Burkholderia Pseudomallei Kynurenine Formamidase Plays a Role in the 2 Regulation of Phenotypes Associated with Chronic Disease

ORE Open Research Exeter

TITLE Burkholderia pseudomallei kynB plays a role in AQ production, bioﬁlm formation, bacterial swarming and persistence

AUTHORS Butt, A; Halliday, N; Williams, P; et al.

JOURNAL Research in Microbiology

DEPOSITED IN ORE 16 March 2017

This version available at

http://hdl.handle.net/10871/26619

Open Research Exeter makes this work available in accordance with publisher policies.

A NOTE ON VERSIONS

The version presented here may diﬀer from the published version. If citing, you are advised to consult the published version for pagination, volume/issue and date of publication 1 Burkholderia pseudomallei kynurenine formamidase plays a role in the 2 regulation of phenotypes associated with chronic disease

3 Aaron Butt1, Nigel Halliday2, Felipe Cia4 Paul Williams2, Gregory Bancroft4 Helen Atkins3, 4 Richard W Titball1*

5 6 ABSTRACT

7 Kynurenine formamidase (KynB) forms part of the kynurenine pathway which metabolises 8 tryptophan to anthranilic acid. This metabolite can be used for downstream production of HAQ 9 signalling molecules that control virulence in Pseudomonas aeruginosa. Here we investigate 10 the role of kynB in the production of H(M)AQs and virulence potential of B. pseudomallei 11 K96243, the causative agent of melioidosis. Deletion of kynB resulted in reduced H(M)AQ 12 production, increased biofilm formation, decreased swarming and increased tolerance to 13 ciprofloxacin. Addition of spent media isolated from wild type B. pseudomallei or adding 14 exogenous anthranilic acid could complement some of these phenotypes. This study suggests 15 the kynurenine pathway is a critical source of anthranilic acid and signalling molecules that 16 regulate phenotypes which are associated with chronic disease.

18 Burkholderia pseudomallei is a motile, Gram-negative, environmental bacterium that resides 19 in soil and stagnant water as a saprophyte in south east Asia and northern Australia [1]. B. 20 pseudomallei is an opportunistic human pathogen and through inhalation or entry via skin 21 abrasions, causes the infectious disease melioidosis [2]. Melioidosis has a mortality rate of up 22 to 44% and is the third most frequent cause of death from infections disease in North East 23 Thailand, despite antibiotic intervention [3]. The bacteria can also establish an asymptomatic 24 latent infection with symptoms occurring years post exposure, or manifest as a chronic 25 recurring infection with up to 15% of patients relapsing following completion of therapy [4]. 26 Due to its intrinsic resistance to many antibiotics, a high mortality rate, chronic disease 27 manifestations and infection by the airborne route B. pseudomallei is also considered a 28 potential bioterrorism agent [5,6].

29 Quorum sensing systems are involved in cell-to-cell signalling and through secondary 30 metabolites control and regulate many genes associated with virulence. Increasingly, these 31 systems are attractive targets for novel therapeutics [7,8]. Mutation of quorum sensing 32 apparatus in B. pseudomallei results in an increased time to death in mice and hamster models 33 [9,10]. B. pseudomallei possesses 3 luxI and 5 luxR quorum sensing genes and synthesizes 34 a range of signalling molecules, including N-octanoyl-homoserine lactone, N-decanoyl- 35 homoserine lactone, N-(3-hydroxyoctanoyl)-L-homoserine lactone, N-(3-hydroxydecanoyl)-L- 36 homoserine lactone and N-(3-oxotetradecanoyl)-L-homoserine [9]. B. pseudomallei also 37 contain the genes hhqABCDE (hmqABCDE) which are homologous to pqsABCDE from 38 Pseudomonas aeruginosa. In P. aeruginosa, these genes code for a biosynthetic pathway 39 required for the synthesis of 2-alkyl-4(1H)-quinolones (HAQs), 2,4-dihydroxyquinoline (DHQ) 40 and 2-aminoacetophenone (2-AA) [11-13]. Inactivation of HAQ synthesis through deletion of 41 pqsE, or pqsA decreases P. aeruginosa virulence in a mouse acute infection suggesting this 42 is a good target for novel antimicrobial therapeutics [14]. 43 44 Previous analysis of B. pseudomallei culture supernatants revealed that the predominant 45 HAQs in B. pseudomallei are HMAQs. These are HAQs that contain an unsaturated aliphatic 46 side chain that is typically methylated at the 3 position by a methyltransferase encoded by 47 hmqG [15]. HAQ synthesis requires anthranilic acid. In P. aeruginosa, this metabolite is 48 derived via three distinct pathways. Two of the pathways encoded by phnAB or trpEG convert 49 chorismic acid to anthranililc acid, while a third, the kynurenine pathway, converts tryptophan 50 to anthranilic acid [16,17]. B. pseudomallei has no apparent phnAB homologs but does 51 possess the TrpEG and kynurenine pathways. The kynurenine pathway consists of three 52 enzymes encoded by kynA (tryptophan-2,3-dioxygenase), kynB (kynurenine formamidase), 53 and kynU (kynureninase) [18]. The kynurenine pathway has previously been shown as a 54 critical source of anthranilic acid for the production of PQS in P. aeuroginosa and the 55 production of HMAQs in B. thailandensis and Burkholderia ambifaria through the incorporation 56 of radiolabelled tryptophan [15,17]. Subsequently this pathway seems likely to be key in the 57 production of 4-quinolones (and many other secondary metabolites) in B. pseudomallei and 58 be an important source of anthranillic acid for signalling during human infections. [17]. 59 60 In this study we set out to investigate the role of the kynurenine pathway in B. pseudomallei. 61 We show that deletion of kynB results in the loss of HAQ/HMAQs and affects phenotypes 62 which are associated with chronic disease caused by other bacteria. 63

64 MATERIALS AND METHODS

65 Bacterial strains, growth conditions and chemicals

66 Bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were routinely 67 grown in Luria–Bertani (LB) broth at 37°C with shaking (200 rpm) or on LB agar plates at 37°C. 68 Where appropriate, media was supplemented with chloramphenicol 25 μg/ml for selection of 69 the pBHR or pDM4 plasmids. For growth in M9 media with tryptophan or L-kynurenine, these 70 carbon sources were added at a final concentration of 5mM and cultures were grown for 48 71 hours.

72 Construction and complementation of the B. pseudomallei K96243 ∆kynB mutant

73 PCR was used to amplify both 500bp upstream of the kynB gene using primers KynB_1 (5’- 74 GTATCCCGGGAAATCGGAGTAGTGCTTCAG-3’) and KynB_2 5’- 75 GCATGCGTCTTCAGAATTCCATGCCGTTGTCGAAGAAACT-3’) and 500bp downstream 76 using KynB_3 (5’-CGACAACGGCATGGAATTCTGAAGACGCATGCGCGTGCCC-3’ and 77 KynB_4 (5’-CCGTACTAGTGTCGATGAGGCGCAGTTCGT-3’) The PCR product also 78 contained regions of sequence that were specific for the start and end of the kynB gene in 79 order for homologous recombination to occur. The upstream and downstream PCR products 80 were then digested with EcoRI and ligated together to create the knockout cassette. This was 81 then cloned into pDM4 [29] via the XmaI and SpeI restriction sites. The resulting plasmid was 82 transformed into E. coli DH5α λpir cells and then conjugated into B. pseudomallei K96243 to 83 create a merodiploid strain with the aid of the helper strain E. coli pKR2013. Colonies were 84 plated onto LB agar supplemented with 10% (w/v) sucrose to drive sacB expression in pDM4 85 and promote recombination. ΔkynB mutants were confirmed by PCR and sequencing of the 86 resulting DNA fragment. For construction of pBHR-kynB, the kynB gene was PCR amplified 87 using primers kynB_comp_fwd (5’-GGCTCACTAGTATGGACACGATCTGGGAC-3’) and 88 kynB_comp_rv 5’-GCCATGATATCCACGCGCATGCGTCTTC-3’) and cloned into SpeI and 89 EcoRV sites of pBHR4.

90 Biofilm assay

91 Cultures of B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. pseudomallei 92 K96243 ∆kynB/pBHR-kynB were grown on tryptic soy agar (TSA) plates at 37oC for 16 hour 93 and then inoculated into PBS standardising to OD590nm 1.0. Cultures were then diluted 1:2000 94 in fresh tryptic soy broth (TSB) or filter sterilised spent media taken from an overnight culture 95 of B. pseudomallei K96243 grown in TSB. 150µl aliquots were added to a 96-well peg lidded 96 plate (12 wells for each culture) and incubated at 37oC. After 24 hours the peg lid was 97 transferred into fresh or spent TSB media for a further 24 hours. Peg lids were washed in PBS 98 before drying at 65oC for 30 minutes. Peg grown biofilms were stained with crystal violet and 99 the optical density measured at 490nm following 3 x PBS and an ethanol treatment to release 100 the stain for quantification.

101 Swarming assay

102 Overnight cultures of B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. 103 pseudomallei K96243 ∆kynB/pBHR-kynB were diluted to OD590nm 1.0 in either LB or filter 104 sterilised spent media taken from an overnight culture of B. pseudomallei K96243 grown in 105 LB. 2 µl samples were then spotted onto the centre of swarming plates (5 g/l bacto agar, 8 g/l 106 nutrient broth N°2, 0.5%(w/v) glucose). Plates were then incubated for 24 hours at 37oC and 107 the distance swarmed measured with a ruler.

108 Persister assay

109 Overnight cultures of B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. 110 pseudomallei K96243 ∆kynB/pBHR-kynB were standardised to OD590nm 0.2 in LB or filter 111 sterilised spent media taken from an overnight culture of B. pseudomallei K96243 grown in 112 LB. 500 µl of culture was then mixed with 500 µl of 400 µg/ml (200 x MIC) ciprofloxacin in LB 113 in a well of 24 well plate and incubated for 24 hours at 37oC. Where appropriate anthranilic 114 acid was also added to the persister assays at the indicated concentrations. After incubation, 115 cells were washed in LB and plated onto LB agar or LB agar supplemented with 116 chloramphenicol for enumeration.

117 Siderophore activity

118 Overnight cultures of B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. 119 pseudomallei K96243 ∆kynB/pBHR-kynB were harvested by centrifugation. 3 µl of the 120 supernantant was then spotted in triplicate onto CAS agar plates and incubated for 24 hours 121 at 37oC. Removal of iron from the agar was seen with a blue to orange colour change. 122 Siderophore activity was measured by determining the area of this orange halo.

123 Protease activity

124 B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. pseudomallei K96243 125 ∆kynB/pBHR-kynB were grown in LB for 24 hours and then 1.5 ml aliquots were harvested by 126 centrifugation. 500 µl of the supernantant added to 500 µl of 5mg/ml azocasesin. The samples 127 were then incubated in a heat block for 30 mins at 37oC. 1 ml of 3.2% TCA was then added 128 and samples centrifuged at 13,000 rpm for 5 minutes. A 500 µl was then added to 500 µl of 129 0.5M NaOH. The optical density was then recorded at OD450nm. Protease activity was 130 obsserved as red to yellow colour change.

131 Metabolite extraction

132 5 ml overnight cultures of B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. 133 pseudomallei K96243 ∆kynB/pBHR-kynB were standardised to OD590nm 2.0. Samples were 134 then aliquoted into 1 ml fractions and cells were harvested by centrifugation for 7 minutes at 135 13,000 rpm. The supernatant was transferred to a fresh tube and 1 ml of acidified ethyl acetate 136 (0.01% acetic acid) was added and mixed vigorously for 1 min. The organic upper layer was 137 then transferred to a fresh tube. The lower solvent layer was then re-extracted as above and 138 the two organic phases were combined. The cell pellet was re-suspended in 1 ml of methanol 139 and incubated at room temperature for 10 minutes to lyse the cells. The samples were then 140 centrifuged at 13,000 rpm for 7 minutes to remove cell debris and the supernatant transferred 141 to a fresh tube. Both ethyl acetate (supernatant) and methanol extracted (cells) samples were 142 then air dried to completeness and stored at -20oC until mass spec analysis.

143 Mass spectrometry analysis

144 Dried samples were re-dissolved in 100 µl of methanol. Undissolved material was removed by 145 centrifugation and the clarified sample transferred to an LC vial prior to analysis. 10µl injections 146 of each sample were used.

147 The HPLC system used was a Waters 2795 separations module. The column oven was 148 maintained at 35°C. The HPLC Column used was a Phenomenex Gemini C18 column (3.0 149 µm, 100 x 3.0 mm) with an appropriate guard column. Mobile phase A was water +0.1% (v/v) 150 formic acid, and mobile phase B methanol +0.1% (v/v) formic acid. The flow rate throughout 151 the chromatographic separation was 450 µl/min. The binary gradient began initially at 10% B 152 and ran isocratically for the first 1 min before increasing linearly to 99% B in 9.5 min. The 153 gradient was then maintained at 99% B for 5 min. A decrease to 10% B occurred over 1 min, 154 and stayed at this composition for 4 min. Total run time per sample was 20 min.

155 The MS system used was a Micromass Quattro Ultima triple quadrupole mass spectrometer 156 equipped with an electro spray ionisation (ESI) interface. Instrument control, data collection 157 and analysis were conducted using Masslynx software. The electrospray settings were: cone 158 gas: 160 L/hr, desolvation gas: 760 L/hr, source temperature: 150°C, desolvation temperature: 159 350°C.

160 MS analysis was conducted under positive electrospray conditions (+ES) with the MS 161 operating in precursor ion scan mode. The scan range was m/z 80-450 with the collision 162 energy being ramped between 20-35 eV during each scan. Precursor scans of m/z= 173.1 163 were used for detecting methyl-AQs, with precursors of m/z=159.1 used for detecting AQs.

164 C57BL/6 infection study

165 Groups of 8 or 10 female C57BL/6 mice (6-8 week-old; Harlan Laboratories, Bicester, Oxon, 166 UK) were used throughout the studies. All animal experiments were performed in accordance 167 with the guidelines of the Animals (Scientific Procedures) Act of 1986 and were approved by 168 the local ethical review committee at the London School of Hygiene and Tropical Medicine. 169 For each infection, aliquots were thawed from frozen bacteria stocks and diluted in pyrogen- 170 free saline (PFS). Prior to intranasal (i.n.) infection, mice were anesthetized intraperitoneally 171 with ketamine (50mg/kg; Ketaset; Fort Dodge Animal, Iowa, USA) and xylazine (10 mg/kg; 172 Rompur; Bayer, Leverkusen, Germany) diluted in PFS. Challenge was performed 173 administering a total volume of 50 µl i.n. containing approximately 200 or 2500 colony forming 174 units of B. pseudomallei K96243 wild type or isogenic kynB mutant. Infection dose was 175 confirmed as described elsewhere [19]. Control uninfected mice received 50 µl of PFS.

176

177 RESULTS

178 kynB (BPSL0848) encodes a functional kynurenine formamidase

179 To confirm that BPSL0848 encodes a functional kynurenine formamidase, B. pseudomallei 180 K96243, B. pseudomallei ∆kynB and B. pseudomallei ∆kynB/pBHR-kynB strains were grown 181 in M9 media supplemented with either typtophan or kynurenine as the sole carbon source. 182 When the strains were inoculated into M9 with tryptophan, (the substrate for the kynurenine 183 pathway), the wild type and complemented strains were able to grow, whereas no growth was 184 seen with the ∆kynB mutant (Fig 1a). In contrast, when the strains were inoculated into M9 185 with kynurenine (the product for kynurenine formamidase) the medium was growth permissive 186 for all strains (Fig 1b).

187

188 Deletion of ∆kynB resulted in the production of fewer HAQs

189 Overnight cultures of B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. 190 pseudomallei K96243 ∆kynB/ pBHR-kynB were harvested by centrifugation and the 191 supernatant and cell pellets were separated and metabolites were solvent extracted. Samples 192 were then analysed by LC-MS looking for families of saturated, unsaturated and saturated 193 methyl HAQs (Table 2). The data revealed that wild type B. pseudomallei K96243 and the 194 complement strain contained 3 saturated methyl HAQs, 2 saturated HAQs and 2 unsaturated 195 HAQ types. 2 out of the 3 methyl HAQs were absent in the ∆kynB mutant and the third had a 196 reduced peak area. The 2 saturated HAQs were also absent from the ∆kynB mutant as was 197 one unsaturated HAQ. The other unsaturated HAQ had a reduced peak area in the mutant.

198

199 Deletion of kynB significantly increased biofilm production

200 Since KynB converts kynurenine into anthranilic acid, a precursor for signalling molecules 201 such as HHQ and 2-AA, we assessed the effect of deleting kynB on biofilm production. B. 202 pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. pseudomallei K96243 ∆kynB/ 203 pBHR-kynB were grown for 48 hours in TSB at 37oC. Peg lids inserted into the cultures were 204 used for the growth of biofilms and this was quantified after crystal violet staining (Fig 2). When 205 the wild type and complemented strains were grown in TSB the OD490nm following crystal 206 violet staining was approximately 0.1-0.15. In contrast the OD490nm for B. pseudomallei 207 K96243 ∆kynB was significantly greater at 0.30 (p<0.0001).

208

209 Deletion of kynB significantly decreased bacterial swarming

210 Next, the effect of deleting kynB on swarming motility was evaluated. Stationary phase 211 cultures of B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. pseudomallei 212 K96243 ∆kynB/ pBHR-kynB were standardised to an OD590nm 1.0 and then spotted onto 213 swarming agar plates and incubated for 24 hours at 37oC. On average the wild type and 214 complemented strains swarmed approximately 35 and 25 mm respectively from the point of 215 inoculation (Fig 3a). In contrast the ∆kynB mutant strain swarmed significantly less at 216 approximately 15 mm (p<0.05 or p<0.0001). The mutant also had a different swarming pattern, 217 forming small branch like structures that were absent in the wild type and complemented 218 strains (Fig 3b).

219

220 Deletion of kynB increased tolerance to ciprofloxacin

221 The effect of deleting kynB on antibiotic tolerance was then tested. Firstly, stationary phase 222 cultures of B. pseudomallei K96243 or B. pseudomallei K96243 ∆kynB were standardised to 8 223 an OD590nm 0.1 (10 CFU/ml) and incubated with a range of ciprofloxacin concentrations for 224 24 hours to determine the MIC. The MIC of ciprofloxacin for both strains was approximately 2 225 µg/ml (Fig S1). Subsequently, B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. 8 226 pseudomallei K96243 ∆kynB/ pBHR-kynB were standardised to an OD590nm 0.1 (10 CFU/ml) 227 and incubated with 100 x MIC (200 µg/ml) ciprofloxacin for 24 hours. On enumeration of 228 surviving bacteria, there were approximately 10-fold more B. pseudomallei K96243 ∆kynB 229 compared to the wild type and complement strains (Fig 4a, ~10-3 compared to ~10-4 ). This 230 difference was significant (p<0.0001 or p<0.001).

231 To determine whether the difference in antibiotic tolerance between B. pseudomallei K96243 232 and B. pseudomallei K96243 ∆kynB was due to a difference in the persister cell populations, 233 these strains were treated with 100 x MIC ciprofloxacin and cell numbers determined 234 periodically over 30 hours. A biphasic kill curve was observed for both strains and the 235 population of antibiotic survivors after 24 and 30 hours was greater for the ∆kynB mutant 236 compared to wild type (Fig 4b).

237 Treating B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. pseudomallei K96243 238 ∆kynB/ pBHR-kynB with 100 x MIC ceftazidime (200 µg/ml) resulted in no significant difference 239 in survival frequencies on enumeration. All three strains showed approximately 10% survival 240 (data not shown). 241

242 Deletion of kynB did not affect siderophore activity, protease activity or virulence in 243 C57BL6 mice

244 Spot plating standardised overnight cultures of the B. pseudomallei strains onto CAS agar 245 plates revealed no difference in siderophore activity. Likewise, a colorimetric assay to detect 246 protease activity in spent media isolated from B. pseudomallei K96243, B. pseudomallei 247 K96243 ∆kynB or B. pseudomallei K96243 ∆kynB/pBHR-kynB showed no difference in 248 absorbance (data not shown).

249 Infection of C57BL/6 mice with B. pseudomallei K96243 or B. pseudomallei K96243 ∆kynB 250 resulted in similar patterns of survival. After challenge with approximately 3300 CFU of wild 251 type or 2,200 CFU of the ∆kynB mutant all mice had died by 4 days post infection. After 252 challenge with 60 CFU of wild type or 145 CFU of the ∆kynB mutantsimilar numbers of mice 253 had died at the termination of the study on day 170 (data not shown).

254

255 Addition of spent media from B. pseudomallei K96243 can partially suppress the 256 hyperbiofilm phenotype of ∆kynB but had no effect on the antibiotic tolerance or 257 swarming phenotypes

258 B. pseudomallei K96243 or B. pseudomallei K96243 ∆kynB were inoculated into a 96- well 259 plate and grown for 48 hours in either TSB or spent TSB media that had been isolated from a 260 culture of wild type B. pseudomallei K96243. Peg lids inserted into the cultures were used to 261 cultivate the biofilms. Biofilm growth was quantified by crystal violet staining and measuring 262 the optical density. When the wild type strain was grown in either TSB or spent media the 263 OD490nm following crystal violet staining was approximately 0.1-0.15 (Fig 5a). In contrast the 264 OD490nm for B. pseudomallei K96243 ∆kynB varied depending on the growth media. In fresh 265 TSB media the OD490nm was approximately 0.35, whereas in spent media the optical density 266 was significantly reduced to 0.25 (p<0.0001).

267

268 When B. pseudomallei K96243 or B. pseudomallei K96243 ∆kynB were resuspended in spent 269 media from K96243 and spotted onto swarming agar, the bacterial strains swarmed to a similar 270 distance as the corresponding strain in fresh media (Fig 5b). Likewise, resuspending the 271 strains in K96243 spent media before ciprofloxacin treatment had no effect on persister 272 frequency compared to fresh LB (Fig 5c).

273

274 Addition of exogenous anthranilic acid reduced the hyperbiofilm phenotype but 275 increased bacterial persistence.

276 Next we asked whether supplementing B. pseudomallei ∆kynB with anthranilic acid (the end 277 product of the kynABU pathway) could complement the biofilm and persister phenotypes. 278 Firstly B. pseudomallei K96243 or B. pseudomallei K96243 ∆kynB were inoculated into a 96- 279 well plate and grown for 48 hours in either TSB or TSB media supplemented with 0, 1 or 10 280 mM anthranilic acid. Addition of 1 mM anthranilic acid to the wild type strain had no effect on 281 biofilm growth compared to a non-supplemented control, whereas addition of 10 mM was able 282 to partially reduce biofilm growth (fig 6a). In comparison, addition of anthranilic acid to the 283 ∆kynB mutant was able to significantly reduce biofilm formation in a step wise manner 284 compared to the non-supplemented ∆kynB control (p<0.0001). 10 mM anthranilic acid was 285 able to repress biofilm formation of the ∆kynB mutant to wild type levels. 286 Next overnight cultures of B. pseudomallei K96243 or B. pseudomallei K96243 ∆kynB were 287 standardised to OD590nm 0.1 and incubated with 100 x MIC ciprofloxacin and either 0, 1 or 10 288 mM anthranilic acid for 24 hours (fig 6b). Following incubation cells were washed and plated 289 onto LB agar plates for enumeration. When cells were treated with ciprofloxacin only, persister 290 frequencies for both wild type and ∆kynB were similar to previous persister assays. Addition 291 of anthranilic acid significantly increased persister frequencies of the wild type strain in a 292 relative manner, i.e. 10-fold more anthranilic acid resulted in approximately 10-fold more 293 persisters. Increasing the concentration of anthranilic acid also increased persister 294 frequencies for the ∆kynB mutant. Supplementation of 10 mM anthranilic acid increased both 295 B. pseudomallei and B. pseudomallei K96243 ∆kynB persister frequencies to comparable 296 levels at around 5 x 10-2.

297

298 DISCUSSION

299 There are many manifestations of melioidosis in humans, ranging from acute to chronic 300 disease and to a latent and often inapparent infection which can spontaneously re-appear as 301 overt disease [2] . Chronic infection accounts for approximately 11% of patients and is 302 characterised by symptoms lasting >2 months [2,20]. The treatment of disease can be 303 difficult because of the high degree of resistance to many antimicrobial drugs. Even following 304 apparently successful drug intervention, relapse occurs in 10-30% of cases [20]. Recurrent 305 disease is a occurs in 6-13 % of patients. After primary exposure, sometimes accompanied 306 with symptomatic disease, the infection becomes latent, only to appear as symptomatic 307 disease up to several decades later [2,21]. The intrinsic resilience of B. pseudomallei to 308 harsh environments such as nutrient depletion, salt concentrations and acidic environments 309 may also have important ramifications in establishing chronic disease in humans [22,23].

310 As with B. pseudomallei, P. aeruginosa infections can also be difficult to treat. In P. 311 aeruginosa the bacterium is able to switch between phenotypes associated with chronic and 312 phenotypes associated with acute disease. During acute infection, cells express a wide 313 variety of virulence factors including type III secretion systems, flagella and QS-regulated 314 virulence factors such as proteases, elastases, phenazines, and toxins that allow the 315 establishment of infection. [24-26]. In this state the bacteria are more susceptible to antibiotic 316 treatment. Conversely, the onset of chronic disease is associated with the development of 317 biofilm, hypermutability, conversion to mucoidy, acquisition of resistance and suppression of 318 the expression of acute phase virulence factors and the bacteria are less susceptible to 319 antibiotic treatment [27-29]. This switch from acute to chronic phase involves the RetS- 320 LadS-GacSA-Rsm regulatory cascade, with RetS and LadS having opposing effects on the 321 GacS sensor kinase [30-32].

322 The onset of chronic disease caused by many other bacterial species is associated with 323 biofilm formation. For example, venous leg ulcers have been associated with 324 Staphylococcus aureus biofilms [33] while biofilms play a key role in recurrent urinary tract 325 infections caused by uropathogenic Escherichia coli [34]. The cells in these biofilms are 326 resistant to antibiotics, partially as a consequence of the physical constraints on the ability of 327 antibiotics and antimicrobials to fully penetrate and kill all the cells [35,36] and partly 328 because biofilms are also rich in drug-tolerant persister cells [37]. Biofilms and persister cells 329 may be important reservoirs in chronic and recurring melioidosis.

330 The kynurenine pathway plays a role in regulating a range of phenotypes associated with 331 chronic disease including biofilm formation, swarming and persistence. Biofilm formation and 332 swarming are inversely correlated in P. aeruginosa and this is also our finding in B. 333 pseudomallei [38]. The ∆kynB mutant of B. pseudomallei produced more biofilm than wild type 334 and swarmed less, producing branched like structures. Previous studies with P. aeruginosa 335 have shown that the pqs operon was important in controlling these phenotypes [39] [40] [41]. 336 Deletion of pqsH decreased biofilm density, while incubation of the mutant with exogenous 337 PQS increased biofilm density [39]. However, previous studies have shown that B. 338 pseudomallei does not produce PQS [15,42] and we found no evidence that the K96243 strain 339 produces HHQ. Our data suggests that other HAQ(s) regulate biofilm and swarming in B. 340 pseudomallei K96243.

341 Our results also showed that the B. pseudomallei ∆kynB mutant was more tolerant to 342 ciprofloxacin due to an increase in the persister population but there was no difference in the 343 frequency of ceftazidime persisters. Previous studies suggest that ceftazidime reveals 344 persisters with anaerobic-like metabolism, whereas ciprofloxacin reveals persisters with 345 reduced metabolism [43,44]. Quorum sensing can also influence the appearance of 346 ciprofloxacin persisters; deletion of the HAQ synthesis gene pqsA in P. aeruginosa increased 347 ciprofloxacin tolerance, while overexpressing HAQs increased susceptibility [45].Other 348 quorum sensing molecules have been linked to persistence in other bacteria. For example, 349 the addition of pyocyanin, paraquat or acyl homo serine-lactone significantly increased 350 persister numbers of logarithmic phase P. aeruginosa [46] while indole increased persistence 351 in E. coli [47]. It therefore seems possible that increase in frequency of ciprofloxacin persister 352 cells reflect the disruption of signalling cascades in the kynB mutant.

353 The kynurenine pathway can feed anthranilic acid into the HAQ/HMAQ quorum signalling 354 pathway and deletion of kynB reduced the level of these signalling molecules in the bacterial 355 culture supernatant.

356 Since the ∆kynB mutant produced fewer HAQ/HMAQs, we attempted to complement the 357 mutant by addition of spent media isolated from wild type cultures or chemically complement 358 with the kynurenine pathway product anthranilic acid. Previous assays have shown that 359 supplementing exogenous anthranilic acid to kynurenine pathway mutants can restore the 360 production of HAQ compounds [17]. Both spent media and anthranilic acid reduced hyper 361 biofilm production in the ∆kynB mutant, with 10 mM anthranilic acid restoring production to the 362 same level as the wild type. This suggests that both anthranilic acid and HAQ/HMAQ 363 production are important in biofilm regulation.

364 Spent media from wild type cultures was unable to complement the reduced swarming or 365 increased ciprofloxacin persister phenotypes. Failure to complement these phenotypes maybe 366 due to HAQ/HMAQs in the supplemented spent media being below a threshold needed to 367 elicit a response in these assays. However, anthranilic acid complementation also failed to 368 reduce the perister frequencies of the ∆kynB mutant. This suggests that the persister 369 phenotype is not due to the loss of anthranilic acid or HAQ/HMAQ production. Paradoxically, 370 at the highest concentration of anthranilic acid tested (10 mM) the persister frequency of both 371 wild type and ∆kynB were both increased to a similar level. Clearly there is complex interplay 372 between different regulatory mechanisms controlling persistence that warrants further study.

373 Quorum sensing pathways are attractive targets for the design of novel therapeutics in many 374 bacteria, as they offer an alternate approach to treat infection by targeting virulence rather 375 than through the inhibition of bacterial growth.

376 Deletion of kynB did not affect virulence of B. pseudomallei in a C57BL/6 mice. 377 Thepathogenesis of experimental melioidosis in this strain of mice is reported to shown some 378 similarities to chronic human disease [48]. Importantly, our results indicate that the phenotypes 379 we have investigated, and which are associated with long-term infections caused by other 380 bacteria, do not appear to be associated with chronic disease caused by B. pseudomallei. 381 However, it is important to highlight that it is not known whether the C57BL/6 model of disease 382 reflects the long-term and often clinically insignificant form of melioidosis which can re-appear 383 periodically. The development of a murine model of latent infection would allow the role of 384 KynB in long trem infections to be further investigated.

385

386

387 REFERENCES

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(2007) Effects of iron on DNA 481 release and biofilm development by Pseudomonas aeruginosa. Microbiology 153: 1318-1328. 482 41. Ha DG, Merritt JH, Hampton TH, Hodgkinson JT, Janecek M, et al. (2011) 2-Heptyl-4-quinolone, a 483 precursor of the Pseudomonas quinolone signal molecule, modulates swarming motility in 484 Pseudomonas aeruginosa. J Bacteriol 193: 6770-6780. 485 42. Diggle SP, Lumjiaktase P, Dipilato F, Winzer K, Kunakorn M, et al. (2006) Functional genetic analysis 486 reveals a 2-Alkyl-4-quinolone signaling system in the human pathogen Burkholderia 487 pseudomallei and related bacteria. Chem Biol 13: 701-710. 488 43. Hemsley CM, Luo JX, Andreae CA, Butler CS, Soyer OS, et al. (2014) Bacterial drug tolerance under 489 clinical conditions is governed by anaerobic adaptation but not anaerobic respiration. 490 Antimicrob Agents Chemother 58: 5775-5783. 491 44. Zhang L, Chiang WC, Gao Q, Givskov M, Tolker-Nielsen T, et al. (2012) The catabolite repression 492 control protein Crc plays a role in the development of antimicrobial-tolerant subpopulations 493 in Pseudomonas aeruginosa biofilms. Microbiology 158: 3014-3019. 494 45. Haussler S, Becker T (2008) The pseudomonas quinolone signal (PQS) balances life and death in 495 Pseudomonas aeruginosa populations. Plos Pathog 4: e1000166. 496 46. Moker N, Dean CR, Tao J (2010) Pseudomonas aeruginosa increases formation of multidrug- 497 tolerant persister cells in response to quorum-sensing signaling molecules. J Bacteriol 192: 498 1946-1955. 499 47. Vega NM, Allison KR, Khalil AS, Collins JJ (2012) Signaling-mediated bacterial persister formation. 500 Nat Chem Biol 8: 431-433. 501 48. Leakey AK, Ulett GC, Hirst RG (1998) BALB/c and C57Bl/6 mice infected with virulent Burkholderia 502 pseudomallei provide contrasting animal models for the acute and chronic forms of human 503 melioidosis. Microb Pathog 24: 269-275. 504 49. Milton DL, O'Toole R, Horstedt P, Wolf-Watz H (1996) Flagellin A is essential for the virulence of 505 Vibrio anguillarum. J Bacteriol 178: 1310-1319.

506

507 508 Table 1 Bacterial strains used or created in this study

509 Bacterial Strain Genotype/ comments Source

E. coli DH5α λpir ΔlacU169(ΦlacZΔM15), Lab strain collection recA1, endA1, hsdR17, thi-1, gyrA96, relA1, λpir phage lysogen

E. coli DH5α (pRK2013) ΔlacU169(ΦlacZΔM15), Lab strain collection recA1, endA1, hsdR17, thi-1, gyrA96, relA1, pRK2013 (KmR oriColE1 RK2-Mob+ RK2-Tra+

B. pseudomallei K96243 Clinical isolate Lab strain collection

B. pseudomallei K96243 K96243 derivative. This study ΔkynB Unmarked deletion ΔkynB

Plasmid Comments Source

pBHR-kynB camR This study

pDM4 camR [49]

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525 526

527

528

529

530

531

532

533

534

535

536 Table 2. Peak areas of H(M)AQs following LC-MS analysis of extracted supernantant and whole 537 bacterial cells. ND= not detected.

Supernatant Extracts Cell Extracts

Saturated Methyl AQs R [M+H]+ K96 ΔKynB Comp K96 ΔKynB Comp

C5H11 230 ND ND ND ND ND ND

C6H13 244 ND ND ND ND ND ND

C7H15 258 7862 ND 6343 6058 ND 2144

C8H17 272 ND ND ND 1133 ND ND

C9H19 286 38934 1230 21153 65947 727 37570

C10H21 300 4353 ND 1988 12076 ND 6404

Supernatant Extracts Cell Extracts Saturated AQs R [M+H]+ K96 ΔKynB Comp K96 ΔKynB Comp

C5H11 216 ND ND ND ND ND ND

C6H13 230 ND ND ND ND ND ND

C7H15 244 ND ND ND ND ND ND

C8H17 258 ND ND ND ND ND ND

C9H19 272 2411 ND 6996 3344 ND 1732

C10H21 286 2659 ND 4202 ND ND ND

C11H23 300 ND ND ND ND ND ND

Unsaturated AQs Supernatant Extracts Cell Extracts R [M+H]+ K96 ΔKynB Comp K96 ΔKynB Comp

C5H9 214 ND ND ND ND ND ND

C6H11 228 ND ND ND ND ND ND

C7H13 242 ND ND 1345 ND ND ND C8H15 256 ND ND ND ND ND ND

C9H17 270 22564 1250 43537 21279 464 10201

C10H19 284 2438 ND 2477 5035 ND 1748

C11H21 298 ND ND ND ND ND ND 538

539

540

541

542

543

544 A) B)

0 .5 * * 0 .5

0 .4 0 .4

m m

n 0 .3

5 D

0 .2 D 0 .2

O O

0 .1 0 .1

0 .0 0 .0

3 B B 3 B 4 n n 4 B 2 y n 6 y 2 y n k k 6 k y 9  - 9 k K 4  - R K 4 H R B H /p B B /p n B y n k y  k  S tr a in S tr a in 545

546 Fig 1. Growth of B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. pseudomallei 547 K96243 ∆kynB ∆kynB/pBHR-kynB strain in M9 media with A) 5mM Tryptophan as the only carbon 548 source B) 5mM L-kynurenine as the only carbon source. Cultures were grown for 48 hours at 549 37oC, 200rpm in 3ml volumes. Data shown are the average of two biological replicates. Error 550 bars show SEM. *=p<0.05 following one way Anova, Tukey post-test.

551

552

553

554

555

556

557 558

559

560

561

562

0 .4 **** ****

0 .3

n 0

9 0 .2

D O 0 .1

0 .0

6 B B 9 n n K y y k k  - R H B /p B n y k  S tr a in 563 564 Fig 2. Biofilm production following growth of B. pseudomallei K96243, B. pseudomallei K96243 565 ∆kynB or B. pseudomallei K96243 ∆kynB ∆kynB/pBHR-kynB on peg lids for 48 hours. Biofilm 566 was quantified by measuring crystal violet released following ethanol treatment. Data shown is 567 the average of two biological repeats. Error bars show SE. ****= p<0.001 following one way 568 Anova, Tukey post-test.

569

570

571

572

573

574

575

576

577 578

579

580 A) **** * 581 4 0

582 )

m (

583 3 0 d

584 e

m r

a 2 0

585 w

586 c

n 1 0 a

587 t

i D 588 0

3 B B 4 n n 589 2 y y 6 k k 9  - K 4 R 590 H B /p 591 B B) n y k 592  S tr a in 593

594

595

596 K96243 ∆kynB ∆kynB/pBHR-kynB 597

598

599 Fig 3. Swarming of motility of B. pseudomallei K96243, B. pseudomallei K96243 ∆kynB or B. 600 pseudomallei K96243 ∆kynB ∆kynB/pBHR-kynB on swarming agar for 24 hours at 37 oC. A) 601 Distance migrated. Data shown are the average of five biological repeats. **** = p<0.0001, 602 *=p<0.05 following One way Anova, Tukey post-test. Error bars represent SEM. B) 603 representative images

604

605

606

607

608

609 610

10-2 10 9 **** *** 8 y 10 -3 K96243 c 10 n 7 e 10 kynB

q 10-4 6

/ 10

r 5 -5 F 10

t 10

s i 10 4

r -6

e 10 10 3

10-7 10 2 0 10 20 30 40 3 B B 4 n n 62 y y Time (hours) 9 k -k K  R H B A) B) /p B n 611 ky  612

Strain 613

614

615

616 Fig 4.A) Persister frequency following incubation of B. pseudomallei K96243, B. pseudomallei 617 K96243 ∆kynB or B. pseudomallei K96243 ∆kynB/ pBHR-kynB with 100 X MIC ciprofloxacin (200 618 µg/ml) for 24 hours at 37oC. Persister frequency was calculated as CFU post antibiotic treatment 619 divided by CFU pre-treatment. Data shows the average of three biological replicates. ****= 620 p<0.0001, ***=p<0.001.Error bars show SEM. B) Number of culturable cells of B. pseudomallei 621 K96243 or B. pseudomallei K96243 ∆kynB over a 30 hour treatment with 100 x Mic ciprofloxacin.

622

623

624

625

626

627

628

629

630

631

632 A) B) 633 4 0 0 .4 **** ) F r e s h L B 634 m

F r e s h L B m

S p e n t m e d ia ( 3 0 S p e n t m e d ia 635 d

0 .3 e

m a

n 636 2 0

0 w

9 0 .2

4 e

D 637

c O

n 1 0 a

0 .1 t

i D 0

0 .0 3 B 4 n 2 y 6 B 6 k 9 n 9 K y  k K  S tr a in s tra in C)

1 0 -2

F r e s h L B y

c S p e n t m e d ia -3

n 1 0

q e

r -4

f 1 0

t i

s -5

r 1 0

e P

1 0 -6

3 B 4 n 2 y 6 k 9  K

S tr a in

638

639 Fig 5. Complementation of B. pseudomallei K96243 and B. pseudomallei K96243 ∆kynB with the 640 addition of spent media isolated from cultures of B. pseudomallei K96243. A) Biofilm formation 641 following crystal violet staining. B) swarming distance C) Persister frequency following 24 hour 642 ciprofloxacin treatment. Data are representative from at least three biological replicates. ****= 643 p<0.0001, ***=p<0.001, **=p<0.01. Error bars show SEM.

644

645

646

647

648

649

650

651 652 A) B)

0 m M

) h

t 1 .5 -1 1 m M

w 0 m M 1 0

o **

r 1 0 m M

1 m M

c t

-2 n e 1 0 m M *** l 1 .0 1 0 ****

**** e

o i

u **** **

e r

a -3

f t

1 0

r y

0 .5 e

(

s -4 0

r 1 0

4 P

D 0 .0

O -5 3 B 1 0 4 n 2 y 6 3 B k 4 9  n K 2 y 6 k 9  K s tra in 653 s tra in 654 Fig 6. Complementation of B. pseudomallei K96243 and B. pseudomallei K96243 ∆kynB with 655 exogenous anthranilic acid. A) Biofilm formation quantified by measuring the optical density 656 following crystal violet staining. B) Persister frequency following 24 hours treatment with 100 X 657 MIC ciprofloxacin. ****= p<0.0001, ***=p<0.001, **=p<0.01. Error bars show SEM following two- 658 way Anova, Tukey post-test. Data is the average of at least 2 biological repeats.

659

660

661

662

663

664

665

666

667

668

669

670

671

672

673 674

675

1.5 676 K96243 677 kynB 1.0 678 m

0 679 9

680 O 0.5

681

682 0.0 6 8 4 2 6 8 4 2 1 5 5 0 5 2 6 3 1 0. .2 683 2 1 0

684 Figure Ciprofloxacin concentrationg/ml S1. MIC 685 determination of B. 686 pseudomallei 687 K96243 or B. pseudomallei K96243 ∆kynB treated with a range of ciprofloxacin concentrations for 688 24 hours. MIC was determined as the concentration of ciprofloxacin before an increase in optical 689 density was observed

690

691

692

693