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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_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 [21–24]. Ala69 is located on the edge of the interface in close proximity of these residues.
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pone.0163458.g003: 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 [21–24]. Ala69 is located on the edge of the interface in close proximity of these residues.

Mentions: A total of two mutations were identified in this gene from two isolates. The mutation at position 589,311 (amino acid position 110) is a synonymous mutation while the mutation at 589,187 is a non-synonymous mutation. The wild-type triplet encodes Ala at position 69 in the corresponding protein. The mutation found results in a Glu at this position. One of the closest structural templates for modeling of FTL_0601 is a crystal structure of DesI from Streptomyces venezuelae (PDB chain: 2po3_A). The level of sequence identity between FTL_0601 and 2po3_A is 29%. Another identified structural template with a similar level of sequence identity is GDP-perosamine synthase from Caulobacter crescentus (PDB chain: 3dr4_C). Both templates belong to the same aspartate aminotransferase superfamily. The functional units of enzymes in this group are typically formed as homodimers with an extensive subunit/subunit interface [21]. Construction of a structural model of FTL_0601 and comparative analysis with these two proteins suggests that the corresponding interface in FTL_0601 is formed by the following segments: Lys8-Asn32, N58-Arg68, F83-Asn93, Ile188-E189, F207-Ile212, and Ile218-F230. In Fig 3 examples of critical residues (colored as yellow sticks) found within these segments were shown: interacting residues Arg68 and Ser92, and highly conserved residues Thr60 and Asn224 which are involved in stabilizing interaction between ligand and protein [22]. The detected mutation associated with Cipro resistance is located at Ala69; in immediate proximity to these critical residues (Fig 3). Recently published studies [23, 24]on structural analysis of active sites of different aminotransferases suggests that the observed differences in residues in close proximity to functionally critical residues may be crucial for enzyme function, substrate binding and specificity. Results from the StralSV analysis indicate that the Ala69 position can absorb substitutions with different types of amino acids without significant conformational change of the backbone structure (terminal part of the α-helix). The list of observed diversity of amino acids at the corresponding position in homologous proteins is provided in the Table 2.


Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F . tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing
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 [21–24]. Ala69 is located on the edge of the interface in close proximity of these residues.
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Related In: Results  -  Collection

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Show All Figures
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pone.0163458.g003: 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 [21–24]. Ala69 is located on the edge of the interface in close proximity of these residues.
Mentions: A total of two mutations were identified in this gene from two isolates. The mutation at position 589,311 (amino acid position 110) is a synonymous mutation while the mutation at 589,187 is a non-synonymous mutation. The wild-type triplet encodes Ala at position 69 in the corresponding protein. The mutation found results in a Glu at this position. One of the closest structural templates for modeling of FTL_0601 is a crystal structure of DesI from Streptomyces venezuelae (PDB chain: 2po3_A). The level of sequence identity between FTL_0601 and 2po3_A is 29%. Another identified structural template with a similar level of sequence identity is GDP-perosamine synthase from Caulobacter crescentus (PDB chain: 3dr4_C). Both templates belong to the same aspartate aminotransferase superfamily. The functional units of enzymes in this group are typically formed as homodimers with an extensive subunit/subunit interface [21]. Construction of a structural model of FTL_0601 and comparative analysis with these two proteins suggests that the corresponding interface in FTL_0601 is formed by the following segments: Lys8-Asn32, N58-Arg68, F83-Asn93, Ile188-E189, F207-Ile212, and Ile218-F230. In Fig 3 examples of critical residues (colored as yellow sticks) found within these segments were shown: interacting residues Arg68 and Ser92, and highly conserved residues Thr60 and Asn224 which are involved in stabilizing interaction between ligand and protein [22]. The detected mutation associated with Cipro resistance is located at Ala69; in immediate proximity to these critical residues (Fig 3). Recently published studies [23, 24]on structural analysis of active sites of different aminotransferases suggests that the observed differences in residues in close proximity to functionally critical residues may be crucial for enzyme function, substrate binding and specificity. Results from the StralSV analysis indicate that the Ala69 position can absorb substitutions with different types of amino acids without significant conformational change of the backbone structure (terminal part of the α-helix). The list of observed diversity of amino acids at the corresponding position in homologous proteins is provided in the Table 2.

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.