracellular pathogen. In addition, we interrogated our screening results to find novel host-targeted therapeutics against intracellular S. aureus. We found that silencing the human gene TRAM2 resulted in a significant reduction of intracellular bacterial load while host cell viability was restored, showing its importance during intracellular infection. Furthermore, TRAM2 is an interactive partner of the endoplasmic reticulum SERCA pumps and treatment with the SERCA-inhibitor Thapsigargin halted intracellular MRSA survival. Our results suggest that Thapsigargin could be repurposed to tackle S. aureus host cell infection in combination with conventional antibiotics.

Highlights

During patient colonization, Staphylococcus aureus is able to invade and proliferate within human cells to evade the immune system and last resort drugs such as vancomycin
We infected shRNA-HeLa cells with the clinically relevant S. aureus NCTC 13626 strain to investigate the effect of silencing specific genes on intracellular MRSA infection; vancomycin was added after one hour of incubation to kill extracellular bacteria[17], and uninfected shRNA-HeLa cells were used as negative control
By calculating the ratio between the occurrence of all shRNA constructs targeting the same gene found in S. aureus-infected cells versus the uninfected condition (Supplementary Table 1), we identified 5,674 shRNAs that were under-represented after S. aureus infection, whereas 9,997 shRNAs were over-represented when compared to uninfected cells (Supplementary Fig. 2)

Summary

Introduction

Staphylococcus aureus is able to invade and proliferate within human cells to evade the immune system and last resort drugs such as vancomycin. There are different strategies available to determine host factors hijacked by intracellular pathogens, combining genomics, computational biology, proteomics and transcriptomics[10]. These strategies could lead to the identification or development of new host-targeted treatments to combat intracellular infections. Loss-of-function phenotypic analysis, such as RNA interference (RNAi), is a common procedure to identify host genes or proteins that are necessary for the pathogen internalization, growth or survival within mammalian cells[11,12,13]. The availability of commercial shRNA libraries constitutes a powerful tool that allows large-scale loss-of-function genetic screenings in mammalian cells[14,16]

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