Proceedings of the 10th International Colloquium on Paratuberculosis

#179 Rhodanine agents active against non-replicating intracellular Mycobacterium avium
subspecies paratuberculosis
Tim J Bull, Richard Linedale, John Hermon-Taylor, Jason Hinds,St George's University <strong>of</strong> London, United
Kingdom; King's College London, United Kingdom
Introduction and Objective: The capacity for long term persistence inside macrophages and o<strong>the</strong>r cell lineages
combined with <strong>the</strong> ability to enter a non-replicating viable (NRV), <strong>of</strong>ten non-culturable state, allows MAP
to develop paucimicrobial chronic infection states leading to disease. Therapy is difficult because <strong>the</strong> lack <strong>of</strong>
bacterial division, diminution <strong>of</strong> cell wall antigens and low transcriptomic turnover in NRV drastically reduces
<strong>the</strong> effectiveness <strong>of</strong> conventional antibiotics. In addition, MAP can manipulate host intracellular killing mechanisms
and disrupt pathogen antigen presentation leading to dysregulation <strong>of</strong> host cell mediated clearance. In
this study we evaluate a novel anti-MAP agent Rhodanine(D157070) that aims to re-assert <strong>the</strong> normal surveillance
and activation <strong>of</strong> host cell defense mechanisms by inhibiting MAP from releasing anti-reactive nitrogen
intermediates.
Materials and Methods: Culturable viability (CV) and viable persistence (VP) <strong>of</strong> MAP K10 in a variety
macrophage models and culture alone as a control, with and without Rhodanine(D157070) treatment was
monitored over a 7 day period. CV was measured using conventional colony counting. VP was determined
as <strong>the</strong> degree <strong>of</strong> ribosomal turnover represented by <strong>the</strong> qPCR DNA:RNA ratio <strong>of</strong> a 102bp pre16SrRNA leader
sequence present as single copy number (DNA) but cleaved and degraded during ribosomal assembly (RNA).
Full MAP genome transcriptomic pr<strong>of</strong>iles were also obtained during treatment using <strong>the</strong> ParaTBtools MAPAC
microarray.
Results: Rhodanine(D157070) did not kill MAP in conventional extracellular culture but transcriptome pr<strong>of</strong>iles
were altered. Rhodanine(D157070) was highly active against intracellular MAP in macrophage infection
models increasing MAP killing as measured by both CV and VP assays. Rhodanine(D157070) alone showed
no toxicity to cell lines.
Conclusion: Rhodanine(D157070) does not kill MAP directly but can enter host cells and induce inhibition
<strong>of</strong> MAP derived processes that normally decrease effectiveness <strong>of</strong> intracellular killing mechanisms. This
agent thus shows good promise as a <strong>the</strong>rapy directed at non-replicating intracellular MAP infection.
#206 The role <strong>of</strong> TcrXY, a two component system in Mycobacterium avium subspecies
paratuberculosis
Michelle Pinto, Lucy Mutharia, University <strong>of</strong> Guelph, Canada
Bacteria are able to survive in environments that experience continuous fluctuation in both physical and chemical
aspects by adapting to <strong>the</strong>se changes using sensory-response mechanisms such as Two Component Systems.
These systems involve autophosphorylation <strong>of</strong> membrane histidine protein kinase receptors on detection
<strong>of</strong> certain stimuli and phosphoryl transfer to a cytoplasmic response regulator, which generally acts as a transcription
regulator. Mycobacterium avium subspecies paratuberculosis (MAP), <strong>the</strong> etiological agent involved
in Johne’s disease in ruminants, has ten putative two component systems in addition to one orphan histidine
protein kinase and five orphan response reglulators. The TcrXY system has been studied in M. tuberculosis
and infection assays using a partial deletion construct resulted in hypervirulence in SCID mice. The tcrXY promoter
in MAP_K10 was used to generate promoter: lacZ fusions and used in in vitro β-galactosidase assays in
Mycobacterium avium subsp. hominisuis 104 (MAA104). The tcrXY promoter was induced in acidic conditions
(pH 4.5 to 5.5). It is possible that TcrXY may be responsive to changes in pH in <strong>the</strong> environment in addition to
what it faces in <strong>the</strong> gut and feces <strong>of</strong> <strong>the</strong> host as well as in phagosomes. A MAP tcrXY gene knockout will be
characterized by investigating <strong>the</strong> survival <strong>of</strong> <strong>the</strong> mutants in macrophages and when exposed to acidic gradients
with <strong>the</strong> goal <strong>of</strong> identifying <strong>the</strong> role that TcrXY plays in virulence and pathogenesis. The recently described
conditionally replicating shuttle phasmid pHAE87 is used to generate <strong>the</strong> TcrXY mutants.
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