Visualizing Pneumococcal Infections in the Lungs of Live Mice Using Bioluminescent Streptococcus pneumoniae Transformed with a Novel Gram-Positive lux Transposon

Abstract

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.Streptococcus pneumoniae is the leading cause of invasive bacterial disease in the very young and the elderly and is the bacterium most responsible for community-acquired pneumonia in the developed world (31). It can behave as a transient commensal, colonizing the nasopharynx of 40% of healthy adults and children, with no adverse effects (2). Children carry this pathogen in the nasopharynx asymptomatically for about 4 to 6 weeks, often carrying several serotypes at a time (13, 33). Occasionally, perhaps in conjunction with a viral infection (9), one of these strains gives rise to a symptomatic pneumococcal infection, including sinusitis, otitis media, pneumonia, and meningitis (12, 13, 16).Antibiotic treatment of pneumococcal infections has been less effective in recent years with the increased occurrence of multidrug-resistant strains of S. pneumoniae. About one-third to one-half of pneumococci recovered from humans are at least partially resistant to penicillin, which may occur in addition to resistance to a number of other common antibiotics (1, 3). These factors, plus the ability of the pneumococcus to transfer genes for resistance, encapsulation, and virulence via transformation (12), make it imperative to develop a better understanding of the mechanism by which pneumococci cause disease. Probably the best way to enhance this process is to develop a better animal model.In 1995, Contag et al. (7) showed that it was possible to monitor disease processes in living animals using b...

Bacterial vectors for imaging and cancer gene therapy: a review

“…This is exemplified by the decrease in luminescence that has been observed when many luxexpressing bacterial species, such as Bifidobacterium, 38 enter stationary phase during in vitro growth. [46][47][48] Bioluminescent bacteria have been used extensively to study a diverse range of biological processes such as infection, 49 quorum sensing 50 and gene expression. 51 However, the employment of luciferase or lux tagged bacterial strains as gene delivery vehicles for the treatment of cancer is an emerging field.…”

“…For example, strains of E. coli rendered metabolically dependent on diaminopimelic acid (DPI) have been generated, permitting maintenance of plasmids featuring DPI-producing genes as plasmid loss lead to bacterial cell death. 68,69 Transposon, 46,70 transducing bacteriophages, 42 temperature sensitive vectors 71 and bacteriophage integrase systems [72][73][74] have been developed to integrate genes into the chromosomes of selected bacteria. However, it is important to stress that the maintenance and expression of high levels of recombinant DNA may place an unwelcome metabolic burden microorganisms.…”

Reporter systems forin vivotracking of lactic acid bacteria in animal model studies

“…28,29 To circumvent the problem, Gram-positive ribosomal binding sites were cloned into the lux operon. 30 A vast array of reporter systems is available ( Tables 2 and 3). The most popular include bioluminescence with bacterial lux genes (lux), firefly and click beetle luciferase (luc), and fluorescence recorded in the case of green and red proteins.…”

“…2) originates from a Photorhabdus sp. 30,54 Bacterial luciferase is encoded by the structural genes luxA and luxB. Luciferase is thus a heterodimer composed of 2 different polypeptides known as a and b, respectively.…”

“…66 The bacterial luciferase system is predominantly used when bacterial infection models are studied. 30,67 An example of this is the cloning of the P. luminescence lux operon into infectious pathogens such as E. coli, Citrobacter rodentium, Listeria monocytogenes and Salmonella spp [20][21][22][23] Pathogenicity of the labeled bacteria remained unaffected and they were visualized throughout infection. Bacteria labeled with the lux genes were detected in the GIT of mice (Fig.…”