Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.

Animal studies with Streptococcus pneumoniae have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive lux transposon cassette, Tn4001 luxABCDE Km(r), that allows random integration of lux genes onto the bacterial chromosome was constructed. The cassette was designed so that the luxABCDE and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome. S. pneumoniae D39 was transformed with plasmid pAUL-A Tn4001 luxABCDE Km(r), and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical S. pneumoniae isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the lux construct was highly stable even after several days in vivo. We believe that this lux transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence.

Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct.

Strains of Staphylococcus aureus were transformed with plasmid DNA containing a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria. S. aureus cells containing this novel lux construct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37 degrees C and do not require exogenous aldehyde substrate, thus allowing S. aureus infections in living animals to be monitored by bioluminescence. Two strains of S. aureus 8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoring S. aureus infections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies.

Apoptosis is induced by transforming growth factor-beta 1 within 5 hours in regressing liver without significant fragmentation of the DNA.

In previous studies we showed that transforming growth factor-beta 1 induces apoptosis in hepatocyte cultures and regressing livers, the mature form being more potent than the transforming growth factor-beta 1 latency-associated protein. In this study we addressed the question of whether apoptosis can be induced within a short time after administration of transforming growth factor-beta 1. Five hours after a single intravenous injection of 25 micrograms mature transforming growth factor-beta 1/kg body weight, apoptosis is augmented ninefold in the regressing rat liver. A second preceding application induces no further augmentation. Transforming growth factor-beta 1 latency-associated protein shows no effect with either regimen. Morphological evaluation shows that 5 hr after injection of transforming growth factor-beta 1 nearly all apoptotic bodies are already engulfed by their neighbor cells. After homogenization of the transforming growth factor-beta 1-treated livers, the condensed apoptotic bodies are not destroyed and remain in the nuclear pellet. No DNA fragmentation into oligosomes could be detected after purification of the DNA from the nuclear pellet and application to conventional gel electrophoresis. Application of in situ nick translation, which allows detection of DNA single- and double-strand breaks in individual apoptotic bodies, also revealed no substantial fragmentation of the DNA in apoptotic bodies. These studies show that transforming growth factor-beta 1 is able to induce apoptosis within a rather short time and also suggest that in vivo digestion of the DNA does not lead to chromatin condensation.

Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease.

A current hypothesis holds that chromatin fragmentation into oligonucleosomal patterns is an early event during apoptosis. In contrast, induction of apoptosis in cultured hepatocytes by TGF-beta 1 was not associated with DNA fragmentation into oligonucleosomes in hepatocyte monolayers and apoptotic fragments. For a more rigorous test of the hypothesis we performed a number of experiments. We compared nuclear changes resulting from TGF-beta 1 with those induced by Ca2+, a known activator of endonuclease. The morphology of apoptotic and Ca(2+)-treated nuclei was different as judged by DNA staining with Hoechst 33258. Likewise, electron microscopy of apoptotic nuclei showed characteristic condensation of the chromatin as well as dissolution of the nucleolar structure and nuclear fragmentation, changes not seen after Ca2+ treatment, after three hours of incubation. Analysis of DNA fluorescence of nuclei by FACS revealed that treatment with Ca2+ reduced the signal by 20%. In contrast, nuclei from TGF-beta 1-treated hepatocytes did not exhibit a reduced signal and after sorting by FACS, apoptotic nuclei remained in the 2N and 4N fractions. The absence of detectable DNA fragmentation in apoptotic nuclei was further verified by in situ nick translation, not only in hepatocytes but also in a mouse lymphoma cell line. From these findings we conclude that activation of an endonuclease is not an early event on the pathway to morphologically recognizable apoptosis.

Induction of apoptosis in cultured hepatocytes and in the regressing liver by transforming growth factor-beta 1 occurs without activation of an endonuclease.

In previous studies in vivo apoptotic liver cells were found to be positive for transforming growth factor-beta 1 (TGF-beta 1). In hepatocyte cultures TGF-beta 1 induced rounding up and fragmentation of the cells into multiple vesicles. As revealed by the DNA specific stain H33258 the chromatin of these cells condensed and segregated into masses at the nuclear membrane, followed by nuclear fragmentation. Ultrastructurally the cytoplasm was well preserved as demonstrated by the presence of intact cell organelles. These features strongly suggest that occurrence of apoptosis. Furthermore we administered TGF-beta 1 in vivo using an experimental model in which regression of the liver was initiated by a short preceding treatment with the hepatomitogen cyproterone acetate (CPA). Two doses of 1 nM TGF-beta 1/kg each augmented the incidence of apoptotic hepatocytes 5-fold. These studies strongly suggest that TGF-beta 1 is involved in the initiation of apoptosis in the liver In TGF-beta 1 treated hepatocytes both from the liver and monolayer culture no DNA fragmentation into oligosomes could be detected. Comparison of nuclei in which endonuclease was activated by Ca2+ with apoptotic nuclei revealed no obvious similarities, as demonstrated by FACS analysis, H33258 staining and electron microscopy. Thus, apoptosis induced by a growth inhibitor obviously occurs without activation of an endonuclease.