Figure 1.
Proposed pathway of 4-androstene-3,17-dione (AD) degradation via β-oxidation of methylhexahydroindanone propionate intermediates 3aα-H-4α(3′-propionic acid)-7aβ-methylhexahydro-1,5-indanedione (HIP) and 3aα-H-4α(3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5OH-HIP) by Rhodococcus equi.
Adapted from [35], [46]–[47]. Numbers represent the following proposed enzymatic steps of β-oxidation of HIP: 1) ATP dependent HIP-CoA transferase, 2) HIP-CoA 5-reductase, 3) acyl-CoA dehydrogenase, 4) 2-enoyl-CoA hydratase, 5) 3-hydroxyacyl-coA dehydrogenase, 6) 3-ketoacyl-CoA thiolase. Dashed lines indicate multiple steps.
Figure 2.
Growth curves of wild type, mutant and complemented mutant strains of R. equi RE1 in mineral medium supplemented with 4-androstene-3,17-dione (AD) or 3aα-H-4α(3′-propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5OH-HIP) as a sole carbon and energy source.
Panels A and B show growth curves of wild type strain (diamonds) and mutants strains RE1ΔipdAB (squares), RE1ΔipdA2B2 (triangles), RE1ΔipdABΔipdA2B2 (crosses), RE1ΔfadE30 (asterisks) and RE1ΔfadA6 (circles) in MM+AD and MM+5OH-HIP, respectively. Panels C and D show growth curves of complemented strains of RE1ΔipdAB (diamonds) and RE1ΔfadE30 (squares) in MM+AD and MM+5OH-HIP, respectively. Curves represent averages of two independent experiments. Error bars represent standard deviations. Media with AD are turbid; therefore culture protein content was measured instead of optical density.
Figure 3.
Gas chromatography profiles showing the formation of methylhexahydroindanone propionate intermediates during whole cell biotransformations of 4-androstene-3,17-dione (AD) by mutant strains of R. equi RE1 at T = 120 hours.
Methylhexahydroindane-1,5-dione propionate (HIP) and 5-hydroxy-methylhexahydro-1-indanone propionate (5OH-HIP) accumulation in cell cultures of RE1ΔipdABΔipdA2B2 and RE1ΔfadE30, respectively. No accumulation is observed in cell cultures of RE1ΔipdAB. Lower panel shows the GC profiles of HIP (200 mg/L) and 5OH-HIP (50 mg/L) as authentic samples. Abbreviations: 1,4-androstadiene-3,17-dione (ADD), 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione (3-HSA), progesterone (50 mg/L) internal standard (I.S.).
Figure 4.
Macrophage infection assays of the human monocyte cell line U937 with R. equi strains.
Macrophage cell suspensions were infected with wild type virulent strain R. equi RE1 or mutant strains RE1ΔipdAB, RE1ΔipdA2B2, RE1ΔipdABΔipdA2B2, RE1ΔfadE30 and RE1ΔfadA6. The numbers of intracellular bacteria were determined by plate counts in duplicate following macrophage lysis. The data represents the averages for at least three independent experiments. Error bars represent standard deviations. Panel A shows the results for attenuated mutant strains RE1ΔipdAB, RE1ΔipdABΔipdA2B2 and RE1ΔfadE30. Avirulent strain R. equi 103- was used as a control. Panel B shows the results for non-attenuated mutant strains RE1ΔipdA2B2 and RE1ΔfadA6. Statistically, mutant strains RE1ΔipdAB (P<0.02), RE1ΔipdABΔipdA2B2 (P<0.01) and RE1ΔfadE30 (P<0.01) were significantly attenuated compared to parent strain RE1. Panel C shows the results (duplicates) with complemented mutant strains of RE1ΔfadE30 and RE1ΔipdAB. Wild type RE1, strain 103+, and mutant strains RE1ΔipdAB and RE1ΔfadE30 were included as controls.
Figure 5.
Intratracheal challenge of 3 to 5-week-old foals. Foals (mean of n = 3) were challenged intratracheally with mutant R. equi RE1ΔipdAB (7.1×106 CFU; squares) or wild type RE1 (4.3×106 CFU; diamonds).
Panel A shows rectal temperatures. Panel B shows numerical clinical scores. Error bars represent standard deviation.
Figure 6.
Serum antibody titer against R. equi of 3 to 5-week-old foals (n = 3) at day of intratracheal challenge (T = 0 days) and 3-weeks post-challenge (T = 21 days) with mutant R. equi RE1ΔipdAB (7.1×106 CFU; grey bars) or wild type RE1 (4.3×106 CFU; white bars).
Values represent mean ± standard deviation (error bars).
Table 1.
Pulmonary consolidation and re-isolation of R. equi of 3 to 5-week-old foals (n = 3) challenged intratracheally with wild type strain RE1 (4.3×106 CFU) or mutant strain RE1ΔipdAB (7.1×106 CFU).
Figure 7.
Serum antibody titer against R. equi of foals (n = 4) immunized orally (grey bars) at T = 0 and T = 14 days with attenuated R. equi strain RE1ΔipdAB (5×107 CFU) and challenged at T = 28 days with R. equi strain 85F (5×106 CFU).
The serum antibody titer of unvaccinated control foals (n = 4) are shown in white bars. Bars represent mean titers at day of vaccination (T = 0), at day of booster vaccination (T = 14), at day of intratracheal challenge (T = 28) and 20 days post-challenge (T = 48). Error bars represent standard deviation.
Figure 8.
Oral immunization and subsequent intratracheal challenge of foals.
Foals (2 to 4-week-old) vaccinated with RE1ΔipdAB (squares) and non-vaccinated controls (diamonds) (mean of n = 4) were challenged intratracheally with virulent strain R. equi 85F (5×106 CFU). Panel A shows rectal temperatures. Panel B shows numerical clinical scores. Statistically, rectal temperatures (P<0.005) and clinical scores (P<0.0001) were significantly different in vaccinates compared to the non-vaccinated control foals. Error bars represent standard deviation.
Figure 9.
Histopathology of lung tissue of vaccinates versus non-vaccinated foals following intratracheal challenge with wild type R. equi.
Lung specimen of a vaccinated foal showing normal airways (bronchi and bronchioli), blood vessels and alveoli at (A) 25x and (B) 200x magnification. Typical pyogranuloma (5 mm diameter) observed in lung specimens of non-vaccinated control foals at (C) 25x and (D) 200x magnification. The centre of the pyogranuloma consists of necrotic debris, neutrophils and toxic neutrophils with complete loss of lung architecture.
Table 2.
Lung weights and percentage pulmonary consolidation per lobe of vaccinated and unvaccinated (control) 2 to 4-week-old foals (n = 4).
Table 3.
Quantitative re-isolation of R. equi 85F from lung lobes of vaccinated and unvaccinated (control) 2 to 4-week-old foals (n = 4).