Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence

PLOS ONE, Nov 2019

Bioluminescence has been harnessed for use in bacterial reporter systems and for in vivo imaging of infection in animal models. Strain Xen35, a bioluminescent derivative of Streptococcus pneumoniae serotype 4 strain TIGR4 was previously constructed for use for in vivo imaging of infections in animal models. We have shown that strain Xen35 is less virulent than its parent TIGR4 and that this is associated with the expression of the genes for bioluminescence. The expression of the luxA-E genes in the pneumococcus reduces virulence and down regulates the expression of the pneumococcal pilus.

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Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence

January Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence Jenny A. Herbert 0 1 Andrea M. Mitchell 0 1 Ryan Ritchie 1 Jiangtao Ma 1 Kirsty Ross- Hutchinson 1 Timothy J. Mitchell 0 1 0 Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham , Birmingham , United Kingdom , 2 Institute of Infection , Immunity and Inflammation , College of Medical, Veterinary and Life Sciences, University of Glasgow , Glasgow , United Kingdom , 3 Technology Hub Manager, Infrastructure and Facilities , College of Medical and Dental Sciences, University of Birmingham , Birmingham , United Kingdom 1 Editor: Jens Kreth, Oregon Health & Science University , UNITED STATES Bioluminescence has been harnessed for use in bacterial reporter systems and for in vivo imaging of infection in animal models. Strain Xen35, a bioluminescent derivative of Streptococcus pneumoniae serotype 4 strain TIGR4 was previously constructed for use for in vivo imaging of infections in animal models. We have shown that strain Xen35 is less virulent than its parent TIGR4 and that this is associated with the expression of the genes for bioluminescence. The expression of the luxA-E genes in the pneumococcus reduces virulence and down regulates the expression of the pneumococcal pilus. - Data Availability Statement: All primary data has been deposited in NCBI Bioproject database (Accession Number: PRJNA421796). All other data are within the paper and its Supporting Information files. Funding: Work was funded using a PhD studentship for Dr Jenny Herbert from the Biotechnology and Biological Sciences Research Council (BBSRC). Competing interests: The authors have declared that no competing interests exist. Introduction S. pneumoniae is normally found as part of the commensal flora in the nasopharynx of humans and is also the leading cause of bacterial pneumonia. Pneumococcal infection can also manifest as meningitis, septicaemia and otitis media. To study pneumococcal disease a well characterised murine model is widely used [ 1, 2 ]. Recent advances in the development of fluorescent and bioluminescent reporters have made it possible to visualise disease progression in animals in real time. This methodology also reduces animal numbers used. Bioluminescence is generated by a chemical reaction which releases energy in the form of light [3]. This occurs naturally in several organisms. The bioluminescence reaction consists of oxidation of reduced flavin mononucleotide (FMNH2) and a long chain aldehyde (RCHO) to produce light, flavin mononucleotide (FMN), fatty acids (RCOOH) and water. Five genes are required for all bioluminescent reactions in all bacterial species [ 4 ]. These genes are luxA/luxB, which encode the luciferase enzyme and luxC-E, which encode the enzymes required for synthesis of the substrate for the luciferase [ 4 ]. Oxygen is also required for the enzymatic reaction to take place. Cloning of the lux genes into non-native organisms to make them bioluminescent has enabled this chemical reaction to be utilised as a tool to visualise bacteria. S. pneumoniae is naturally competent making genetic manipulation of most strains relatively easy [ 5, 6 ]. To study pneumococcal disease progression in vivo using bioluminescence, a panel of bioluminescent pneumococcal strains, that are now commercially available, were constructed [7±9]. Initial construction was performed by placing luxA-E amplified from Photorhabdus luminescens (formerly known as Xenorhabdus luminescens) and a kanamycin resistance cassette onto a promoterless transposon, which was randomly inserted into the genome of serotype 2 strain D39 [ 7, 8 ]. Bacteria were screened for levels of bioluminescence and the most highly expressing strain was designated Xen7. The site of insertion of the lux genes was mapped and shown to be in the gene coding for a hypothetical protein of unknown function (Gene number SPD_1717). This mutation was then transferred into a panel of S. pneumoniae strains by transformation using genomic DNA isolated from Xen7 [ 8 ]. Transformation was initially performed into a serotype 4 strain TIGR4, which was the first strain of S. pneumoniae to undergo complete whole genome sequencing [ 10 ]. Transformation of TIGR4 with Xen7 DNA and selection of luminescent mutants gave rise to Xen35, which had luxA-E inserted into SP_1914 (the TIGR4 equivalent of SPD_1717). The stable integration of these genes into Xen35 removed the need for use of antibiotics in vivo to maintain a plasmid expressing these genes. The Xen strains and other bioluminescent strains have been used in a number of studies to evaluate virulence of S. pneumoniae in vivo [8, 9, 11±14]. A recent study showed that a constitutive expression of the genes required for bioluminescence in Citrobacter rodentium resulted in a competitive disadvantage in a mouse model of infection and impaired growth in minimal medi (...truncated)


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Jenny A. Herbert, Andrea M. Mitchell, Ryan Ritchie, Jiangtao Ma, Kirsty Ross-Hutchinson, Timothy J. Mitchell. Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence, PLOS ONE, 2018, Volume 13, Issue 1, DOI: 10.1371/journal.pone.0189426