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Microorganisms
2024 May 31;126:. doi: 10.3390/microorganisms12061120.
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Exploring the Role of a Putative Secondary Metabolite Biosynthesis Pathway in Mycobacterium abscessus Pathogenesis Using a Xenopus laevis Tadpole Model.
Miller NJ
,
Dimitrakopoulou D
,
Baglia LA
,
Pavelka MS
,
Robert J
.
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Mycobacterium abscessus (Mab) is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within Mab, which may be a key factor in its pathogenesis. This novel pathway is encoded in a gene cluster spanning MAB_0284c to 0305 and is related to Streptomyces pathways, producing the secondary metabolites streptonigrin and nybomycin. We constructed an in-frame deletion of the MAB_0295 (phzC) gene and tested it in our Xenopus laevis animal model. We have previously shown that X. laevis tadpoles, which have functional lungs and T cells, can serve as a reliable comparative model for persistent Mab infection and pathogenesis. Here, we report that tadpoles intraperitoneally infected with the ∆phzC mutant exhibit early decreased bacterial loads and significantly increased survival compared with those infected with WT Mab. ∆phzC mutant Mab also induced lower transcript levels of several pro-inflammatory cytokines (IL-1β, TNF-α, iNOS, IFN-γ) than those of WT Mab in the liver and lungs. In addition, there was impaired macrophage recruitment and decreased macrophage infection in tadpoles infected with the ∆phzC mutant, by tail wound inoculation, compared to those infected with the WT bacteria, as assayed by intravital confocal microscopy. These data underline the relevance and usefulness of X. laevis tadpoles as a novel comparative animal model to identify genetic determinants of Mab immunopathogenesis, suggesting a role for this novel and uncharacterized pathway in Mab pathogenesis and macrophage recruitment.
R21 AI166114 NIAID NIH HHS , R24-AI059830 and R21 5R21AI166114 National Institute of Allergy and Infectious Diseases at the National Institutes of Health, P30 ES001247 NIEHS NIH HHS
Figure 1. Putative secondary metabolite pathway in M. abscessus. The cluster of genes (MAB_0284c to MAB_0305) is shown at the top, with the MAB ORF number designations from the genome of M. abscessus ATTC 19977. Embedded within the cluster are MAB_0330c to MAB_0303, also known as the adqRABC cluster, which has been shown to be responsible for the degradation of quinolone signaling molecules from P. aeruginosa [31]. The MAB_0284c to MAB_0290 genes are syntenic with those in the streptonigrin pathway genes (stnG to stnL), as shown below the Mab cluster. MAB_0290 and MAB_0295 to MAB_0298 are also syntenic with the streptonigrin genes, except that they are inverted in Mab and include two additional genes, a transcriptional regulator (MAB_0291, cdaR) and a putative carboxyesterase (MAB_0294). Mab gene products that are homologous to those in the calcimycin and benzoxazole pathways are indicated (* or #). Genes unique to the Mab cluster are shown in bold: MAB_0291 (cdaR), MAB_0294 (putative carboxyesterase), MAB_0299 (nbaD, a putative aminohydrolase), MAB_0304 (hypothetical), and MAB_0305 (putative aminotransferase). Predicted functions of some of the gene products are color-coded in the key.
Figure 2. Survival of adult frogs and tadpoles following ip inoculation with either WT Mab or ∆phzC Mab. (A) One-year-old frogs (six per group) were (ip) inoculated with 5 × 105 CFU of WT or ∆phzC Mab and monitored for 50 days for survival. (B) The bacterial loads in livers of adult frogs from A, determined at the end of the experiment. (C) Two-week-old tadpoles (st. 44–45, 10 per group), were inoculated with 5 × 105 CFU of WT or ∆phzC Mab. As a negative control, tadpoles were injected with APBS. Survival was monitored for 50 days for survival. Statistical differences between groups were assessed by a log-rank (Mantel–Cox) test (* p > 0.05; ** p > 0.001).
Figure 3. Changes in bacterial loads in tadpoles following (ip) inoculation with WT or ∆phzC Mab. Three-weeks-old tadpoles (stage 55; 10 per group) were (ip) inoculated with 5 × 105 CFU of either WT or ∆phzC Mab. Colony assays was performed on liver lysates at 1 and 14 days post-infection. One tadpole per condition was also tested 2 h post-infection to control the initial infection load. Statistical differences between the groups was determined by t-test (* p > 0.05).
Figure 4. Change in relative gene expression of tadpole livers and lungs at 14 days post-infection with WT or ∆phzC Mab. Three-week-old tadpoles (stage 55; 10 per group) were (ip) inoculated with 5 × 105 CFU of either WT or ∆phzC Mab, or injected with APBS as a negative control (Control). RNAs from the collected livers and lungs were used to determine gene expression for TNF-α, IL-1β, IFN-I, iNOS, and IFN-γ. Gene relative quantification (RQ) was determined as the fold increase relative to the GAPDH endogenous control, normalized to the lowest expression level. Bars represent standard deviations (n = 10 animals). Statistical differences between the groups were determined by one-way ANOVA and Tukey’s post hoc test. (**) p > 0.001; (*) p > 0.05.
Figure 5. Visualization by intravital confocal microscopy of macrophage recruitment and infection in representative tadpoles 1 day after tail wound inoculation. DsRed WT (A–C) or ∆phzC Mab (D–F) were inoculated using a biopsy punch in the tailmuscle of mpeg:GFP transgenic tadpoles (stage 56). Infiltration of mpeg1+ macrophage (green) and mycobacteria-infected macrophages (red+ green+) were monitored in live tadpoles at 1 dpi using confocal microscopy (Leica DMi8 Inverted Microscope). GFP only (A,D), DsRed only (B,E), and overlayed (C,F) signals are shown. Size bar: 10 μm.
Figure 6. Visualization of macrophage recruitment and infection following 1, 3, and 7 days following tadpoletail wound inoculation using intravital confocal microscopy. DsRed WT (A–C) or ∆phzC (D–F) Mab were inoculated with a biopsy punch in the tailmuscle of mpeg:GFP transgenic tadpoles (stage 56). GFP mpeg1+ macrophage (green) and DsRed mycobacteria-infected macrophages (red+ green+) were monitored in live tadpoles at 1, 3, and 7 dpi by confocal microscopy (Leica DMi8 Inverted Microscope). Overlayed (red+/green+) signal is shown. Infected macrophages are circled. Size bar: 10 μm.
Figure 7. Volume ratio of DsRed Mab determined by confocal microscopy using the Imaris microscopy image analysis software to calculate the total volume of DsRed bacteria versus the total volume of GFP-expressing macrophages. The volume ratio was calculated for each tadpoletail wound from sections that totaled between ~1–2 square millimeters. N = 5 per group. (*) p > 0.05.
