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Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.
Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus’ ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.
Streptococcus pneumoniae (S. pneumoniae, the pneumococcus) is one of the principal causes of morbidity and mortality globally. Up until recently, close to 100 serotypes of S. pneumoniae have been discovered1,2,3,4,5,6,7. Yearly, invasive pneumococcal disease (IPD) claims around 700,000 deaths, of children younger than 5 years old8. S. pneumoniae is the major cause of bacterial pn....
1. Generation of the mutating amplicon by SOE-PCR23 and amplification of the spectinomycin cassette
The protocol described here starts by using PCR to amplify the left and right homology arms, whilst simultaneously deleting 191 bp from the middle region of the ply gene. While performing the PCR a BamHI site is introduced at the 3’ of the left homology arm and at the 5’ end of the right homology arm (Figure 1A). This is followed by PCR-SOE where left and right homology arms are fused into one amplicon (Figure 1B). This SOE-PCR amplicon is t.......
Streptococcus pneumoniae, in particular serotype 1, continues to be a global threat causing invasive pneumococcal disease and meningitis. Despite the introduction of various vaccines that should be protective against serotype 1, in Africa, this serotype is still capable of causing outbreaks that lead to high morbidity and mortality13. The ability to genetically manipulate this serotype is of critical importance because of its clinical relevance. The method described in this study allows t.......
We would like to thank the Meningitis Trust and the MRC for providing funding for this work.
....Name | Company | Catalog Number | Comments |
AccuPrime Pfx DNA polymerase | Invitrogen | 12344024 | Used for amplification of the fragments |
Ampicillin sodium salt | Sigma Aldrich | A9518 | Used for bacterial selection on stage 1(pSD1) |
Blood Agar Base | Oxoid | CM0055 | Used to plate S. pneumoniae transformants |
Bovine Serum Albumine | sigma | 55470 | used for S. pneumoniae Transformation |
Brain Heart Infusion | Oxoid | CM1135 | used to grow S. pneumoniae cells |
Calcium Chloride Cacl2 | Sigma | 449709 | used for S. pneumoniae Transformation |
Competence stimulating peptide 1 | AnaSpec | AS-63779 | used for S. pneumoniae Transformation |
Luria Broth Agar | Gibco | 22700025 | used for plating and selection of pSD1 and pSD2 |
Luria Broth Base (Miller's formulation) | Gibco | 12795027 | used for plating and selection of pSD1 and pSD2 |
Monarch Gel Extraction Kit | NEB | T1020S | Used to extract the bands from the DNA gel |
Monarch Plasmid Miniprep Kit | NEB | T1010S | Used to extract plasmid from the cells |
pGEM T-easy | Promega | A1360 | used as suicide plasmid |
S.O.C. | Invitrogen | 15544034 | used for recovery of cells after transformation |
Sodium Hydroxide (NaOH) | Sigma | S0899 | used for S.pneumoniae Transformation |
Spectinomycin Hydrochloride | SigmaAldrich | PHR1426 | Used for bacterial selection |
Subcloning Efficiency DH5α Competent Cells | Invitrogen | 18265017 | used for the creation of pSD1 and pSD2 |
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