An In Vitro Cell Culture Model for Pyoverdine-Mediated Virulence

Abstract

Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that utilizes a wide-range of virulence factors to cause acute, life-threatening infections in immunocompromised patients, especially those in intensive care units. It also causes debilitating chronic infections that shorten lives and worsen the quality of life for cystic fibrosis patients. One of the key virulence factors in P. aeruginosa is the siderophore pyoverdine, which provides the pathogen with iron during infection, regulates the production of secreted toxins, and disrupts host iron and mitochondrial homeostasis. These roles have been characterized in model organisms such as Caenorhabditis elegans and mice. However, an intermediary system, using cell culture to investigate the activity of this siderophore has been absent. In this report, we describe such a system, using murine macrophages treated with pyoverdine. We demonstrate that pyoverdine-rich filtrates from P. aeruginosa exhibit substantial cytotoxicity, and that the inhibition of pyoverdine production (genetic or chemical) is sufficient to mitigate virulence. Furthermore, consistent with previous observations made in C. elegans, pyoverdine translocates into cells and disrupts host mitochondrial homeostasis. Most importantly, we observe a strong correlation between pyoverdine production and virulence in P. aeruginosa clinical isolates, confirming pyoverdine’s value as a promising target for therapeutic intervention. This in vitro cell culture model will allow rapid validation of pyoverdine antivirulents in a simple but physiologically relevant manner.

Box C/D small nucleolar ribonucleoproteins regulate mitochondrial surveillance and innate immunity

“…In the Liquid Killing assay, host death occurs due to the production of the siderophore pyoverdine, which is secreted by the bacterium to obtain iron [ 26 , 58 ]. Pyoverdine enters host tissue and removes iron from mitochondria, causing sufficient damage to inflict death [ 59 , 60 ]. This damage also activates the ESRE mitochondrial surveillance network, which is important for host defense [ 20 ].…”

Development and Characterization of High-Throughput Caenorhabditis elegans – Enterococcus faecium Infection Model

“…In the case of P. aeruginosa , growth on agar (or in the host) leads to high bacterial density and key involvement of quorum-sensing pathways ( Feinbaum et al., 2012 ). In liquid, growth is limited, quorum sensing has no role, and production of the siderophore pyoverdine drives a lethal host intoxication ( Kirienko et al., 2015 ; Kang et al., 2018 ; Kang and Kirienko, 2020 ).…”

Unconventional Animal Models in Infectious Disease Research

“…In the case of P. aeruginosa, growth on agar (or in the host) leads to high bacterial density and key involvement of quorumsensing pathways (Feinbaum et al, 2012). In liquid, growth is limited, quorum sensing has no role, and production of the siderophore pyoverdine drives a lethal host intoxication (Kirienko et al, 2015;Kang et al, 2018;Kang and Kirienko, 2020). A recently published analysis of the role of Candida albicans adhesins in virulence using a liquid-based C. elegans model (Rosiana et al, 2021) reveals a third possibility.…”