Microbiological, histological, immunological, and toxin response to antibiotic treatment in the mouse model of Mycobacterium ulcerans disease

Abstract

Mycobacterium ulcerans infection causes a neglected tropical disease known as Buruli ulcer that is now found in poor rural areas of West Africa in numbers that sometimes exceed those reported for another significant mycobacterial disease, leprosy, caused by M. leprae. Unique among mycobacterial diseases, M. ulcerans produces a plasmid-encoded toxin called mycolactone (ML), which is the principal virulence factor and destroys fat cells in subcutaneous tissue. Disease is typically first manifested by the appearance of a nodule that eventually ulcerates and the lesions may continue to spread over limbs or occasionally the trunk. The current standard treatment is 8 weeks of daily rifampin and injections of streptomycin (RS). The treatment kills bacilli and wounds gradually heal. Whether RS treatment actually stops mycolactone production before killing bacilli has been suggested by histopathological analyses of patient lesions. Using a mouse footpad model of M. ulcerans infection where the time of infection and development of lesions can be followed in a controlled manner before and after antibiotic treatment, we have evaluated the progress of infection by assessing bacterial numbers, mycolactone production, the immune response, and lesion histopathology at regular intervals after infection and after antibiotic therapy. We found that RS treatment rapidly reduced gross lesions, bacterial numbers, and ML production as assessed by cytotoxicity assays and mass spectrometric analysis. Histopathological analysis revealed that RS treatment maintained the association of the bacilli with (or within) host cells where they were destroyed whereas lack of treatment resulted in extracellular infection, destruction of host cells, and ultimately lesion ulceration. We propose that RS treatment promotes healing in the host by blocking mycolactone production, which favors the survival of host cells, and by killing M. ulcerans bacilli.

Figure 1. After reaching plateau in bacillary numbers, M. ulcerans-infected mouse footpads continue to increase in swelling.

Swelling first became apparent at day 20 after infection. For the first 3 weeks, the number of cultivable organisms increased from 3.29±0.33 log₁₀ on day 3 to 4.26±0.08 log₁₀ on day 7, 4.73±0.42 log₁₀ on day 13, 5.05±0.19 log₁₀ on day 20, and 6.02±0.13 log₁₀ on day 24, the first day of treatment. CFU remained at approximately this level for the remainder of the experiment in untreated mice. Swelling averaged ∼grade 1 at day 27 after infection, nearly grade 2 at day 34, grade 2.67 at day 40, 3.42 at day 48, 3.75 at day 55, 3.58 at day 63, and 3.75 again at day 78. Data based on 3 mice per group per time point. Red lines emphasize the period between the onset and augmentation of swelling and the plateau in CFU.

Figure 2. Swelling and CFU Reduction after RIF-STR treatment.

After 3 days of RIF-STR (RS) treatment (day 27 after infection), swelling averaged grade 1, and then declined to 0.61±0.33, 0.27±0.18, 0.14±0.18, 0, 0.06±0.17, and 0 on days 34, 40, 48, 44, 63, and 78, respectively. CFU also declined from 6.02±0.09 log₁₀ on day 27, and then 3.83±0.56 log₁₀, 2.82±0.49 log₁₀, 0.52±0.45 log₁₀, 0.2±0.35 log₁₀, 0, and 0 on days 34, 40, 48, 44, 63, and 78, respectively. For comparison, CFU per footpad for untreated controls (black line) show the impact of antibiotic treatment on bacterial burden. Data based on 3 mice per group per time point.

Figure 3. M. ulcerans in mouse footpads, before (A, 1–5) and after (B, 1–5) treatment with Rifampin-Streptomycin.

A1: AFB are detectable in the dermis 3 days after infection with little or no cellular infiltrate. 500× Magnification A2: At day 7, the migration of inflammatory cells and phagocytosis has begun. 500× Magnification A3: By day 13, AFB are primarily associated with inflammatory and phagocytic cells. 1000× Magnification A4: At day 20, the extracellular phase of M. ulcerans infection has begun, coinciding with the onset of footpad swelling. 1000× Magnification A5: At day 27, shortly after the initiation of antibiotic treatment, a mix of intracellular and extracellular AFB is observed. 500× Magnification In panel B, untreated footpad lesions are shown on the left and treated footpads are shown on the right. B1: At day 34 after infection and after 10 days of treatment, M. ulcerans bacilli in treated mice are primarily associated with host cells whereas the bacilli are outside of cells in untreated mice; the cells are either not recruited or may have been destroyed by the mycolactone toxin. 500×, inset 1000×, Magnification, B2: At day 40, bacilli are found in extracellular masses in untreated mice but in mice treated for 16 days, the bacilli are still associated with host cells and stain less solidly. 500×, inset 1000×, Magnification B3: At day 48, after 24 days of treatment, the bacilli have lost solid staining and have become beaded in appearance. 500×, inset 1000×, Magnification B4: At day 55 after infection, M. ulcerans bacilli are apparent in the superficial dermis and epidermis and are being shed from the ulcerated lesions of untreated mice. 500×, inset 1000×, Magnification B5: 3 months after treatment completion, the organisms are uncultivable but non-solid staining bacilli are still present in the footpads. 500× Magnification.

Figure 4. Cytokine production responses to concanavalin A or mycobacterial protein before and after Rifampin-Streptomycin treatment.

The dotted line indicates the levels in uninfected control (UIC) mice. Responses by untreated mice are indicated by solid bars and those by rifampin-streptomycin (RS) treated mice are indicated by hatched bars. Responses of splenocytes stimulated with Concanavalin A (ConA) are in the left column and with mycobacterial culture filtrate proteins (CFP) in the right column for representative cytokines, from top to bottom, IL-2, IFN-γ, IL-5, IL-17, and TNF-α. Data based on 3 mice per group per time point.

Figure 5. Cytotoxic activity of lipids extracted from footpads of mice without treatment or treated with rifampin-streptomycin.

Cytotoxicity of lipid extracts was measure by the MTT assay after incubation with HELF cells for 48 hrs. Cytotoxicity is expressed relative to that induced by synthetic mycolactone measured concurrently. Data based on 3 mice per group per time point. By day 63 there were statistically significant differences between the infected right hind footpad (RHFP, black solid circles) and the contralateral (LHFP, solid diamonds, p<0.02) and footpads of the rifampin-streptomycin (Abx) treated mice (RHFP, red open circles; LHFP, red open diamonds p<0.05).

Figure 6. Mycolactone detection by mass spectrometry in mouse footpads.

A. EPI Mass spectra indicated a propensity of authentic mycolactone A/B to form a sodium adduct (m/z 765.7, [M+Na+]). Fragment ions at m/z 429.6 and 359.5 correspond to the core lactone and polyketide side chains, respectively. B. Mycolactone A/B detected in footpad extracts displayed similar mass spectra and also contained a number of other ions (e.g. m/z 659.6 and 747.7) that were present in authentic reference material. C and D. Extracted ion chromatograms (XIC) for m/z 429.6, which was selected for quantification in LC-MRM analyses of tissue extracts. Standard mycolactone is shown in C and a footpad sample in D. The sensitivity of the assay (<10 pg) was such that it was possible to measure mycolactone in the tissue samples. E. Mycolactone (ML) was detectable as early as day 13 after infection and before the onset of visible swelling in mouse footpads. No ML was detectable in the contralateral footpad of either the RIF-STR-treated or the untreated control mice. Data based on pooled lipid extracts from 3 mice per group per time point.