1 The Francisella tularensis FabI enoyl-ACP-reductase gene is

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The Francisella tularensis FabI enoyl-ACP-reductase gene is essential to 1 bacterial viability and is expressed during infection 2

3 Luke C. Kingrya, b, c, Jason E. Cummingsa,b, Kerry W. Brookmana,b, Gopal R. 4 Bomminenid, Peter J. Tonged, and Richard A. Slaydena, b, c * 5 6 a Rocky Mountain Regional Center of Excellence, b Department of Microbiology, 7 Immunology and Pathology, and c Cellular and Molecular Biology, Colorado State 8 University, Fort Collins, CO 80523 9 d Institute for Chemical Biology & Drug Discovery, Department of Chemistry, 10 Stony Brook University, Stony Brook, New York 11794-3400 11 12 13 Running Title: Essentiality of F. tularensis enoyl-ACP-reductase FabI 14 15 Keywords: fatty acid, drug target, FAS-II, tularemia, Francisella tularensis 16 17 *Author to whom correspondence should be addressed: Richard A. Slayden, 18 Department of Microbiology, Immunology and Pathology, Colorado State 19 University, Fort Collins, CO 80523-1682. Phone: 970-491-2902, Email: 20 [email protected] 21

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01957-12 JB Accepts, published online ahead of print on 9 November 2012

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Abstract 24 Francisella tularensis is classified as a Category A priority pathogen and 25 causes fatal disseminated disease in humans upon inhalation of less than 50 26 bacteria. Although drugs are available for treatment, they are not ideal because 27 of toxicity and delivery, and in some cases patients relapse upon withdrawal. We 28 have an ongoing program to develop novel FAS-II FabI enoyl-ACP reductase 29 enzyme inhibitors for Francisella and other select agents. To establish ftFabI in 30 F. tularensis as a clinically relevant drug target we demonstrated that fatty acid 31 biosynthesis and FabI activity is essential for growth even in the presence of 32 exogenous long chain lipids, and that ftfabI is not transcriptionally altered in the 33 presence of exogenous long chain lipids. Inhibition of ftFabI or fatty acid 34 synthesis results in loss of viability that is not rescued by exogenous long chain 35 lipid supplementation. Importantly, whole genome transcriptional profiling of F. 36 tularensis with DNA microarrays from infected tissues revealed that ftfabI and de 37 novo fatty acid biosynthetic genes are transcriptionally active during infection. 38 This is the first demonstration that the FabI enoyl-ACP-reductase enzyme 39 encoded by F. tularensis is essential and not bypassed by exogenous fatty acids, 40 and that de novo fatty acid biosynthetic components encoded in F. tularensis are 41 transcriptionally active during infection in the mouse model of tularemia. 42 43


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44 Introduction 45 Although the incidence of the disease tularemia is low and drugs are 46 available for treatment, interest in the causative agent Francisella tularensis 47 remains due to its highly infectious nature and because of the possibility that F. 48 tularensis could be used deliberately as a bioterror agent. Currently treatment of 49 tularemia consists of a single course of streptomycin augmented with a 50 fluoroquinolone if needed (5). While the emergence of naturally resistant strains 51 is rare, there is a need for new chemotherapeutics to treat acute disseminated 52 infections due to the fact that current therapies must be administered early during 53 infection for positive disease outcome. Further, due to the low LD50 (<50 CFU) of 54 F. tularensis antibiotic treatments that result in 100 percent clearance of the 55 bacteria are required (5, 22, 27). 56

The bacterial fatty acid synthesis pathway (FAS-II) is unique from the 57 mammalian fatty acid biosynthesis pathway and therefore has emerged as an 58 attractive pathway for novel therapeutic intervention. As a rate-limiting step in 59 the FAS-II system, the enoyl-ACP reductase enzyme FabI has been a prime 60 example of an enzyme that can be targeted for clinically relevant drug discovery. 61 As part of our broad-spectrum drug discovery program, we have developed a 62 series of lead compounds with activity against FAS-II enoyl-ACP reductases of 63 Mycobacterium tuberculosis and priority pathogens F. tularensis, Burkholderia 64 pseudomallei and Yersinia pestis (6, 13, 15). Lead compounds have been 65 shown to inhibit ftFabI enoyl-ACP reductase activity in vitro, and demonstrate 66 efficacy against F. tularensis in the mouse model of infection (6, 15). Since drug 67


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efficacy in host models only indirectly demonstrates the essentiality of the ftfabI 68 gene product because of possible off target effects, there is a need to directly 69 determine the requirement for ftFabI for de novo synthesis of fatty acids and 70 activity during infection. 71

Recently it has been reported that there is a significant decrease in 72 expression and production of FAS-II enzymes encoded by Streptococcus 73 agalactiae in the presence of exogenous fatty acids. This observation indicates 74 that this bacterium can utilize host lipids to circumvent de novo fatty acid 75 biosynthesis (4). Based on this observation it has been argued that FAS-II is not 76 a suitable antimicrobial target for gram-positive organisms because fatty acid 77 synthesis is not essential in the host environment. However, it has also been 78 demonstrated that fatty acid synthesis is required to maintain viability of the 79 gram-positive organism Staphylococcus aureus and the differences observed in 80 the essentiality of the enoyl-reductase step of FAS-II may be dependent on the 81 ability of pathogens to subsist on exogenously supplied fatty acids (1). The 82 heterogeneity of these observations suggests that there are organism specific 83 capabilities in terms of nutrient uptake and utilization that may rely heavily on 84 evolutionary background, the metabolic capacity, and the pathobiological niche 85 that an organism occupies (4, 26, 31). 86

While the utilization of host lipids and the extent to which host lipids can 87 be used appear to be more relevant to gram-positive organisms and applicable to 88 specific species of gram-positive bacteria, it is important to evaluate FAS-II 89 essentiality on a pathogen-by-pathogen basis. Gram-negative bacteria are 90


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known to be able to uptake exogenous lipids and utilize them as intermediate 91 substrates for the synthesis of phospholipids, however unique bacterial lipids 92 cannot be obtained directly from host sources, thus requiring biosynthesis by the 93 bacterium. Notably, F. tularensis is characterized by hydroxyl fatty acids (24) 94 and the major lipid A form being tetraacylated with 3-hydroxyoctadecanoic acid, 95 3-hydroxyhexadecanoic acid, hexadecanoic acid, and tetradecanoic acid (28). 96 Although the unique lipid components of F. tularensis are unlikely to be supplied 97 by the host and incorporated as intermediates without biosynthesis, as part of a 98 rigorous academic drug discovery program there is a need to determine if ftFabI 99 is essential under infectious conditions in order to efficiently translate in vitro 100 potency to in vivo efficacy, and to substantiate this protein as a clinically relevant 101 drug target. 102

To experimentally substantiate ftFabI as a clinically relevant drug target for 103 the treatment of tularemia, we set out to verify that ftFabI activity is essential for 104 growth, and that ftfabI expression is not influenced by the presence of 105 exogenously supplied mixture of long chain lipids, and that ftfabI is expressed 106 during infection. We report here that de novo biosynthesis of fatty acids is 107 essential in vitro based on the inability to obtain an auxotrophic strain in the 108 presence of exogenous fatty acids. The presence of exogenous fatty acids do 109 not alter the transcriptional activity of fatty acid biosynthetic genes or ftfabI, and 110 whole genome transcriptional profiling of F. tularemia in infected tissues showed 111 that ftfabI and other fatty acid biosynthetic genes were active during infection. In 112 addition, the minimal inhibitory concentration of known ftFabI lead inhibitors and 113


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fatty acid biosynthesis inhibitors was unaffected in vitro by supplementation with 114 a mixture of long chained lipids to the culture media. Together, the inability to 115 engineer an ftfabI conditional mutant and the transcriptional profile observed 116 during infection demonstrate the essentiality of ftfabI to bacterial viability and 117 confirm the FAS-II pathway as a clinically relevant drug target for the priority 118 pathogen F. tularensis. 119 120


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Materials and Methods 121 Bacterial strains and growth. 122

F. tularensis Schu4 and F. tularensis LVS were provided by Dr. J. 123 Petersen (Centers for Disease Control, Fort Collins, CO). In general, F. 124 tularensis Schu4 and LVS strains were cultured in Mueller-Hinton broth (BD) 125 modified with 0.025% ferric pyrophosphate (Sigma-Aldrich), 2% IsoVitaleX (BD), 126 0.1% glucose (Sigma-Aldrich) at 37 °C with constant shaking overnight. For the 127 fatty acid supplementation studies, F. tularensis was grown in modified Mueller-128 Hinton broth supplemented with 1% lipid mixture containing 2 μg/ml arachidonic 129 and 10 μg/ml each linoleic, linolenic, myristic, oleic, palmitic and, stearic acid 130 (Invitrogen). 131 Drug treatment studies. 132

The minimal inhibitory concentration of ketoacyl-ACP synthase (FabF) 133 inhibitors thiolactomycin and cerulenin and enoyl-ACP reductase (FabI) inhibitors 134 triclosan and SBPT04 were assessed (as previously described [6]) against 135 bacteria grown either in the presence or the absence of the exogenous lipid 136 mixture. 137 The effect of exogenous long chain lipids on the recovery of treated F. 138 tularensis was assessed by treating F. tularensis with either 1x MIC triclosan 139 (0.062 mg/L) or 1x MIC SBPT04 (0.25 mg/L) for 18hrs. Then the concentration 140 of each drug was reduced by a 1:1000 dilution into Mueller-Hinton broth with or 141 without 1% lipid mixture supplement. Viable bacterial growth was determined by 142 plating serial 10-fold dilutions at 0, 2, 4, 6, and 8 hours post antibiotic exposure. 143


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144 Construction and manipulation of fabI merodiploid, deletion, and 145 conditional alleles. 146

Plasmids used in this study were recombinant derivatives of F. tularensis 147 chromosomal integration vectors pMP812 and pMP815, (14) provided by Dr. H.P. 148 Schweitzer, Colorado State University. Cloning and strain maintenance were 149 carried out in E. coli DH5α (Life Technologies) in Luria Bertani broth (Becton 150 Dickinson). Phusion High-Fidelity DNA polymerase, restriction endonucleases, 151 site directed mutagenesis and other DNA modifying enzymes were supplied by 152 New England Biolabs, and genomic, plasmid, and amplicon DNAs were purified 153 with reagents manufactured by Qiagen. Oligonucleotides were synthesized by 154 Integrated DNA Technologies. 155

All electroporations, transformations of sucrose-competent F. tularensis 156 LVS and Schu4 with pMP815- or pMP812-based allelic exchange constructs, 157 and subsequent generation of primary and secondary recombinants were 158 performed as described by LuVollo et al, 2009 (14). The ftfabI merodiploid strain 159 was constructed as follows. Two fragments, a 202 bp element containing the 160 rpsL promoter and the 783 bp ftfabI CDS with 126 bp downstream sequence, 161 were amplified from F. tularensis Schu4 DNA and cloned into the blaB integration 162 vector pMP815. A 5x his tag was engineered into the construct by site directed 163 mutagenesis. The ΔftfabI was created using two non-sequential fragments with 164 extensions providing overlapping ends were amplified from the ftfabI genomic 165 region of LVS DNA. The two fragments were combined in a second amplification 166


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and were cloned into sacB suicide plasmid pMP812 (14) to form a 356 bp 167 deletion of fabI promoter and 5' CDS sequences flanked by 535 bp upstream and 168 759 bp downstream. The ftfabI conditional mutation allele created from a 169 segment of Schu4 ftfabI genomic region including 435 bp upstream and 437 bp 170 downstream of sequence coding residue T241 was cloned into pMP812. The 171 T241 codon was converted from ACT to TTT by site directed mutagenesis to 172 create T241F, orthologous to the fabI S241F mutation in temperature sensitive E. 173 coli JP1111 (2) 174 175 F. tularensis mouse model of infection and isolation of bacterial RNA from 176 infected tissues. 177 Six week-old female C57BL/6 mice were purchased from Jackson Laboratories, 178 Bar Harbor, Maine. All mice were housed in sterile micro-isolator cages in the 179 laboratory animal resources facility or in the Regional Biocontainment Laboratory 180 BSL-3 facility at Colorado State University (Fort Collins, CO) and provided water 181 and food ad libitum. All research involving animals was conducted in accordance 182 with the Animal Care and Use Committee approved animal guidelines and 183 protocols. Mice were infected with F. tularensis Schu4 via the intranasal (i.n.) 184 route. Mice were anesthetized with ketamine-xylazine and 10 μL inoculum (50-185 100 CFU) administered to each of the nares in sequential droplets allowing mice 186 to inhale the fluid (20 μL total). Infected mice were monitored for morbidity twice 187 daily and were euthanized at pre-determined endpoints. 188


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Lungs and spleen from infected mice were homogenized in TRIzol 189 (Invitrogen) and total RNA was extracted using phenol/chloroform and ethanol 190 precipitation. Samples were DNAseI (Fermentas) treated to remove genomic 191 DNA contaminants. Total RNA was depleted for ribosomal RNA and poly-192 adenylated RNA using the Ribominus Mouse and Bacterial Modules and the 193 poly-T dynabead module (Invitrogen). The resulting samples, enriched for F. 194 tularensis RNA were pooled and converted to cDNA (Superscipt III, Invitrogen) 195 and amplified using klenow fragment (Fermentas) and random hexamer primers 196 (3 μg/μL, Invitrogen, Carlsbad, CA) in the presence of Cy3 coupled dUTPs (GE), 197 at 37 °C for 2 hours. Amplified cDNA was purified using a PCR purification kit 198 (Qiagen). 199 200 In vivo transcriptional analysis. 201 Custom 70mer, cDNA oligo sets designed from the full assembled genome of F. 202 tularensis Schu4 (Operon Biotechnologies) were printed in house using a Bio-203 Rad Chipwriter Pro microarray printer on poly-amine coated glass microarray 204 slides (Array-it). Slides were post processed by UV crosslinking, blocked with a 205 10% BSA (Sigma-Aldrich), 3X SSC blocking buffer, rinsed with water and dried. 206 Labeled cDNA was combined with 1:1:0.2 Formamide:20X SSC:10% SDS 207 hybridization buffer, 10 μg yeast tRNA (Sigma) heated to 98oC for 2 min, cooled 208 and applied under a coverslip placed above the array on the glass slide. Arrays 209 were hybridized at 42 °C for 16 hours in the dark. Arrays were washed in a 210 decreasing SSC gradient and dried. 211


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Arrays were scanned using the Genepix Pro 4000B scanner and Genepix 212 software. Raw intensity values were normalized across technical triplicates and 213 analyzed for present or absence based on fluorescence intensity. Genes were 214 considered present if the fluorescence intensity was greater than 2 times the 215 background fluorescence intensity (SNR of 2), corresponding to the lowest 216 detectable signal (GEO accession number GSE39871). Gene lists were grouped 217 by functional annotation to determine biochemical pathways that were over-218 represented in the data set. 219

Genes of interest that were observed as expressed during infection using 220 arrays were confirmed using quantitative real time PCR. Briefly, cDNA synthesis 221 from total RNA was carried out using First Strand cDNA Synthesis Kit 222 (Invitrogen). 1 μg of total RNA was combined with random hexamer and heated 223 for 5 min. 10 μL of buffered enzyme mix (2 μL 10X buffer, 4 μL MgCl2 (6 mM), 224 2 μL DTT (0.1 M), 1 μL RNAse out, and 1 μL superscriptTM) was added and 225 incubated at 25 °C for 10 min, 50 °C for 50 min, and 85 °C for 5 min. Platinum 226 SYBR Green qPCR Supermix-UDG (Invitrogen) was combined with gene specific 227 primers (5 nmol) and 50 ng of template (cDNA) and run in triplicate on an IQ5 228 thermocycler (Bio-Rad). Resulting data from each condition was compared to 229 uninfected controls in an independent fashion using the ΔCT method. 230 231 232 233 234


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Results 235 Exogenous long chain lipids do not alter transcriptional response of fatty 236 acid biosynthetic components 237 It is known that the transcription and production of components within a 238 biosynthetic pathway is responsive and relative to their utilization and 239 requirement for growth. Therefore, the transcriptional profile resulting from 240 growth in different nutrient conditions and environments can be used for 241 comparison as well as assessing the involvement of specific metabolic processes 242 (e.g. fatty acid synthesis and β-oxidation) that respond respectively if fatty acids 243 are utilized as intermediate substrates for macromolecular synthesis. 244 Accordingly, the transcriptional activity of F. tularensis grown in defined media 245 and in media supplemented with a mixture of long chain lipids was assessed 246 using F. tularensis whole genome microarrays. Analyses of the global 247 transcriptional responses revealed a concordance of 84% between bacteria 248 grown in fatty acid supplemented media compared to bacteria grown in control 249 media (variance <0.05, differentially expressed greater than 1.5-fold). 250 Specifically, fatty acid biosynthesis genes accA, accB, accC and accD encoding 251 the malonyl-CoA synthase and fabF, fabG, fabH, fabI and fabZ encoding FAS-II 252 components were not differentially expressed and neither was FTT0910 253 encoding a putative phospholipase, FTT0941c encoding a putative 254 esterase/lipase, fadD1, fadD2, acbP, which encode the β-oxidation components 255 annotated in the F. tularensis genome (Figure 1A, B). The resulting 94% 256 concordance in the transcriptional responses of 23 ORFs involved in fatty acid 257


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metabolism between bacteria grown with exogenous fatty acid supplementation 258 as compared to control bacteria indicates that exogenous host lipids do not 259 drastically affect the metabolic flux of F. tularensis FAS-II and β-oxidation. 260 261 ftfabI is essential for growth even in the presence of exogenous fatty acids 262 Although the fatty acid biosynthetic pathway is attractive for drug 263 discovery and is the target for the anti-tuberculosis drug isoniazid that targets the 264 enoyl-ACP reductase, InhA in M. tuberculosis (21), and inhibitors of the 265 Staphylococcus aureus FabI enzyme (saFabI) have in vivo activity (23, 25, 30, 266 33), we wanted to establish that ftFabI was essential for growth. We have 267 developed long residence time inhibitors of InhA (17, 36), saFabI (39) and the F. 268 tularensis FabI enzyme (ftFabI) (15), one of which (SBPT04) demonstrates 100% 269 efficacy, controls dissemination and prevents relapse in an animal model of 270 tularemia (6). However, a recent report raised the possibility that at least in some 271 bacteria, de novo fatty acid synthesis may not be required for growth in the 272 presence of fatty acids, which are thought to be present in the host environment 273 (4). Therefore, to confirm that ftfabI is essential for growth even when supplied 274 with exogenous fatty acids we employed molecular approaches to assess 275 essentiality of ftfabI and de novo fatty acid biosynthesis in the presence of 276 exogenous long chain lipids. Based on the rational that F. tularensis utilizes 277 exogenous lipids then it should be possible to create a fatty acid auxotrophic 278 strain with lipid supplementation. After performing a population screen in the 279 absence and presence of exogenously supplied fatty acids it was determined that 280


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availability of fatty acids cannot rescue a native ftfabI mutant. After multiple 281 attempts, we were unable to engineer a stable ftfabI knockout even in the 282 presence of a second copy of ftfabI and we were not able to create a 283 temperature sensitive conditional mutant strain. The inability to develop a stable 284 merodiploid-complemented knockout or a conditional mutant is consistent with 285 our observations with other bacterial pathogens and those reported in M. 286 tuberculosis, that expression and production of enoyl-ACP reductases are tightly 287 controlled and the bacterium does not readily tolerate manipulation of ftfabI (34). 288 Specifically, the fact that we were unable to engineer a fatty acid auxotrophic 289 strain or a stable merodiploid-compliment strain substantiates that ftfabI is 290 essential and its activity is not bypassed by the availability of exogenous long 291 chain lipids. 292 293 Inhibition of FtFabI is gene dosage dependent. 294

To confirm that the molecular target for triclosan and SBPT04 is ftFabI, a 295 gene dosage drug sensitivity study was performed. Since high level of 296 expression of ftfabI is not well-tolerated, low level expression was used to 297 demonstrate a correlation between ftfabI expression and increase in MIC. 298 Expression of ftfabI approximately 3.5 fold (Figure 2A) resulted in a 2.4 fold and 299 2.3 fold increase in the MIC’s of triclosan and SBPT04 respectively (Figure 2B). 300 Importantly, the gene dosage associated increase in MIC demonstrates that the 301 molecular target for triclosan and SBPT04 in F. tularensis is ftFabI, which further 302


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substantiates that F. tularensis cannot utilize exogenous fatty acids to bypass 303 ftFabI inhibition. 304 305 Inhibition of ftFabI and fatty acid biosynthesis is unaffected by exogenous 306 long chain lipids. 307

To directly assess whether exogenous long chain lipids alter the sensitivity 308 of ftFabI inhibitory compounds, we tested the minimal inhibitory concentration of 309 the known FabI inhibitors, triclosan and SBPT04 (6), along with known FabF 310 inhibitors thiolactomycin and cerulenin as positive controls. The ability of F. 311 tularensis to utilize exogenous long chain lipids to circumvent ftFabI inhibition 312 was evaluated by determining the MIC and post-treatment rescue. 313

The MIC of the ftFabI inhibitors triclosan and SBPT04 was determined in 314 the presence of long chain lipids. FabF inhibitors thiolactomycin and cerulinin 315 were tested as positive controls for inhibition of fatty acid synthesis to assure that 316 the observed effects was not due to off target effects or drug binding by 317 exogenous lipids. There was no difference in inhibitory activity of FabI or FabF 318 inhibitors when tested with exogenous long chain lipids supplementation when 319 compared to un-supplemented control (Table 2). 320

Similarly, exogenous long chain lipids did not reverse the inhibitory activity 321 of triclosan or SBPT04. Supplying exogenous long chain lipids for up to 8 hours 322 post drug did not alter the recovery and growth of bacteria treated for 18 hours. 323 These results are consistent with the inability to obtain an auxotrophic strain of F. 324


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tularensis and indicate that F. tularensis requires de novo fatty acid biosynthesis 325 and cannot subsist solely on freely available lipids. 326 327 The F. tularensis FAS-II pathway is transcriptionally active in infected 328 tissues. 329

Although bacteria grown in defined culture medium displayed a 330 transcriptional response that indicated that they could not utilize exogenously 331 supplied fatty acids for fatty acid synthesis, it is still necessary to assess whether 332 the transcriptional profile of bacteria from infected tissues show a pattern of fatty 333 acid gene expression consistent with active de novo fatty acid biosynthesis. 334 Accordingly, the whole bacterial genome transcriptional response of F. tularensis 335 obtained from infected host tissues was assessed using F. tularensis whole 336 genome DNA microarrays. We have studied the host response to infection with 337 F. tularensis and dissemination to secondary sites and have determined that 338 infection of the spleen is an important indicator of disease progression and 339 mortality (12), and thus is an important consideration for drug discovery and 340 development of clinically relevant agents. Accordingly, total RNA from spleen 341 samples was harvested 120 hours post infection and pooled from two mice for 342 microarray analysis. Array data was subjected to a threshold cutoff based on 343 signal to noise ratios to identify F. tularensis Schu4 ORFs that were present, thus 344 discriminating between F. tularensis ORFs that are transcriptionally active in 345 infected tissue. 346


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Genes that had a transcriptional signal to noise ratio of 2 or greater were 347 deemed to be present, thus implicating activity of a metabolic pathway. In 348 addition, triplicate arrays allowed the elimination of any feature that was flagged 349 as poor quality on any of the three arrays to increase stringency of calling active 350 metabolism. Overall, a total of 1,022 F. tularensis Schu4 ORFs were identified 351 as transcriptionally active in the spleens of infected mice (Table S2). Gene lists 352 were subjected to functional enrichment analysis to determine pathways that had 353 a disproportionally high number of genes with a particular ontological 354 classification. Overall, the transcriptional activity of F. tularensis from infected 355 spleens included basic metabolic pathways of amino acid, protein biosynthesis, 356 carbon metabolism via the TCA cycle, as well as fatty acid biosynthesis (Table 1). 357

Although, transcriptional activity identified by microarray provided an 358 overall picture of the metabolic activities of F. tularensis in infected tissues, it was 359 necessary to validate the microarray analysis, and specifically the expression of 360 the F. tularensis encoded FAS-II system because of the nature of measuring 361 transcriptional activity in complex biological samples. Accordingly, FAS-II gene 362 expression was assessed using quantitative real time RT-PCR at 96 hours post 363 infection in both the lungs and spleen of F. tularensis Schu4 infected mice for 364 confirmation. Analysis of qRT data revealed expression of all F. tularensis 365 annotated FAS-II pathway genes in both the lung and spleen of infected mice 366 (Figure 3), thus validating the transcriptional data obtained by microarray and 367 further substantiating that ftfabI encodes a clinically relevant drug target. 368 369


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370 Discussion 371

The development of new drugs has numerous hurdles, including 372 verification that the target of interest is necessary under conditions associated 373 with disease, and is therefore a clinically relevant drug target. Clinically relevant 374 targets for drug discovery can be experimentally defined as those proteins that 375 are essential, whose functions are not bypassed by available exogenous 376 nutrients, and are active during, and at sites of, the infection. Recently, there has 377 been a debate about the essentiality of the bacterial FAS-II system based on the 378 ability of some bacteria to incorporate exogenous lipids thereby bypassing de 379 novo fatty acid synthesis rendering the pathway conditionally non-essential and 380 thus, a poor target for the development of novel therapeutics (4). It has been 381 shown however, that this phenomenon is a result of the ability of individual 382 species of bacteria to uptake and utilizes available nutrients (1, 26). Notably, 383 there have been numerous publications that have substantiated enoyl-ACP 384 reductases as valid drug targets (8-10, 16, 20, 21, 35, 38). 385

Because we have a drug discovery effort targeting enoyl-ACP reductases 386 there is a need to demonstrate that FabI is a clinically relevant drug target in F. 387 tularensis, despite the fact that we have previously shown the efficacy of novel 388 FabI inhibitors both in vitro and in vivo (3, 6, 16). To further substantiate FabI as 389 a relevant drug target, we investigated the expression and essentiality of ftfabI in 390 the presence of exogenously supplied fatty acids, evaluated if exogenous fatty 391 acids affect the potency of known FabI inhibitors, and determined if the F. 392 tularensis encoded FAS-II system is transcriptionally active during infection. 393


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Transcriptional profiling of F. tularensis grown in the presence of 394 exogenous lipids and recovered from the spleen revealed that the expression of 395 fatty acid biosynthetic genes are unaffected by availability of lipids and are 396 transcriptionally active at the site of infection. This response indicates that 397 despite the availability of lipids supplied exogenously or as part of host 398 environment de novo fatty acid biosynthesis is still necessary to synthesize the 399 full complement of fatty acids required for the F. tularensis cell wall. In 400 combination, these transcriptional studies demonstrate that de novo fatty acid 401 synthesis is not bypassed by availability of lipids and potential short chain fatty 402 acid intermediates. Consistent with the lack of transcriptional response to the 403 availability of lipids, an auxotrophic mutant could not be created. This further 404 substantiates that exogenous lipids cannot be readily used to circumvent fatty 405 acid biosynthesis. 406

Although, an auxotrophic mutant could not be created, a ftfabI knockout 407 was pursued in a ftFabI-merodiploid strain. Even though a second copy of ftfabI 408 was present, generation of a stable ftfabI knockout strain of F. tularensis proved 409 unobtainable. Further investigation via gene dosage expression studies revealed 410 that artificial expression of ftfabI and production of ftFabI was not well tolerated. 411 These studies demonstrated that only low-level expression was stable. This 412 observation is consistent with observations in M. tuberculosis that the tbfabI, inhA 413 is not induced equivalently to other fatty acid biosynthetic genes (34). These 414 studies demonstrate that FabI activity under native control is essential, which is 415


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consistent with transposon mutant studies that did not identify FabI as a gene 416 that could tolerate transposon insertion (7, 11, 18, 19, 29, 32, 37). 417 We are currently testing novel FabI inhibitors against F. tularensis as well 418 as priority pathogens Y. pestis and B. pseudomallei and the medically important 419 pathogen M. tuberculosis in an effort to develop broad-spectrum antibiotics that 420 target the FAS-II system of bacterial pathogens. Therefore, we determined if the 421 availability of exogenous lipids altered the minimal inhibitory activity of a known 422 FabI inhibitor and a novel FabI inhibitor developed in our drug discovery program 423 (3, 6, 15, 16). It was shown that there was no difference in the MIC of FabI 424 inhibitors when compared to testing without exogenous long chain lipids. In 425 addition, the inhibitory activity of the positive control fatty acid inhibitors cerulenin 426 and thiolactomycin that target the keto-acyl ACP synthase were also unaffected 427 by the availability of lipids. The MIC studies in the presence of exogenous lipids 428 further substantiates that FabI in F. tularensis is a clinically relevant drug target. 429

Importantly, we showed ftfabI transcriptional activity both in the presence 430 of exogenously supplied long chain lipids and in vivo during infection signifying 431 ftfabI and de novo fatty acid biosynthesis have an essential metabolic role during 432 in the host and during infection. These data demonstrate the importance of 433 establishing essentiality of a drug target as well as clinical relevance based on 434 being actively expressed during the infection and therefore readily targetable for 435 therapeutic intervention. As the development of broad-spectrum 436 chemotherapeutic targeting FAS-II advances, work with priority pathogens can 437 be translated to medically important infectious diseases that affect a much larger 438


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number of patients. Together, this information can be used to substantiate fatty 439 acid biosynthesis as well as ftFabI as a clinically relevant drug target in F. 440 tularensis. 441

442 443 Acknowledgements 444 This work was supported by RP006 of the Rocky Mountain Regional Center of 445 Excellence funded by NIH, NIAID grant AI065357 (R.A.S. & P.J.T.). 446


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447 References 448 1. Balemans, W., N. Lounis, R. Gilissen, J. Guillemont, K. Simmen, K. 449

Andries, and A. Koul. 2010. Essentiality of FASII pathway for 450 Staphylococcus aureus. Nature 463:E3; discussion E4. 451

2. Bergler, H., G. Hogenauer, and F. Turnowsky. 1992. Sequences of the 452 envM gene and of two mutated alleles in Escherichia coli. Journal of 453 general microbiology 138:2093-2100. 454

3. Boyne, M. E., T. J. Sullivan, C. W. amEnde, H. Lu, V. Gruppo, D. 455 Heaslip, A. G. Amin, D. Chatterjee, A. Lenaerts, P. J. Tonge, and R. A. 456 Slayden. 2007. Targeting fatty acid biosynthesis for the development of 457 novel chemotherapeutics against Mycobacterium tuberculosis: evaluation 458 of A-ring-modified diphenyl ethers as high-affinity InhA inhibitors. 459 Antimicrob Agents Chemother 51:3562-3567. 460

4. Brinster, S., G. Lamberet, B. Staels, P. Trieu-Cuot, A. Gruss, and C. 461 Poyart. 2009. Type II fatty acid synthesis is not a suitable antibiotic target 462 for Gram-positive pathogens. Nature 458:83-86. 463

5. Dennis, D. T., T. V. Inglesby, D. A. Henderson, J. G. Bartlett, M. S. 464 Ascher, E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer, M. Layton, S. 465 R. Lillibridge, J. E. McDade, M. T. Osterholm, T. O'Toole, G. Parker, T. 466 M. Perl, P. K. Russell, and K. Tonat. 2001. Tularemia as a biological 467 weapon: medical and public health management. JAMA 285:2763-2773. 468


http://jb.asm.org/

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23

6. England, K., C. am Ende, H. Lu, T. J. Sullivan, N. L. Marlenee, R. A. 469 Bowen, S. E. Knudson, D. L. Knudson, P. J. Tonge, and R. A. Slayden. 470 2009. Substituted diphenyl ethers as a broad-spectrum platform for the 471 development of chemotherapeutics for the treatment of tularaemia. J 472 Antimicrob Chemother 64:1052-1061. 473

7. Gallagher, L. A., E. Ramage, M. A. Jacobs, R. Kaul, M. Brittnacher, 474 and C. Manoil. 2007. A comprehensive transposon mutant library of 475 Francisella novicida, a bioweapon surrogate. Proc Natl Acad Sci U S A 476 104:1009-1014. 477

8. Heath, R. J., J. R. Rubin, D. R. Holland, E. Zhang, M. E. Snow, and C. 478 O. Rock. 1999. Mechanism of triclosan inhibition of bacterial fatty acid 479 synthesis. J Biol Chem 274:11110-11114. 480

9. Heath, R. J., S. W. White, and C. O. Rock. 2002. Inhibitors of fatty acid 481 synthesis as antimicrobial chemotherapeutics. Appl Microbiol Biotechnol 482 58:695-703. 483

10. Heath, R. J., Y. T. Yu, M. A. Shapiro, E. Olson, and C. O. Rock. 1998. 484 Broad spectrum antimicrobial biocides target the FabI component of fatty 485 acid synthesis. J Biol Chem 273:30316-30320. 486

11. Kawula, T. H., J. D. Hall, J. R. Fuller, and R. R. Craven. 2004. Use of 487 transposon-transposase complexes to create stable insertion mutant 488 strains of Francisella tularensis LVS. Appl Environ Microbiol 70:6901-6904. 489

12. Kingry, L. C., R. M. Troyer, N. L. Marlenee, H. Bielefeldt-Ohmann, R. A. 490 Bowen, A. R. Schenkel, S. W. Dow, and R. A. Slayden. 2011. Genetic 491


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24

identification of unique immunological responses in mice infected with 492 virulent and attenuated Francisella tularensis. Microbes Infect 13:261-275. 493

13. Liu, N., J. E. Cummings, K. England, R. A. Slayden, and P. J. Tonge. 494 2011. Mechanism and inhibition of the FabI enoyl-ACP reductase from 495 Burkholderia pseudomallei. J Antimicrob Chemother 66:564-573. 496

14. LoVullo, E. D., C. R. Molins-Schneekloth, H. P. Schweizer, and M. S. 497 Pavelka, Jr. 2009. Single-copy chromosomal integration systems for 498 Francisella tularensis. Microbiology 155:1152-1163. 499

15. Lu, H., K. England, C. am Ende, J. J. Truglio, S. Luckner, B. G. Reddy, 500 N. L. Marlenee, S. E. Knudson, D. L. Knudson, R. A. Bowen, C. Kisker, 501 R. A. Slayden, and P. J. Tonge. 2009. Slow-onset inhibition of the FabI 502 enoyl reductase from francisella tularensis: residence time and in vivo 503 activity. ACS Chem Biol 4:221-231. 504

16. Lu, H., and P. J. Tonge. 2008. Inhibitors of FabI, an enzyme drug target 505 in the bacterial fatty acid biosynthesis pathway. Acc Chem Res 41:11-20. 506

17. Luckner, S. R., N. Liu, C. W. am Ende, P. J. Tonge, and C. Kisker. 507 2010. A slow, tight binding inhibitor of InhA, the enoyl-acyl carrier protein 508 reductase from Mycobacterium tuberculosis. J. Biol. Chem. 285:14330-509 14337. 510

18. Maier, T. M., M. S. Casey, R. H. Becker, C. W. Dorsey, E. M. Glass, N. 511 Maltsev, T. C. Zahrt, and D. W. Frank. 2007. Identification of Francisella 512 tularensis Himar1-based transposon mutants defective for replication in 513 macrophages. Infect Immun 75:5376-5389. 514


http://jb.asm.org/

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25

19. Maier, T. M., R. Pechous, M. Casey, T. C. Zahrt, and D. W. Frank. 2006. 515 In vivo Himar1-based transposon mutagenesis of Francisella tularensis. 516 Appl Environ Microbiol 72:1878-1885. 517

20. McMurry, L. M., P. F. McDermott, and S. B. Levy. 1999. Genetic 518 evidence that InhA of Mycobacterium smegmatis is a target for triclosan. 519 Antimicrob Agents Chemother 43:711-713. 520

21. Mdluli, K., R. A. Slayden, Y. Zhu, S. Ramaswamy, X. Pan, D. Mead, D. 521 D. Crane, J. M. Musser, and C. E. Barry, 3rd. 1998. Inhibition of a 522 Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid. 523 Science 280:1607-1610. 524

22. Miller, S. D., M. B. Snyder, M. Kleerekoper, and C. H. Grossman. 1989. 525 Ulceroglandular tularemia: a typical case of relapse. Henry Ford Hosp 526 Med J 37:73-75. 527

23. Miller, W. H., M. A. Seefeld, K. A. Newlander, I. N. Uzinskas, W. J. 528 Burgess, D. A. Heerding, C. C. Yuan, M. S. Head, D. J. Payne, S. F. 529 Rittenhouse, T. D. Moore, S. C. Pearson, V. Berry, W. E. DeWolf, Jr., P. 530 M. Keller, B. J. Polizzi, X. Qiu, C. A. Janson, and W. F. Huffman. 2002. 531 Discovery of aminopyridine-based inhibitors of bacterial enoyl-ACP 532 reductase (FabI). J. Med. Chem. 45:3246-3256. 533

24. Nichols, P. D., W. R. Mayberry, C. P. Antworth, and D. C. White. 1985. 534 Determination of monounsaturated double-bond position and geometry in 535 the cellular fatty acids of the pathogenic bacterium Francisella tularensis. 536 Journal of clinical microbiology 21:738-740. 537


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

26

25. Park, H. S., Y. M. Yoon, S. J. Jung, C. M. Kim, J. M. Kim, and J. H. 538 Kwak. 2007. Antistaphylococcal activities of CG400549, a new bacterial 539 enoyl-acyl carrier protein reductase (FabI) inhibitor. J. Antimicrob. 540 Chemother. 60:568-574. 541

26. Parsons, J. B., M. W. Frank, C. Subramanian, P. Saenkham, and C. O. 542 Rock. 2011. Metabolic basis for the differential susceptibility of Gram-543 positive pathogens to fatty acid synthesis inhibitors. Proc Natl Acad Sci U 544 S A 108:15378-15383. 545

27. Penn, R. L., and G. T. Kinasewitz. 1987. Factors associated with a poor 546 outcome in tularemia. Arch Intern Med 147:265-268. 547

28. Phillips, N. J., B. Schilling, M. K. McLendon, M. A. Apicella, and B. W. 548 Gibson. 2004. Novel modification of lipid A of Francisella tularensis. Infect 549 Immun 72:5340-5348. 550

29. Qin, A., and B. J. Mann. 2006. Identification of transposon insertion 551 mutants of Francisella tularensis tularensis strain Schu S4 deficient in 552 intracellular replication in the hepatic cell line HepG2. BMC Microbiol 6:69. 553

30. Ramnauth, J., M. D. Surman, P. B. Sampson, B. Forrest, J. Wilson, E. 554 Freeman, D. D. Manning, F. Martin, A. Toro, M. Domagala, D. E. Awrey, 555 E. Bardouniotis, N. Kaplan, J. Berman, and H. W. Pauls. 2009. 2,3,4,5-556 Tetrahydro-1H-pyrido[2,3-b and e][1,4]diazepines as inhibitors of the 557 bacterial enoyl ACP reductase, FabI. Bioorg. Med. Chem. Lett. 19:5359-558 5362. 559


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

27

31. Schiebel, J., A. Chang, H. Lu, M. V. Baxter, P. J. Tonge, and C. Kisker. 560 2012. Staphylococcus aureus FabI: inhibition, substrate recognition, and 561 potential implications for in vivo essentiality. Structure 20:802-813. 562

32. Schulert, G. S., R. L. McCaffrey, B. W. Buchan, S. R. Lindemann, C. 563 Hollenback, B. D. Jones, and L. A. Allen. 2009. Francisella tularensis 564 genes required for inhibition of the neutrophil respiratory burst and 565 intramacrophage growth identified by random transposon mutagenesis of 566 strain LVS. Infect Immun 77:1324-1336. 567

33. Seefeld, M. A., W. H. Miller, K. A. Newlander, W. J. Burgess, W. E. 568 DeWolf Jr, P. A. Elkins, M. S. Head, D. R. Jakas, C. A. Janson, P. M. 569 Keller, P. J. Manley, T. D. Moore, D. J. Payne, S. Pearson, B. J. Polizzi, 570 X. Qiu, S. F. Rittenhouse, I. N. Uzinskas, N. G. Wallis, and W. F. 571 Huffman. 2003. Indole Naphthyridinones as Inhibitors of Bacterial Enoyl-572 ACP Reductases FabI and FabK. J. Med. Chem. 46:1627-1635. 573

34. Slayden, R. A., R. E. Lee, and C. E. Barry, 3rd. 2000. Isoniazid affects 574 multiple components of the type II fatty acid synthase system of 575 Mycobacterium tuberculosis. Mol Microbiol 38:514-525. 576

35. Stewart, M. J., S. Parikh, G. Xiao, P. J. Tonge, and C. Kisker. 1999. 577 Structural basis and mechanism of enoyl reductase inhibition by triclosan. 578 J Mol Biol 290:859-865. 579

36. Sullivan, T. J., J. J. Truglio, M. E. Boyne, P. Novichenok, X. Zhang, C. 580 F. Stratton, H.-J. Li, T. Kaur, A. Amin, F. Johnson, R. A. Slayden, C. 581 Kisker, and P. J. Tonge. 2006. High Affinity InhA Inhibitors with Activity 582


http://jb.asm.org/

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28

against Drug-Resistant Strains of Mycobacterium tuberculosis. ACS Chem. 583 Biol. 1:43-53. 584

37. Tempel, R., X. H. Lai, L. Crosa, B. Kozlowicz, and F. Heffron. 2006. 585 Attenuated Francisella novicida transposon mutants protect mice against 586 wild-type challenge. Infect Immun 74:5095-5105. 587

38. Ward, W. H., G. A. Holdgate, S. Rowsell, E. G. McLean, R. A. Pauptit, 588 E. Clayton, W. W. Nichols, J. G. Colls, C. A. Minshull, D. A. Jude, A. 589 Mistry, D. Timms, R. Camble, N. J. Hales, C. J. Britton, and I. W. 590 Taylor. 1999. Kinetic and structural characteristics of the inhibition of 591 enoyl (acyl carrier protein) reductase by triclosan. Biochemistry 38:12514-592 12525. 593

39. Xu, H., T. J. Sullivan, J. Sekiguchi, T. Kirikae, I. Ojima, W. Mao, F. L. 594 Rock, M. R. K. Alley, F. Johnson, S. G. Walker, and P. J. Tonge. 2008. 595 Mechanism and Inhibition of saFabI, the Enoyl Reductase from 596 Staphylococcus aureus. Biochemistry 47:4228-4236. 597

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601 Figure 1: F. tularensis FAS-II and β-oxidation pathways are unaffected by 602 supplementation of exogenous fatty acids in vitro. F. tularensis LVS was grown 603 in standard media and in the presence of 3% fatty acid supplement. RNA from 604 three biological replicates was labeled and hybridized to a total of nine individual 605 F. tularensis full genome microarrays. Transcriptional analysis reveals the (A) 606 fatty acid and (B) β-oxidation pathways are not differentially regulated by fatty 607 acid supplementation. Horizontal lines indicate the minimum threshold cutoff for 608 genes to be considered differentially regulated. 609 610 Figure 2: Gene dosage drug sensitivity study. Low-level over expression of 611 ftfabI correlates with an increase in MIC of both Triclosan and SBPT04. A. The 612 transcriptional activity of the fabI merodiploid strain of F. tularensis in comparison 613 to wildtype. B. Evaluation of MIC90 of wildtype F. tularensis and a fabI 614 merodiploid F. tularensis strain in the presence of PT04, triclosan and 615 doxycycline. 616 617 Figure 3: F. tularensis requires de novo fatty acid biosynthesis during infection 618 of the lung and spleen 96 hours post-infection. Total RNA was isolated from the 619 lungs and spleen of mice 96 hours post-infection with F. tularensis Schu4. Gene 620 specific primers run against RNA from infected and uninfected mouse tissue and 621 compared to generate deltaCT values. Gene expression patterns in both the 622 lung (A) and spleen (B) indicate the necessity of F. tularensis de novo 623 biosynthesis pathway during infection. 624


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625 Table 1: Expression profiling of the F. tularensis transcriptome during infection 626 reveals the metabolic requirements to cause disseminated disease. Total RNA 627 was isolated from the spleens of mice 120 hours post-infection with F. tularensis 628 Schu4. Material from two mice was pooled and enriched for bacterial transcripts 629 and converted to labeled cDNA and hybridized to Francisella full genome cDNA 630 microarrays. Gene lists were interrogated for pathways with an overabundance 631 of representative genes. 632 633 Table 2: Supplementation of exogenous fatty acids does not effect inhibition of 634 FabF or FabI. MIC of known FabI inhibitors triclosan and SBPT04 and FabF 635 inhibitors thiolactomycin and cerulinin against F. tularensis strains LVS and 636 Schu4 grown in the presence of a mixture of long chain lipids. 637 638 on A

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Table 1: Expression profiling of the F. tularensis transcriptome during infection 639 reveals the metabolic requirements for growth in the host. 640

Amino Acid Biosynthesis

ID Gene Name Annotation

FTT0588 aroA 3-phosphoshikimate 1-carboxyvinyltransferase

FTT1154c aroB 3-dehydroquinate synthetase

FTT0876c aroC chorismate synthase

FTT1155c aroK shikimate kinase I

FTT1287 cbs cystathionine beta-synthase (cystein synthase)

FTT0640 ilvD Dihydroxy-acid dehydratase

FTT0560c serC phosphoserine aminotransferase

FTT1772c trpA tryptophan synthase alpha chain

FTT1773c trpB tryptophan synthase beta chain

Fatty Acid Biosynthesis


FTT1498c accA Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha

FTT0472 accB Acetyl-CoA carboxylase, biotin carboxyl carrier protein subunit

FTT0372c accD Acetyl-CoA carboxylase beta subunit

FTT1377 fabF 3-oxoacyl-[acyl-carrier-protein] synthase II

FTT1375 fabG 3-oxoacyl-(acyl-carrier-protein) reductase

FTT1373 fabH 3-oxoacyl-[acyl carrier protein] synthase III

FTT0782 fabI Enoyl-[acyl-carrier-protein] reductase (NADH)

FTT0148 FTT0148 fatty acid desaturase

FTT1552 ole1 Delta 9 acyl-lipid fatty acid desaturase

FTT1372 plsX fatty acid/phospholipid synthesis protein lsX

Stress Response


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FTT1769c clpB ClpB protein

FTT1269c dnaK Chaperone protein dnaK (heat shock protein family 70 protein)

FTT0666c FTT0666c Methylpurine-DNA glycosylase family protein

FTT0733 FTT0733 glutathione peroxidase

FTT0846 FTT0846 deoxyribodipyrimidine photolyase

FTT0886 FTT0886 DNA repair protein recN

FTT0986 FTT0986 conserved hypothetical protein

FTT0356 htpG Chaperone Hsp90, heat shock protein HtpG

FTT0862c htpX heat shock protein HtpX

FTT0721c katG Peroxidase/catalase

FTT1736c kdpD two component sensor protein kdpD

FTT1387c ligN DNA ligase

FTT0644c mfd Transcription-repair coupling factor,ATP-dependent

FTT0486 mutL DNA mismatch repair protein

FTT0648c nth Endonuclease III

FTT1518 ogt Methylated-DNA--protein-cysteine methyltransferase

FTT0111 polA DNA polymerase I

FTT0873c radA DNA repair protein radA

FTT0478c recJ Single-stranded-DNA-specific exonuclease

FTT1087c rep ATP-dependent DNA helicase

FTT1112c rpoH RNA polymerase sigma-32 factor

FTT0658 ruvA holliday junction DNA helicase, subunit A

FTT1013c ruvB holliday junction DNA helicase, subunit B

FTT0656 ruvC holliday junction endodeoxyribonuclease


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FTT1578c ung Uracil-DNA glycosylase

FTT0245 usp universal stress protein

FTT1312c uvrA DNA excision repair enzyme, subunit A

FTT0777 uvrC DNA excision repair enzyme, subunit C

FTT0121 uvrD DNA helicase II

FTT0959c xthA Exodeoxyribonuclease III

Protein Biosynthesis


FTT1096c alaS Alanyl-tRNA synthetase

FTT1616 cysS Cysteinyl-tRNA synthetase

FTT0925 fmt Methionyl-tRNA formyltransferase

FTT0316 frr ribosome recycling factor

FTT0323 fusA elongation factor G (EF-G)

FTT0307 gltX Glutamyl-tRNA synthetase

FTT0419 glyQ Glycyl-tRNA synthetase alpha chain

FTT0052 hisS Histidyl-tRNA synthetase

FTT0915c ileS Isoleucyl-tRNA synthetase

FTT0050 infB translation initiation factor IF-2

FTT0818 infC translation initiation factor IF-3

FTT0990 leuS Leucyl-tRNA synthetase

FTT0192 lysU Lysyl-tRNA synthetase

FTT1290 metG Methionyl-tRNA synthetase

FTT1002c pheT Phenylalanyl-tRNA synthetase, beta subunit

FTT0476c poxA Lysyl-tRNA synthetase

FTT0168 prfA peptide chain release factor 1


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FTT0191 prfB peptide chain release factor 2

FTT0118 prfC peptide chain release factor 3

FTT1412 proS Prolyl-tRNA synthetase

FTT0817 thrS Threonyl-tRNA synthetase

FTT0314 tsf protein chain elongation factor EF-Ts

FTT0137 tufA elongation factor Tu (EF-Tu)

FTT0691 tyrS Tyrosyl-tRNA synthetase

FTT0299 valS Valyl-tRNA synthetase

TCA Cycle


FTT0306 fumC fumarate hydratase, Class II

FTT0071c gltA citrate synthase

FTT1526c idh isocitrate dehydrogenase

FTT0074 sdhA succinate dehydrogenase, catalytic and NAD/flavoprotein subunit

FTT0075 sdhB succinate dehydrogenase iron-sulfur protein

FTT0072 sdhC succinate dehydrogenase, cytochrome b556

FTT0077 sucB dihydrolipoamide succinyltransferase (2-oxoglutarate dehydrogenase complex) 641 642


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643 Table 2: Supplementation of exogenous fatty acids does not effect inhibition of 644 FabF or FabI 645 In vitro Activity of FabF and FabI Inhibitors Against F. tularensis

FabF Inhibitor LVS MIC mg/L Schu4 MIC mg/L

- Lipid + Lipid - Lipid + Lipid

TLM 4 4 16 16 Cerulinin 2 2 1 1

FabI Inhibitor LVS MIC mg/L Schu4 MIC mg/L

- Lipid + Lipid - Lipid + Lipid

SBPT04 0.25 0.5 1 1 Triclosan 0.06 0.12 0.25 0.25

646


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Documents

1 The Francisella tularensis FabI enoyl-ACP-reductase gene is