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

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.

Fig 1. The test statistic values for a short region of the DNA gyrase A gene in one of the Cipro resistant F. tularensis isolates.

The log likelihood ratio has a clear peak in this region. Candidate SNP positions were identified by looking for regions of the genome where the log likelihood ratio exceeds a fixed threshold.

Fig 2. 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.

Fig 3. Structural model of FTL_0601 with two mutation positions Ala69, and Asp110 labeled.

Left plot: a ribbon representation of two subunits forming homodimer with mutation positions Ala69 and Asp110 shown as spheres colored in red and blue respectively (the asterisk indicates residue from the second subunit of the dimer). Asp110 is located on the surface of the protein within conserved helical region outside the interface area. Right plot: close-up of the region showing Ala69 located at the end of the helical segment Asn58-Ala69 which is a part of the interface between subunits. Examples of three residue positions within this helical region are shown as yellow sticks and their functional importance can be described based on annotation of corresponding positions in other homologous aminotransferases. In particular: two residues Asn58 and Arg68 from both ends of the helix contribute to the interface formation by interacting with Asn224* and Ser92* respectively, the residues Thr60 and Asn224* are both highly conserved and are involved in stabilizing interaction between ligand and protein [–24]. Ala69 is located on the edge of the interface in close proximity of these residues.

Fig 4. 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.