RESUMEN
Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's Disease (JD), which is endemic to dairy cattle and also incriminated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. JD is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative polymerase chain reaction (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD disease. MAP IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry and TaqMan technology was used in 107 reports. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity.
RESUMEN
Actinorhodin, an antibiotic produced by Streptomyces coelicolor, is exported from the cell by the ActA efflux pump. actA is divergently transcribed from actR, which encodes a TetR-like transcriptional repressor. We showed previously that ActR represses transcription by binding to an operator from the actA/actR intergenic region. Importantly, actinorhodin itself or various actinorhodin biosynthetic intermediates can cause ActR to dissociate from its operator, leading to derepression. This suggests that ActR may mediate timely self-resistance to an endogenously produced antibiotic by responding to one of its biosynthetic precursors. Here, we report the structural basis for this precursor-mediated derepression with crystal structures of homodimeric ActR by itself and in complex with either actinorhodin or the actinorhodin biosynthetic intermediate (S)-DNPA [4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho-[2,3-c]-pyran-3-(S)-acetic acid]. The ligand-binding tunnel in each ActR monomer has a striking hydrophilic/hydrophobic/hydrophilic arrangement of surface residues that accommodate either one hexacyclic actinorhodin molecule or two back-to-back tricyclic (S)-DNPA molecules. Moreover, our work also reveals the strongest structural evidence to date that TetR-mediated antibiotic resistance may have been acquired from an antibiotic-producer organism.