Limits...
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 the open dimeric conformation of DNA binding/cleavage domain of GyrA from FTL_0533.Left plot: the active site residues essential for DNA cleavage Arg121 and Tyr122 are shown in yellow sticks. Right plot close-up of the region outlined by the rounded rectangle shows location of the mutated positions Thr83 and Asp87.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036845&req=5

pone.0163458.g002: Structural model of the open dimeric conformation of DNA binding/cleavage domain of GyrA from FTL_0533.Left plot: the active site residues essential for DNA cleavage Arg121 and Tyr122 are shown in yellow sticks. Right plot close-up of the region outlined by the rounded rectangle shows location of the mutated positions Thr83 and Asp87.

Mentions: A protein structural model of FTL_0533 was constructed to identify the potential structural and functional effects of the two mutations at positions 83 and 87. The N-terminal domain of GyrA from E. coli (PDB chain: 2wl2_B) was used to construct the homology model. The percentage of amino acid sequence identity between FTL_0533 and 2wl2_B is 65%. Structural analysis of created models showed that the mutations at Thr83 and Asp87 are located on the fragment 83–90 which is a short helical region in close proximity to the active site residues Arg121 and Tyr122 (Fig 2). A recent study of the gyrA crystal structure from E. coli showed that the substitutions in the region 81 to 84 and at position 87 in F. turlarensis would likely impact drug binding [20].


Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F . tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing
Structural model of the open dimeric conformation of DNA binding/cleavage domain of GyrA from FTL_0533.Left plot: the active site residues essential for DNA cleavage Arg121 and Tyr122 are shown in yellow sticks. Right plot close-up of the region outlined by the rounded rectangle shows location of the mutated positions Thr83 and Asp87.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0163458.g002: Structural model of the open dimeric conformation of DNA binding/cleavage domain of GyrA from FTL_0533.Left plot: the active site residues essential for DNA cleavage Arg121 and Tyr122 are shown in yellow sticks. Right plot close-up of the region outlined by the rounded rectangle shows location of the mutated positions Thr83 and Asp87.
Mentions: A protein structural model of FTL_0533 was constructed to identify the potential structural and functional effects of the two mutations at positions 83 and 87. The N-terminal domain of GyrA from E. coli (PDB chain: 2wl2_B) was used to construct the homology model. The percentage of amino acid sequence identity between FTL_0533 and 2wl2_B is 65%. Structural analysis of created models showed that the mutations at Thr83 and Asp87 are located on the fragment 83–90 which is a short helical region in close proximity to the active site residues Arg121 and Tyr122 (Fig 2). A recent study of the gyrA crystal structure from E. coli showed that the substitutions in the region 81 to 84 and at position 87 in F. turlarensis would likely impact drug binding [20].

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.