Bacterial thymidine kinase as a non-invasive imaging reporter for Mycobacterium tuberculosis in live animals - PubMed (original) (raw)

Bacterial thymidine kinase as a non-invasive imaging reporter for Mycobacterium tuberculosis in live animals

Stephanie L Davis et al. PLoS One. 2009.

Abstract

Background: Bacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2'deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-[(125)I]-iodouracil ([(125)I]-FIAU), a nucleoside analog substrate for bacterial thymidine kinase (TK). Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis.

Methodology/principal findings: We engineered a M. tuberculosis strain with chromosomally integrated bacterial TK under the control of hsp60 -- a strong constitutive mycobacterial promoter. [(125)I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis P(hsp60) TK strain was evaluated in experimentally-infected BALB/c and C3HeB/FeJ mice using the thigh inoculation or low-dose aerosol infection models. M. tuberculosis P(hsp60) TK strain actively accumulated [(125)I]FIAU in vitro. Growth characteristics of the TK strain and susceptibility to common anti-tuberculous drugs were similar to the wild-type parent strain. M. tuberculosis P(hsp60) TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested.

Conclusion: We have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals. This tool will allow real-time pathogenesis studies in animal models of TB and has the potential to simplify preclinical studies and accelerate TB research.

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Conflict of interest statement

Competing Interests: The authors Sanjay Jain (lead inventor), Gyanu Lamichhane, Sridhar Nimmagadda, Martin Pomper and William Bishai are co-inventors for a US provisional patent USPA # 61/181,813, ‘Real-time evaluation of Mycobacterium tuberculosis bacterial burden in animal models of TB’ filed by Johns Hopkins University. The authors Sanjay Jain (lead inventor), Stephanie Davis and Nicholas Be are co-inventors for a US provisional patent USPA # 61/187,461, ‘Biosafety Level (BSL)-3 Life-support Cell for Studying Live Animals’ filed by Johns Hopkins University. Note that these inventions will be available to other academic and non-profit institutions for non-commercial research purposes, at no profit to the inventors or JHU.

Figures

Figure 1

Figure 1. Characteristics of the Mycobacterium tuberculosis Phsp60 TK strain.

A. Equal numbers of M. tuberculosis H37Rv (wild-type) or M. tuberculosis Phsp60 TK strains were incubated with 1 µCi/ml of [125I]FIAU at 37°C in Middlebrook 7H9 liquid broth for 6 and 24 hours. At each specified time-point equal aliquots were withdrawn from the cultures and washed 3 times to remove the media. Each pellet was resuspended in PBS in 1.5 ml Eppendorf tubes and disinfected with Lysol overnight. The activity for each Eppendorf was measured using a gamma counter. M. tuberculosis Phsp60 TK strain actively accumulates [125I]FIAU in vitro. Mean uptake activity is 6086 (±536) and 8011 (±3233) counts per minute (cpm) at 6 and 24 hours for the M. tuberculosis Phsp60 TK strain which is significantly more than 549 (±50) and 615 (±260) cpm for the wild-type parent strain (p<0.03). B. We evaluated the in vivo growth characteristics of M. tuberculosis Phsp60 TK strain and compared it to the wild-type parent strain. Both strains grow similarly in the lungs of C3HeB/FeJ mice after a low-dose aerosol infection. C. Using TK specific primers and colony PCR, the TK gene is reliably amplified from all the randomly picked M. tuberculosis Phsp60 TK colonies (n = 13) obtained from lung homogenates 8-weeks after an aerosol infection (panel B). +ve and –ve refer to the positive (pGS400H) and negative (M. tuberculosis wild-type) controls respectively.

Figure 2

Figure 2. [125I]FIAU-SPECT can detect and localize Mycobacterium tuberculosis Phsp60 TK strain in situ.

M. tuberculosis H37Rv wild-type or Phsp60TK strain (∼108 CFU) were inoculated into either thighs of a BALB/c mouse. 1 mCi of [125I]FIAU was injected via a tail vein injection. Panels A and B show the fused SPECT and CT images 3 and 12 hours after the [125I]FIAU injection. Signal is detected at both time-points in the thigh inoculated with M. tuberculosis Phsp60TK strain but not in the thigh inoculated with the wild-type strain. As expected, SPECT signal is detected in several tissues [liver/gall bladder (L), stomach (St), gastrointestinal tract (GI), urinary bladder (UB)] that either metabolize or excrete FIAU or its iodinated derivatives.

Figure 3

Figure 3. Mycobacterium tuberculosis Phsp60 TK signal localizes to caseating granulomas in lungs of C3HeB/FeJ mice.

A. [125I]FIAU-SPECT/CT images from M. tuberculosis Phsp60TK infected C3Heb/FeJ mice 6-weeks after a low-dose aerosol infection. 3-D co-registered SPECT (blue/red) and CT (yellow) images are also shown. SPECT signal co-localizes to TB granulomas in the lungs of M. tuberculosis Phsp60TK infected mouse (arrows). B. Though extensive lung disease is present, images from M. tuberculosis wild-type infected mouse show no SPECT activity in the lungs. C. _M. tuberculosis Phsp60_TK SPECT signal localizes to a TB granuloma (arrows) in C3Heb/FeJ mice imaged 8-weeks after infection. D. The same lung and TB granuloma (arrow) is shown post-mortem. Ex-vivo radioactivity in the selected granuloma was 5318 counts per minute per milligram of tissue compared with 2364 in lung tissue outside of the granuloma and 856 in lung tissue from the animal infected with M. tuberculosis wild-type strain (St = Stomach).

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