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Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae.

ABSTRACT: The recent availability of bacterial genome sequence information permits the identification of conserved genes that are potential targets for novel antibiotic drug discovery. Using a coupled bioinformatic/experimental approach, a list of candidate conserved genes was generated using a Microbial Concordance bioinformatics tool followed by a targeted disruption campaign. Pneumococcal sequence data allowed for the design of precise PCR primers to clone the desired gene target fragments into the pEVP3 'suicide vector'. An insertion-duplication approach was employed that used the pEVP3 constructs and resulted in the introduction of a selectable chloramphenicol resistance marker into the chromosome. In the case of non-essential genes, cells can survive the disruption and form chloramphenicol-resistant colonies. A total of 347 candidate reading frames were subjected to disruption analysis, with 113 presumed to be essential due to lack of recovery of antibiotic-resistant colonies. In addition to essentiality determination, the same high-throughput methodology was used to overexpress gene products and to examine possible polarity effects for all essential genes.

SUBMITTER: Thanassi JA

PROVIDER: S-EPMC135739 | biostudies-literature | 2002 Jul

REPOSITORIES: biostudies-literature

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Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae.

Thanassi Jane A JA Hartman-Neumann Sandra L SL Dougherty Thomas J TJ Dougherty Brian A BA Pucci Michael J MJ

Nucleic acids research 20020701 14

The recent availability of bacterial genome sequence information permits the identification of conserved genes that are potential targets for novel antibiotic drug discovery. Using a coupled bioinformatic/experimental approach, a list of candidate conserved genes was generated using a Microbial Concordance bioinformatics tool followed by a targeted disruption campaign. Pneumococcal sequence data allowed for the design of precise PCR primers to clone the desired gene target fragments into the pEV ...[more]

PMID: 12136097

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Project description:Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.

Project description:We applied genomic array footprinting (GAF) in the search for genes of S. pneumoniae essential during the establishment and progression of experimental meningitis. Four libraries with a total of 6,000 independent TIGR4 marinerT7 transposon mutants were injected intra-cisternally in rabbits, and cerebrospinal fluid (CSF) was collected after three, nine, and fifteen hours. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 genes of which mutants had become attenuated and eleven genes of which mutants had become enriched during infection. Screening results particularly point to an essential role for capsular polysaccharides (cps), nutrient uptake, and metabolism in meningitis. Detailed study on directed mutants of a subset of sixteen GAF targets in an experimental model of rat meningitis revealed that ten were significantly attenuated or enriched during competitive infection. Furthermore, we showed that mutants of adenylosuccinate synthetase (purA), flavodoxin (fld), and the substrate binding protein (livJ) of a branched chain amino acid ABC-transporter were essential for full blown meningitis in a mono-infection setup of rat meningitis. Overall, the GAF screen revealed the general restraint on nutrients encountered by the pneumococcus in CSF, while, except for cps genes, no ‘classic’ virulence factors appeared to be required for infection. This knowledge will contribute to a better understanding of the molecular pathogenesis of pneumococcal meningitis. The rabbit meningitis model was performed as previously described by Østergaard (PMID: 9661008 ). Briefly, 10E6 CFU of a S. pneumoniae mutant library was suspended in 20 µl beef-broth and injected into cisterna magna of four groups of four outbred New Zealand White rabbits of app. 2.5 kg (in one group one rabbit died). CSF (0.3 ml) samples were repeatedly obtained from the same rabbit by aspiration from indwelling spinal- and arterial cannules. Samples were collected before pleocytosis developed (three hrs), at the time of maximal pleocytosis (nine hrs), and at a time of slowing bacterial growth and abating pleocytosis (fifteen hrs ). Until further processing in the GAF protocol, CSF and inocula were stored with 15% glycerol at -80ºC. The GAF technology was performed as described previously (PMID: 17261526 ). Briefly, stored inocula and CSF obtained during the experimental rabbit meningitis experiments were defrosted, diluted in GM17 medium supplemented with spectinomycin, and grown to mid-log phase. Chromosomal DNA was isolated, digested with AluI endonuclease, and used as a template for in vitro transcription initiated from the T7 promoters of the marinerT7 transposon that is present in each mutant. After removal of template DNA by DNAseI treatment, mutant-specific T7 RNA was reverse transcribed in the presence of fluorescent Cy3/Cy5-labeled dUTP nucleotides. Labeled Cy3/Cy5-cDNA from the CSF samples and oppositely labeled Cy5/Cy3 cDNA from the inocula were combined, purified, and washed by ultrafiltration. The cDNA was hybridized to pneumococcal microarrays containing, 2,087 ORFs of Streptococcus pneumoniae TIGR4 and 70-mer oligo's specific for the non-homologous ORFs in the R6, D39, 23F, INV104B, INV200, OXC141, and G54 strains, all spotted in duplicate (PMID: 18174343) . Dual channel array images were acquired on a GenePix 4200AL microarray scanner (Axon Instruments, Union City, CA). Microarray image files were analyzed with GenePix Pro software (Axon Instruments, Union City, CA). Spots were screened visually to identify those of low quality and removed from the data set prior to analysis. A net mean intensity filter based on hybridization signals obtained with R6-specific spots was applied in all experiments. Slide data were processed and normalized using MicroPreP (PMID: 15907200). Further analysis was performed using a CyberT implementation of the Student’s t test (cybert.microarray.ics.uci.edu). This web-based program lists the ratios of all intra-replicates (duplicate spots) and inter-replicates (different slides), the mean ratios per gene, and standard deviations and (Bayesian) p values assigned to the mean ratios. For identification of conditionally essential genes, only genes with a minimum of 6/8 (for groups of four rabbits) or 5/6 for the group of three rabbits) reliable measurements and a Bayesian p-value < 0.001 were included. Further selection criteria were applied, as only mutants displaying an average fold-change of >2.5 at a minimum of two time-points, or >4.0 at one time-point were accepted as significantly attenuated or enriched. The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo/) under GEO Series accession number ...

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Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae.

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Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae.

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