Experimental demonstration of the possible role of Acanthamoeba polyphaga in the infection and disease progression in Buruli Ulcer (BU) using ICR mice

Abstract

The transmission of Buruli ulcer (BU), caused by Mycobacterium ulcerans (MU), remains puzzling although a number of hypothesis including through bites of infected aquatic insects have been proposed. We report the results of experiments using ICR mice that give credence to our hypothesis that Acanthamoeba species may play a role in BU transmission. We cocultured MU N2 and MU 1615 which expresses red fluorescent protein (RFP) and Acanthamoeba polyphaga (AP), and confirmed infected AP by Ziehl-Neelsen (ZN) staining. We tested for viability of MU inside AP and observed strong RFP signals inside both trophozoites and cysts after 3 and 42 days of coculturing respectively. ICR mice were topically treated, either on shaved intact or shaved pinpricked rumps, with one of the following; MU N2 only (2.25 x 106 colony forming units [CFU] / ml), MU N2:AP coculture (2.96 x 104 CFU: 1.6 x 106 cells/ml), AP only (1.6 x 106 cells/ml), PYG medium and sterile distilled water. Both MU N2 only and MU N2:AP elicited reddening on day (D) 31; edema on D 45 and D 44 respectively, and ulcers on D 49 at pinpricked sites only. To ascertain infectivity and pathogenicity of MU N2 only and MU N2:AP, and compare their virulence, the standard mouse footpad inoculation method was used. MU N2:AP elicited reddening in footpads by D 3 compared to D 14 with MU N2 only of the same dose of MU N2 (2.96 x 104 CFU). ZN-stained MU were observed in both thin sectioned and homogenized lesions, and aspirates from infected sites. Viable MU N2 were recovered from cultures of the homogenates and aspirates. This study demonstrates in ICR mice MU transmission via passive infection, and shows that punctures in the skin are prerequisite for infection, and that coculturing of MU with AP enhances pathogenesis.

Dual Role of Mechanisms Involved in Resistance to Predation by Protozoa and Virulence to Humans

“…Although there is limited experimental data showing that long-term protozoan grazing results in genotypic changes, it has been reported that protozoa can induce the expression of bacterial virulence in the short term, probably due mainly to changes in gene expression. For example, Mycobacterium ulcerans when co-cultured with A. polyphaga infected the footpads of mice much faster than the M. ulcerans only controls ( Azumah et al, 2017 ). S. enterica serotype Typhimurium was shown to enter a hyperinvasive state after passage through A. castellanii ( Carlson et al, 2007 ), and intracellular growth in A. castellanii also induced the invasion and virulence of L. pneumophila in mouse infection models ( Cirillo et al, 1999 ).…”

In Silico Screening of Isocitrate Lyase for Novel Anti-Buruli Ulcer Natural Products Originating from Africa

“…Although its mode of transmission remains poorly understood [ 4 , 5 , 6 ], studies have shown that it occurs in communities where there are slow flowing rivers, streams and water bodies [ 6 ]. Azumah et al have also demonstrated the involvement of Acanthamoeba in the disease transmission and progression in animal experiments [ 7 , 8 ]. Although the disease occurs elsewhere, the majority of cases are in sub-Saharan Africa where, for example, 1676 of the 1864 new cases of Buruli ulcer worldwide in 2016 were reported from this region [ 9 ].…”

The Impact of Protozoan Predation on the Pathogenicity of Vibrio cholerae

“…For example, S. enterica and L. pneumophila recovered after exposure to A. castellanii display hyperinvasive phenotypes during in vivo infection (Cirillo et al, 1994;Rasmussen et al, 2005). Similarly, mice infections performed with Mycobacterium ulcerans previously co-incubated with A. polyphaga led to enhanced pathogenicity (Azumah et al, 2017). In the case of Vibrio spp., it has recently been shown that their release in EFVs to the extracellular environment results in bacterial growth and colonization advantage in vitro and in vivo, respectively (Espinoza-Vergara et al, 2019).…”