Background Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.Methodology/Principal FindingsOne of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.Conclusions/SignificanceWhile much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

The transcriptional regulation of several dozen genes in response to low oxygen tension is mediated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric protein composed of two subunits, HIF-1α and HIF-1β. In the HIF-1α-deficient human leukemic cell line, Z-33, exposed to mild (8% O2) or severe (1% O2) hypoxia, we found significant upregulation of two related heterogenous nuclear ribonucleoproteins, RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), which are highly conserved cold stress proteins with RNA-binding properties. Hypoxia also induced upregulation of RBM3 and CIRP in the murine HIF-1β-deficient cell line, Hepa-1 c4. In various HIF-1 competent cells, RBM3 and CIRP were induced by moderate hypothermia (32°C) but hypothermia was ineffective in increasing HIF-1α or vascular endothelial growth factor (VEGF), a known HIF-1 target. In contrast, iron chelators induced VEGF but not RBM3 or CIRP. The RBM3 and CIRP mRNA increase after hypoxia was inhibited by actinomycin-D, and in vitro nuclear run-on assays demonstrated specific increases in RBM3 and CIRP mRNA after hypoxia, which suggests that regulation takes place at the level of gene transcription. Hypoxia-induced RBM3 or CIRP transcription was inhibited by the respiratory chain inhibitors NaN3 and cyanide in a dose-dependent fashion. However, cells depleted of mitochondria were still able to upregulate RBM3 and CIRP in response to hypoxia. Thus, RBM3 and CIRP are adaptatively expressed in response to hypoxia by a mechanism that involves neither HIF-1 nor mitochondria.

According to World Health Organization estimates, infectious organisms are responsible for approximately one in four deaths worldwide. Animal models play an essential role in the development of vaccines and therapeutic agents but large numbers of animals are required to obtain quantitative microbiological data by tissue sampling. Biophotonic imaging (BPI) is a highly sensitive, nontoxic technique based on the detection of visible light, produced by luciferase-catalysed reactions (bioluminescence) or by excitation of fluorescent molecules, using sensitive photon detectors. The development of bioluminescent/fluorescent microorganisms therefore allows the real-time noninvasive detection of microorganisms within intact living animals. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, reducing the number of animals required to yield statistically meaningful data. In the study of infectious disease, the use of BPI is becoming widespread due to the novel insights it can provide into established models, as well as the impact of the technique on two of the guiding principles of using animals in research, namely reduction and refinement. Here, we review the technology of BPI, from the instrumentation through to the generation of a photonic signal, and illustrate how the technique is shedding light on infection dynamics in vivo.

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Induced hypothermia is the only therapy with proven efficacy to reduce brain damage after perinatal asphyxia. While hypothermia down-regulates global protein synthesis and cell metabolism, low temperature induces a small subset of proteins that includes the RNA-binding protein RBM3 (RNA-binding motif protein 3), which has recently been implicated in cell survival. Here, immunohistochemistry of the developing postnatal murine brain revealed a spatio-temporal neuronal RBM3 expression pattern very similar to that of doublecortin, a marker of neuronal precursor cells. Mild hypothermia (32°C) profoundly promoted RBM3 expression and rescued neuronal cells from forced apoptosis as studied in primary neurons, PC12 cells, and cortical organotypic slice cultures. Blocking RBM3 expression in neuronal cells by specific siRNAs significantly diminished the neuroprotective effect of hypothermia while vector-driven RBM3 over-expression reduced cleavage of PARP, prevented internucleosomal DNA fragmentation, and LDH release also in the absence of hypothermia. Together, neuronal RBM3 up-regulation in response to hypothermia apparently accounts for a substantial proportion of hypothermia-induced neuroprotection. Together, neuronal RBM3 up-regulation in response to hypothermia apparently accounts for a substantial proportion of hypothermia-induced neuroprotection.

Hypoxia and other adverse conditions are commonly encountered by rapidly growing cells. The RNA-binding protein RBM3 (RNA-binding motif protein 3), which is transcriptionally induced by low temperature and hypoxia, has recently been implicated in survival of colon cancer cells by mechanisms involving cyclooxygenase-2 (COX-2) signaling. Immunohistochemically, we found strong RBM3 expression in a variety of malignant and proliferating tissues but low expression in resting and terminally differentiated cells. RBM3 expression in fibroblasts and human embryonal kidney (HEK293) cells subjected to serum deprivation or contact inhibition closely paralleled proliferation rates, assessed by real-time RT-PCR and immunoblotting. siRNA-mediated RBM3 knockdown reduced cell viability and finally led to cell death, which did not involve caspase-3-mediated apoptosis, cell cycle arrest, or COX-2 regulation. In contrast, RBM3 over-expression rescued cells from death under serum starvation. This was associated with increased translation rates, as measured by 14 C serine and 3 H phenylalanine incorporation. Together, RBM3 is a critical factor providing cellular survival advantages in an adverse microenvironment presumably by restoring translation efficacy. We have previously demonstrated that both RBM3 mRNA and protein levels increase in response to hypoxia in a hypoxia inducible factor (HIF)-1-independent fashion (2). RBM3 is also one of the first proteins synthesized in response to cold shock (3,4) and consistently elevated during winter in hibernating animals, such as the ground squirrel (5).An involvement of RBM3 in the regulation of translation has been deduced from increased rates of protein synthesis associated with its over-expression in transfected cells or RBM3 up-regulation after hypothermia (6). Over-expression of RBM3 has also been shown to suppress cell death in cells harboring polyglutamine-huntingtin (7) and to stabilize cyclooxygenase-2 (COX-2) mRNA transcripts (8). Moreover, RBM3 was found to prevent cells from caspase-mediated apoptosis and mitotic catastrophe (9). On the other hand, down-regulation of RBM3 in human colon cancer cells has been reported to cause loss of COX-2 translation and decrease cell growth in culture, which was partially overcome with the COX-2 product, prostaglandin E2 (9).Here, we set out to further characterize the link between RBM3, cell proliferation, and apoptosis. We found that RBM3 expression paralleled proliferation rates in vitro and in vivo. Over-expression of RBM3 rescued human embryonal kidney (HEK293) cells from cell death triggered by serum starvation, and siRNA-mediated RBM3 knockdown-induced cell death occurred without caspase-3 activation. Both artificial up-and down-regulation of RBM3 in HEK293 cells had no effect on COX-2 expression, indicating that other mechanisms are involved that improve cell survival especially under adverse conditions. MATERIALS AND METHODSCell lines, lymphocyte preparation, culture conditions, and treatment.

ObjectivesThe current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains.MethodsA commercial IVIS Kinetic® system and a custom-built laser scanning system with fluorescence molecular tomography (FMT) capability were used to detect fluorescent Mtb in living mice and lungs ex vivo. The resulting images were analysed and the fluorescence was correlated with data from cfu assays.ResultsWe have shown that fluorescent Mtb can be visualized in the lungs of living mice at a detection limit of ∼8 × 107 cfu/lung, whilst in lungs ex vivo a detection limit of ∼2 × 105 cfu/lung was found. These numbers were comparable between the two imaging systems. Ex vivo lung fluorescence correlated to numbers of bacteria in tissue, and the effect of treatment of mice with the antibiotic moxifloxacin could be visualized and quantified after only 9 days through fluorescence measurements, and was confirmed by cfu assays.ConclusionsWe have developed a new and efficient method for anti-tuberculosis drug testing in vivo, based on fluorescent Mtb reporter strains. Using this method instead of, or together with, cfu assays will reduce the time required to assess the preclinical efficacy of new drugs in animal models and enhance the progress of these candidates into clinical trials against human tuberculosis.

These data suggest that BCG vaccination of infants induces specific polyfunctional T-helper-1 and T-helper-17 responses and the ability, in the PBMC compartment, to inhibit the growth of mycobacteria in vitro. We also demonstrate that polyfunctional T-helper-1 cells may play a role in growth inhibition as evidenced by a significant correlation between the two.