Bioluminscent Mycobacterium ulcerans, a tool to study host-pathogen interactions in a murine tail model of Buruli ulcer

Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans. In this study we used a previously reported strain of M. ulcerans, genetically engineered to constitutively produce bioluminescence, to follow the progression of Buruli ulcer in mice using an in-vivo imaging (IVIS®) system. We aimed to characterize a mouse tail infection model for pathogenesis, as well as for pre-clinical vaccine and drug development research for Buruli ulcer. Immune parameters, such as antibody titers and cytokine levels, were determined throughout the course of the infection and histology specimens were examined for comparison with human pathology. Nine out of ten (90%) BALB/c mice infected subcutaneously with 105 M. ulcerans JKD8049 (containing pMV306 hsp16+luxG13) exhibited light emission from the site of infection over the course of the experiment indicating M. ulcerans growth in-vivo. Five out of ten (50%) animals developed clinical signs of disease. Antibody titers were overall low and their onset was late, as measured by responses to both heterogenous (bacterial whole cell lysate) and single antigen (Hsp18) targets. IFN-γ, and IL-10 are reported to play a vital role in host control of Buruli ulcer and these cytokines were elevated in animals with pathology. For mice with advanced pathology, histology revealed clusters of acid-fast bacilli within subcutaneous tissue 300-400 μm beneath the epidermis of the tail, with macrophage infiltration and granuloma-formation resembling human Buruli ulcer. This study has shown the utility of using bioluminescent M. ulcerans and IVIS® in a mouse tail infection model to study Buruli ulcer infection. Author summary Buruli ulcer is one of the so called neglected tropical diseases. It is an infectious disease, mainly occurring in West Africa but also in Australia. It manifests as skin lesion and ulcer. Up to date, the way of transmission is inadequately understood. Also, there is no vaccine to protect against the disease. Buruli ulcer is treatable with a course of antibiotics that need to be given for the duration of two months. More laboratory research is needed to elucidate the mechanism of transmission, develop a vaccine and improve and shorten antibiotic therapy. For this, animal (mouse) models of disease are used. The aim of this study was to refine and improve the mouse tail infection model of Buruli ulcer. For this, we used a genetically modified Mycobacterium ulcerans strain that emits light. After infection of animals, light emitted from the bacteria was read out with an in-vivo imaging (IVIS) camera. This allowed us to monitor the location of bacteria in the living animal over time without the need to kill the animal. We also measured parameters of the immune system such as antibodies and cytokines as a baseline for future studies into immunology, vaccine development and pathology of Buruli ulcer. We successfully improved and characterized the mouse tail infection model in Buruli ulcer with the use of modern technology using light emitting bacteria and the IVIS camera.

80 81 There are several major challenges to control Buruli ulcer. The mode of transmission is not 82 yet completely understood and seems to vary by geographic location, although puncturing 83 injuries after contamination from an environmental source seem to be a major cause and in 84 south east Australia at least, mosquitoes have been linked to transmission [9]. Buruli ulcer 85 is currently treated with an eight-week regimen of rifampin and streptomycin or a regimen 86 where the injectable streptomycin is replaced with clarithromycin after four weeks; a fully 87 oral, eight-week rifampin and clarithromycin regimen has been trialled in humans and 88 current trial analysis is ongoing (ClinicalTrials.gov Identifier: NCT01659437) [10,11].
89 Progressed, larger lesions are often managed with surgical excision of the infected tissue 90 followed by functional repair and skin grafting [12]; a recent study showed that the time-91 point for decision making on whether to intervene surgically or not does not matter for 92 overall healing outcomes [13]. No vaccine is available despite several efforts to employ 93 the BCG-vaccine or to develop novel vaccines [14][15][16][17][18][19][20][21][22][23].

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Images from the ventral and dorsal aspect of the tail were taken. Images were analyzed 171 using Living Image® software. Areas emitting light were defined as regions of interest 172 (ROI). A copy of every ROI was placed next to those areas for background measurement.

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All incubation of ELISA-plates was done in a moisturized container at room temperature.  positive correlation (r 2 = 0.98) between photons/s and CFU/ml ( Fig 1A).

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Establishment of mouse tail infection.

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In order to evaluate virulence and to study murine infection, bioluminescent M. ulcerans 239 was injected subcutaneously into mouse tails. Ten BALB/c mice were inoculated with 248

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To study the course of the infection in terms of bacterial burden measured in 250 bioluminescence, mice were imaged weekly with the IVIS® system. Bioluminescence, 251 measured in emitted photons/s rose exponentially to a maximum of 1x10 7 in week seven 252 (Fig 1D), according to our standard curve, this equals about 5x10 6 CFU/ml ( Fig 1A) and 253 was associated with advanced, severe pathology ( onset of the antibody response was noted (Fig 2A). Antibody titers reached higher levels 277 between week 11 and 17, but overall titers, were low (Fig 2A). The response to M. ulcerans

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Hsp18 and WCL was compared and no statistically significant difference (p > 0.05) was 279 found (Fig 2B). Furthermore, ELISA results in response to WCL at week 8 were compared 280 between animals with severe, moderate and no clinical pathology and no statistically 281 significant difference (p > 0.05) was found (Fig 2C,D). state of the animal (Fig 3).

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In order to validate the model and study the pathology of M. ulcerans, histopathology was 296 performed on skin lesions and compared to those of humans described in the literature.

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Specimens from infected tissue were subjected to histopathological analysis in Ziehl-

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We noticed a decline and plateauing of bioluminescence from week 8 onwards. This 332 phenomenon could be explained by a plateauing of the bacterial growth curve in the lesion 333 and transition into a stationary phase where less of the immunosuppressive toxin 334 mycolactone is produced and partial host control sets in which is reflected to some extent 335 by the rise of antibody titers around that time-point (Fig 2). . The elevated IFN-γ response seen in mice with severe pathology (Fig 3A) 358 can thus be interpreted as an early reaction to a large amount of actively multiplying