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Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F . tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

View Article: PubMed Central - PubMed

ABSTRACT

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

No MeSH data available.


Structural model of FTL_1547 in its dimeric conformation (chains A and B) complexed with DNA.A mutation position Ser465 is colored in red. Ser465 is located next to positions 463, 460, 453 and 456 (colored in orange) that correspond to 471, 468, 464 and 461 from the X-ray structure (PDB chain 4i3h_A) described in [32] as critical functional positions located in the helical region of the C-gate facilitating DNA (T-segment) release.
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pone.0163458.g004: Structural model of FTL_1547 in its dimeric conformation (chains A and B) complexed with DNA.A mutation position Ser465 is colored in red. Ser465 is located next to positions 463, 460, 453 and 456 (colored in orange) that correspond to 471, 468, 464 and 461 from the X-ray structure (PDB chain 4i3h_A) described in [32] as critical functional positions located in the helical region of the C-gate facilitating DNA (T-segment) release.

Mentions: FTL_1547 is a type II DNA topoisomerase enzyme that catalyzes topological rearrangement of double-stranded DNA by generating a transient double-stranded break in one DNA duplex (the ‘G’ or gate segment) and passing another duplex (the ‘T’ or transported segment) through the break before resealing it. It is a known target for Cipro resistance. A SNP at location 1,477,418 was identified by microarray analysis and Illumina sequencing. Only one isolate 18:5:2 from the 11 Cipro resistant isolates contained this mutation. Sanger sequencing results showed that none of the first round isolates tested contained this mutation but the majority of the second round Cipro resistant isolates had this mutation. This suggests that the mutation occurred during the second round of the selection at higher Cipro concentrations. The mutation results in an amino acid change from Ser to Tyr and is located next to positions 463, 460, 453 and 456. These positions correspond to 471, 468, 464 and 461 from the X-ray structure of topoisomerase from Streptococcus pneumoniae (PDB chain: 4i3h_A) and are described in [25] as critical functional positions located in the helical region of the C-gate facilitating DNA (T-segment) release (Fig 4). A second mutation was identified by Illumina sequencing at position 1,477,419, but this mutation was not detected in the analysis of the microarray data.


Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F . tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing
Structural model of FTL_1547 in its dimeric conformation (chains A and B) complexed with DNA.A mutation position Ser465 is colored in red. Ser465 is located next to positions 463, 460, 453 and 456 (colored in orange) that correspond to 471, 468, 464 and 461 from the X-ray structure (PDB chain 4i3h_A) described in [32] as critical functional positions located in the helical region of the C-gate facilitating DNA (T-segment) release.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036845&req=5

pone.0163458.g004: Structural model of FTL_1547 in its dimeric conformation (chains A and B) complexed with DNA.A mutation position Ser465 is colored in red. Ser465 is located next to positions 463, 460, 453 and 456 (colored in orange) that correspond to 471, 468, 464 and 461 from the X-ray structure (PDB chain 4i3h_A) described in [32] as critical functional positions located in the helical region of the C-gate facilitating DNA (T-segment) release.
Mentions: FTL_1547 is a type II DNA topoisomerase enzyme that catalyzes topological rearrangement of double-stranded DNA by generating a transient double-stranded break in one DNA duplex (the ‘G’ or gate segment) and passing another duplex (the ‘T’ or transported segment) through the break before resealing it. It is a known target for Cipro resistance. A SNP at location 1,477,418 was identified by microarray analysis and Illumina sequencing. Only one isolate 18:5:2 from the 11 Cipro resistant isolates contained this mutation. Sanger sequencing results showed that none of the first round isolates tested contained this mutation but the majority of the second round Cipro resistant isolates had this mutation. This suggests that the mutation occurred during the second round of the selection at higher Cipro concentrations. The mutation results in an amino acid change from Ser to Tyr and is located next to positions 463, 460, 453 and 456. These positions correspond to 471, 468, 464 and 461 from the X-ray structure of topoisomerase from Streptococcus pneumoniae (PDB chain: 4i3h_A) and are described in [25] as critical functional positions located in the helical region of the C-gate facilitating DNA (T-segment) release (Fig 4). A second mutation was identified by Illumina sequencing at position 1,477,419, but this mutation was not detected in the analysis of the microarray data.

View Article: PubMed Central - PubMed

ABSTRACT

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

No MeSH data available.