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

Ida L Phillips,Lia Danelishvili,Jamie L Everman,Luiz E Bermudez

doi:10.3390/microorganisms8101571

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.

Highlights

Mycobacterium avium subsp. paratuberculosis (MAP) is an etiologic agent of a chronic and highly contagious granulomatous enteritis known as Johne’s disease
In order to find a solution for control of MAP infection and develop effective treatment, management, and diagnostic strategies, it is essential to understand molecular mechanisms of MAP pathogenesis
Inoculum was prepared in phosphate buffered saline (PBS), passed through a 22-gauge needle, and suspension was allowed to settle for 10 min

Summary

Introduction

Mycobacterium avium subsp. paratuberculosis (MAP) is an etiologic agent of a chronic and highly contagious granulomatous enteritis known as Johne’s disease. Paratuberculosis (MAP) is an etiologic agent of a chronic and highly contagious granulomatous enteritis known as Johne’s disease. The MAP infection primarily effects the domestic and wild ruminants worldwide. The infection generally occurs early in neonatal calves that do not develop disease for several years and later serve as a carrier for MAP infection among healthy herds. In the United States, roughly 68–91% of dairy herds are infected with MAP, causing the biggest losses in the cattle dairy industry [1,2]. Johne’s disease does not currently have a cure and while antibiotic therapy can slow the appearance of disease, historically, it has been shown to be ineffective and uneconomical. In order to find a solution for control of MAP infection and develop effective treatment, management, and diagnostic strategies, it is essential to understand molecular mechanisms of MAP pathogenesis

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Results