FVB/N Mice Spontaneously Heal Ulcerative Lesions Induced by Mycobacterium ulcerans and Switch M. ulcerans into a Low Mycolactone Producer

Abstract

Buruli ulcer, a debilitating disease, is caused by Mycobacterium ulcerans. The incidence of this neglected tropical disease is steadily increasing. As a rule, without treatment, skin ulcers occur and a lengthy healing process may be observed associated with severe functional disabilities. Mouse models are already available to study establishment of lesions or evaluation of therapy but a lack of a suitable animal model, mimicking all clinical stages, in particular the healing process, remains an obstacle to understand the pathophysiology of M. ulcerans infection. M. ulcerans was s.c. inoculated in three consanguine mouse strains, that is, BALB/c and C57BL/6, classically used to study mycobacterial infection, and FVB/N. Strikingly, FVB/N mice, although as sensitive as all other mouse strains with respect to M. ulcerans infection, presented a spontaneous healing after the ulcerative phase despite stable bacterial load, and mycolactone toxin was not detected in the healed tissues. The spontaneous healing process was accompanied by an activation of the innate immune system. The adaptive response initiated by FVB/N mice was not involved in the healing process and did not confer protection against M. ulcerans. Our work highlights the importance of innate immune responses to control M. ulcerans infection. This in vivo model of M. ulcerans infection now paves the way for new avenues of research toward the elucidation of critical stages of this disease, such as the characterization of the regulation of mycolactone production, a better understanding of the pathophysiology of M. ulcerans infection, and the development of new therapeutic strategies.

An Optimized Method for Extracting Bacterial RNA from Mouse Skin Tissue Colonized by Mycobacterium ulcerans

“…The principal challenge in this method was the choice of a lysis system resulting in host-cell lysis without damage to the bacterial cells. We recently demonstrated that the mechanical disruption of tissues had no effect on bacterial viability (Marion et al, 2016b). We therefore disrupted the host cells by mechanical lysis and treated them with proteinase K, to digest and degrade the mouse tissue whilst leaving the bacterial cells intact.…”

“…We recently developed the first mouse model for investigating this phenomenon (Marion et al, 2016b). Our investigations have revealed a strong inhibition of mycolactone synthesis during spontaneous healing.…”

“…Bacterial suspensions were prepared as previously described (Marsollier et al, 2007b; Marion et al, 2016b), with adjustment to 2 × 10 5 acid-fast bacilli/ml for inoculation (50 μl) into the tail of 6-week-old females of the inbred FVB/N mouse strain (Charles River Laboratories, Saint-Germain-Nuelles, France).…”

“…We have developed the first dedicated mouse model for studies of the spontaneous healing process (Marion et al, 2016b). During the characterization of this model, we made an interesting discovery concerning the dynamics of viable bacterial load in healed tissues: the load of cultivable bacilli was found to be both high and stable in the long term (Marion et al, 2016b).…”

“…During the characterization of this model, we made an interesting discovery concerning the dynamics of viable bacterial load in healed tissues: the load of cultivable bacilli was found to be both high and stable in the long term (Marion et al, 2016b). We then demonstrated that mycolactone synthesis was inhibited in healed tissues.…”