Acanthamoeba castellanii as a Screening Tool for Mycobacterium avium Subspecies paratuberculosis Virulence Factors with Relevance in Macrophage Infection

Abstract

The high prevalence of Johne’s disease has driven a continuous effort to more readily understand the pathogenesis of the etiological causative bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), and to develop effective preventative measures for infection spread. In this study, we aimed to create an in vivo MAP infection model employing an environmental protozoan host and used it as a tool for selection of bacterial virulence determinants potentially contributing to MAP survival in mammalian host macrophages. We utilized Acanthamoeba castellanii (amoeba) to explore metabolic consequences of the MAP-host interaction and established a correlation between metabolic changes of this phagocytic host and MAP virulence. Using the library of gene knockout mutants, we identified MAP clones that can either enhance or inhibit amoeba metabolism and we discovered that, for most part, it mirrors the pattern of MAP attenuation or survival during infection of macrophages. It was found that MAP mutants that induced an increase in amoeba metabolism were defective in intracellular growth in macrophages. However, MAP clones that exhibited low metabolic alteration in amoeba were able to survive at a greater rate within mammalian cells, highlighting importance of both category of genes in bacterial pathogenesis. Sequencing of MAP mutants has identified several virulence factors previously shown to have a biological relevance in mycobacterial survival and intracellular growth in phagocytic cells. In addition, we uncovered new genetic determinants potentially contributing to MAP pathogenicity. Results of this study support the use of the amoeba model system as a quick initial screening tool for selection of virulence factors of extremely slow-grower MAP that is challenging to study.

Characterization of Rosculus vilicus sp. nov., a rhizarian amoeba interacting with Mycobacterium avium subsp. paratuberculosis

“…Few studies have related MAP association with amoebae. Among them, it was shown that incubation of MAP with Acanthamoeba polyphaga and Acanthamoeba castellanii, led to a decreasing MAP number in the first few post-internalization days, but increased after an extended period of several weeks (Mura et al, 2006;Whan et al, 2006;Phillips et al, 2020). Our results provide evidence that MAP can also persist for at least for 3 days in association with R. vilicus.…”

Modelling dynamics between free‐living amoebae and bacteria

“…FLA have many innate immunity mechanisms to defend against intracellular microbial infections and facilitate the consumption of these microbes, such as phagosome maturation, lysosomal degradation, acidification, and reactive oxygen species production (Phillips et al, 2020). Furthermore, previous studies have highlighted the similarities between amoebae and macrophages (Al-Antary, 2021; Lertjuthaporn et al, 2022).…”

“…Amoebae may act as catalysts of genetic modification, allowing MAP to develop phenotypes different from those expressed in the culture medium (Partridge, 2005). These modified MAP phenotypes alter antibiotic resistance and virulence (Phillips et al, 2020).…”

“…Species interactions play an important role in driving ecological processes, a perspective that harks back to the earliest studies of ecology (Janzen, 1966; Odum, 1964; Pimm, 1984), including in the case of infectious disease dynamics (Johnson et al, 2015; Tylianakis et al, 2008). Among these interactions, many complex relationships between bacteria and free‐living amoebae (FLA) have only been elucidated relatively recently, despite their widespread existence and significant influence in several host–pathogen systems (Balczun & Scheid, 2017; Gu et al, 2022; Phillips et al, 2020; Rodríguez‐Zaragoza, 1994; Shi et al, 2021). Interactions between FLA and bacteria vary along a continuum from classical predator–prey dynamics, to symbiosis, to host–pathogen dynamics (Balczun & Scheid, 2017; Escoll et al, 2013; Fritsche et al, 1993; Horn & Wagner, 2004; Long et al, 2018; Mirabedini et al, 2022; Phillips et al, 2020; Rodríguez‐Zaragoza, 1994; Samba‐Louaka et al, 2018; Samba‐Louaka et al, 2019; Schmitz‐Esser et al, 2008).…”

Biological characteristics and pathogenicity of Acanthamoeba

“…A. castellanii can also host Legionella ( Swart et al, 2018 ; Nomura et al, 2022 ), Vibrio cholerae , Francisella tularensis ( Abd et al, 2003 ) and the causative agent of Johne’s disease, Mycobacterium avium subsp. paratuberculosis (MAP) ( Phillips et al, 2020 ). These bacteria have been referred to as amoeba-resistant bacteria.…”