Identification and characterization of a metalloprotein involved in

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Identification and characterization of a metalloprotein involved in gallium internalization in Pseudomonas aeruginosa Yu Guo, Wangming Li, Hongyan Li, and wei xia ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00271 • Publication Date (Web): 02 Sep 2019 Downloaded from pubs.acs.org on September 2, 2019

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ACS Infectious Diseases

Yu Guo,a Wangming Li,c Hongyan Lib and Wei Xia*, a a.

MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, China, 510275.

b. Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China. c.

Guangdong Institute of Applied Biological Resources, Guangdong Key Laboratory of Animal Conservation and Resource Utilization/Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Guangzhou 510260, China.

*Corresponding author: [email protected] Keywords: gallium nitrate, iron metabolism, Pseudomonas aeruginosa, metalloprotein, internalization pathway Gallium nitrate (Ganite®) is a potential drug for the treatment of Pseudomonas aeruginosa infection. CRISPR/Cas9-based gene mutagenesis studies reveal that siderophore pyochelin-facilitated uptake and an ABC transporter are two major Ga3+ internalization pathways in P. aeruginosa. Crystal structures reveal that Ga3+ and Fe3+ occupy exactly the same metal site of HitA, a periplasmic iron-binding protein of ABC transporter system. The study provides molecular basis for Ga 3+ internalization by P. aeruginosa and facilitates gallium-based anti-microbial drug development.

Pseudomonas aeruginosa (P. aeruginosa) is a human opportunistic pathogen that is found widely in the environment. The bacterium causes a wide spectrum of diseases such as urinary, burn and respiratory infections.1 Importantly, P. aeruginosa is the main cause of lung decline and death in cystic fibrosis (CF) patients. 2 Due to the intrinsic drug resistance and the formation of biofilms, the infections caused by P. aeruginosa are usually resistant to treatment of multiple antibiotics.3 Particularly, the carbapenem-resistant P. aeruginosa was listed as a critical priority 1 pathogen by the WHO in 2017. Therefore, new therapeutic strategy is urgently needed to tackle drug-resistant P. aeruginosa infections. Iron is an essential nutrition for almost all organisms because it is required by enzymes involved in a series of vital cellular processes.4 Therefore, interfering with P. aeruginosa iron homeostasis may serve as a potential therapeutic strategy for bacterial eradication. Recent studies reveal that gallium could act as a “Trojan horse” to be internalized into bacteria owing to its similarity to iron，such as nearly identical ionic radius. Gallium could subsequently disrupt iron-dependent processes because, unlike iron, it cannot be reduced in physiological conditions. This strategy is successful to inhibit growth and biofilm formation of drug-resistant P. aeruginosa.5, 6 It is proposed that gallium could utilize certain iron-transport pathways for internalization into bacteria. However, P. aeruginosa acquires iron via multiple pathways, including two secreted siderophores pyoverdine (PVD) and pyochelin (PCH),7, 8 FeoB transport system for ferrous ions (Fe2+),9 two characterized haem-uptake systems10 and a classical high affinity iron ABC transporter system (HitABC).11 Recently, a new iron acquisition pathway has also been identified in P. aeruginosa, which involves the outer mem-

brane vesicles, secreted protein TseF and the Pseudomonas quinolone signal (PQS) molecules.12 It remains unknown which iron acquisition systems are involved in gallium uptake. Recent transposon-based mutagenesis results demonstrated that inactivation of hitA gene, which encodes a periplasmic iron-binding protein for ABC transporter, significantly enhanced gallium resistance of P. aeruginosa, implying that HitA protein is involved in gallium uptake.13 However, the detailed mechanism is not fully characterized. To investigate the molecular mechanism of gallium uptake by P. aeruginosa, we utilized CRISPR/Cas9-based genome editing tools to mutate essential genes in P. aeruginosa model strain PAO1 to block each iron acquisition pathway individually.14 Totally 5 different PAO1 mutants were constructed as described in the supporting information. In brief, the PAO1∆hitA mutant was constructed to block the classical high affinity iron acquisition system ABC transporter.15 Two mutants PAO1∆pvdA and PAO1∆pchF abolished the biosynthesis pathways of two siderophores, PVD and PCH, respectively.7 While PAO1∆feoB mutant lost the high affinity inner membrane ferrous ion transporter and PAO1∆tesf mutant abrogated the Tesf-mediated iron acquisition pathway (Fig. S1, S2).12 We first examined the effects of Ga(III) on the growth of wildtype (WT) and different PAO1 mutants. The minimal inhibition concentrations (MICs) of Ga(NO3)3 against different strains in iron-deficient M9 medium were measured at 24 h and 48 h. In line with previous studies, Ga(NO3)3 showed bacteriostatic activity against WT PAO1 with MICs of 7.8 and 15.6 µM for 24 h and 48 h, respectively.5 The same MICs were obtained for PAO1∆pvdA, PAO1∆pchF, PAO1∆feoB and PAO1∆tesf mutants. In contrast, the PAO1∆hitA mutant has significantly increased MIC values of 62.5

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µM for 24 h and 125 µM for 48 h, which are nearly 8-fold higher than those of WT PAO1 (Fig. 1A). Moreover, the bacterial growth was monitored in the presence of Ga(NO3)3 and the OD600 of bacterial cultures were recorded. As shown in Fig S3, the growth of WT PAO1 was inhibited by 8 µM Ga(NO3)3 and no bacterial growth was observed with 32 µM Ga(NO3)3. Similar phenomena were observed for PAO1∆pvdA, PAO1∆pchF, PAO1∆feoB and PAO1∆tesf mutants. In consistent with the MIC results, PAO1∆hitA mutant exhibited remarkably increased resistance to gallium toxicity. The final OD600 of PAO1∆hitA mutant in the presence of 8 µM Ga(NO3)3 reached approximate 90% of that without Ga(NO3)3. Substantial bacterial growth of PAO1∆hitA mutant was observed even in the presence of 32 µM Ga(NO3)3. The hitB gene encodes a cytoplasmic permease, which binds iron delivered from HitA and transports iron across inner membrane. Therefore, disruption of HitB permease should also abrogate gallium internalization. Indeed, hitB knockout or hitAB double-mutant also remarkably increased P. aeruginosa’s tolerance to gallium (Fig. S3 C, D and Fig. S4). Complementation of hitAB double-mutant (S. aureus ΔhitAB) with the hitAB wild-type alleles (pAK1900-hitAB) restored bacterial sensitivity to Ga(NO3)3 (Fig. S3 I), which are consistent with the bacterial colony-formation results in the presence of 8 µM Ga(NO3)3 after 14 h incubation (Fig. 1B). All the results demonstrate that abrogation of the iron ABC transporter significantly enhances P. aeruginosa gallium resistance.

Figure 1. (A) MIC values of Ga(NO3)3 against wild-type (WT) P. aeruginosa and different mutants. (B) Colony-forming unit of P. aeruginosa and different mutants in the presence of 8 µM Ga(NO3)3 after 14 h incubation. All experiments are performed in triplicated and data are presented as mean  sd. * means p< 0.05, ** means p< 0.01, *** means p< 0.001, **** means p< 0.0001.

To further assess the effect of different iron acquisition pathways on gallium uptake, PAO1 mutants were incubated with 2 μM Ga(NO3)3 for 6 h in M9 medium and the intracellular gallium contents were quantified by inductively coupled plasma mass spectrometry (ICP-MS). As shown in Fig. 2A, the intracellular gallium levels in PAO1∆feoB and PAO1∆tesf mutants are similar to that in WT PAO1. Intriguingly, two siderophore mutants PAO1∆pvdA and PAO1∆pchF have different impacts on gallium uptake. PAO1∆pvdA mutant has a slightly higher gallium level, while PAO1∆pchF mutant possesses a lower gallium level compared to WT PAO1. The results are consistent with a previous report that siderophore PCH rather than PVD could potentiate gallium uptake.16 As expected, lower intracellular gallium levels were observed for PAO1∆hitA and PAO1∆hitB mutants, while the lowest

gallium level was found for the PAO1∆hitAB double-mutant, indicating that both proteins are involved in the uptake of Ga3+. Although the biosynthesis of siderophores PVD and PCH are not perturbed in PAO1∆hitAB (Fig. S5), substantial decline of intracellular iron level was also observed in this double-mutant strain (Fig. 2B), implying that the HitABC transporter is the major contributor for iron uptake in P. aeruginosa.

Figure 2. Intracellular gallium contents (A) and iron contents (B) of WT P. aeruginosa and mutants. All experiments are performed in triplicated and data are presented as mean  sd. * means p< 0.05, ** means p< 0.01, *** means p< 0.001.

Furthermore, we also generated pBAD30-hitAB and pET28ahitC constructs and co-transformed the plasmids into E. coli BL21 strain, which lacks the endogenous ABC transporter for iron acquisition. The wild-type and HitABC-reconstituted E. coli were incubated in M9 medium supplemented with 15 μM Ga(NO3)3, 0.01% (w/v) L-arabinose and 0.05 mM IPTG for 6 h. The intracellular gallium and iron levels of the E. coli were measured by ICP-MS. Both the gallium and iron levels in reconstituted E. coli are almost twice the amount of that in WT strain (Fig. S6), indicating that the ABC transporter is responsible for both iron and gallium uptake in P. aeruginosa. HitA is a soluble ferric iron-binding protein located in the bacterial periplasm and binds a single Fe3+ with high affinity. We envisioned that HitA could exert the similar role in Ga3+ uptake process. Indeed, cellular thermal shift assay (CETSA) results showed that the apparent melting temperature (Tm) of intracellular HitA was 42.5 °C. Supplementation of P. aeruginosa bacterial culture with 8 μM Ga(NO3)3 resulted in a substantial shift of the Tm to 45.0 °C, indicative of Ga3+-binding of HitA in vivo (Fig. S7).

Figure 3. (A) Ga3+-binding stoichiometry of HitA determined by ICP-MS coupled with BCA assay. (B) Isothermal titration calorimetry of Ga3+ binding to HitA in the presence of 2 mM citrate. The data were fitted to a oneset-of-sites binding model.


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ACS Infectious Diseases Subsequently, Ga3+-binding capability of HitA was characterized in vitro. Apo-HitA was expressed and purified as described in the supporting information (Fig. S8). Purified apo-HitA was incubated with an excess amount of Ga(NO3)3. After removing the unbound metal ions by desalting column, HitA was identified to bind 1.1 ±0.12 molar equivalence of Ga3+ determined by ICP-MS (Fig. 3A). The binding affinity and stoichiometry between HitA and Ga3+ was subsequently measured by isothermal titration calorimetry (ITC). To prevent Ga3+ hydrolysis, 2 mM citrate was added during ITC experiments. The apparent disassociation constant (KD) was determined to be 2.35 ± 0.41 µM with as a stoichiometry of 0.82 ± 0.05 (Fig. 3B). By taking account of the log constant for gallium citrate of 10.02,17 the binding constant for Ga3+-HitA was calculated to be approximately 4.471015 M-1. Previous studies on HitA homologues from Neisseria and Haemophilus demonstrated that HitA bound Fe3+ strongly with binding constant value around 1018-1019 M-1.18, 19 It is indicated that HitA could probably bind Fe3+ much tightly than Ga3+. Indeed, metal competition results demonstrated that Fe3+ could readily replace Ga3+ from Ga3+-HitA protein. In contrast, no release of Fe3+ from Fe3+-HitA could be observed even in the presence of more than 10 molar equivalents of Ga3+ (Fig. S9). Finally, we resolved the high resolution crystal structures of apo-, Fe3+-bound and Ga3+-bound HitA proteins (Fig. S10). The apo-HitA adopts the typical periplasmic ligand-binding protein (PLBP) fold, which possesses bilobate structure with two domains connected by a number of flexible β-strands.20 The final structure models of apo-HitA and metal-bound HitA contain two chains of HitA in one asymmetric unit. The metal coordination sphere are nearly identical in each chain (Fig. S10). Structural comparison revealed that apo-HitA displayed a 2.9°hinge movement of the two domains relative to the Ga3+-HitA structure (Fig. 4A). The angle of movement is smaller than that observed in Haemophilus influenzae HitA upon metal binding, which is probably due to the crystal packing effect in the apo-HitA structure (Fig. S11).21 Such an “openclosed” domain movement upon metal binding is also observed in human transferrin and lactoferrin.22, 23 Inspection of the Ga3+-HitA crystal structure revealed that the Ga3+-binding site was located at the domain interface with four residues originated from both domains. The Ga3+ coordination site is composed of His39, Glu87, Tyr223, Tyr224, one water molecule and an exogenous phosphate ion, which form an octahedral geometry (Fig. 4B). Importantly, the structure of Ga3+-HitA resembles that of Fe3+-HitA with backbone root-mean-square deviation (RMSD) of 0.268 Å. The coordination sphere of Fe3+ is almost the same as that identified in Ga3+ site, consisting of the four residues (His39, Glu87, Tyr223 and Tyr224), one water molecule and a phosphate ion as the monodentate ligand (Fig. S12). The structural data further confirmed that Ga3+ could mimetic Fe3+ to occupy the metal-binding site of HitA.

Figure 4. Crystal structures of apo-HitA (PDBID: 6IWF) and Ga3+-HitA (PDBID: 6J2S). (A) Residues 29-126 of apo-HitA and Ga3+-HitA structures are superimposed. The angle of the other sub-domain (residues 127-334) between apo- and Ga3+-HitA is calculated to be 2.9°. An arrow shows the movement of hitA hinge upon Ga3+ binding. (B) The coordination of Ga3+ in the Ga3+-HitA structure. The coordination residues are shown in cyan sticks and Ga3+ in gray sphere.

Ga3+ was identified as an inhibitor of P. aeruginosa growth and biofilm formation in vitro.5 Previous studies also showed that some gallium compounds exerted antibacterial activities against a number of human pathogens, including multi-drug resistant CF clinical isolates.24-26 A recent preliminary phase I clinical trial further demonstrated that gallium nitrate exhibited anti-P. aeruginosa activity in a mouse infection model. Moreover, gallium nitrate could improve lung function in patients with CF and chronic P. aeruginosa lung infection without signs of any serious adverse effects, indicative of a potential clinical use of gallium nitrate for treatment of P. aeruginosa infection.6 Although gallium was shown to interfere with iron metabolism in P. aeruginosa, how the metal ion was internalized by the bacteria was not fully understood. Metal susceptibility combined with metal uptake assays demonstrated that disruption of ABC transporter significantly enhanced bacterial resistance to gallium toxicity and decreased intracellular gallium uptake, indicating that the ABC transporter composed of HitABC protein is one of the gallium uptake pathways. Meanwhile, abolishment of pyochelin biosynthesis also attenuated intracellular gallium uptake. It is indicated that pyochelin is also involved in gallium uptake, which is in line with previous studies that pyochelin could form complex with Ga3+.27 Ferripyochelin reductase is required to reduce Fe3+ into Fe2+ to release iron from ferripyochelin (pyochelin-Fe3+).28 However, Ga3+ is a redox-inactive ion. It is probably that Ga3+ cannot be released from pyochelin-Ga3+complex after internalization by P. aeruginosa so that pyochelin-Ga3+ exhibits no toxicity to the bacteria. It is also consistent with the results that pyochelin-deficient P. aeruginosa mutant didn’t exhibit increased gallium resistance compared to WT strain. On the contrary, pyoverdine-deficient P. aeruginosa mutant has higher intracellular Ga3+ level. Although the detailed mechanism for this enhanced Ga3+ uptake capability is unknown, it is clearly indicated that pyoverdine is not directly involved in Ga3+ uptake. The similarity in ionic radius between Fe3+ and Ga3+ (Fe3+ 0.65 Å; Ga3+ 0.62 Å) allows gallium to be an iron substitute in HitA protein. Although the crystal structures revealed that Ga3+- and Fe3+-HitA had exactly the same overall structures and coordination spheres, the binding affinity of Fe3+ to HitA is almost 103-fold higher than that of Ga3+. The different metal binding affinity of Ga3+ and Fe3+ to HitA is probably due to the different metal ion acidity (log KOH=11.32 for Fe3+ compared with 10.91 for Ga3+).29 Previous studies demonstrated that tightly bound Fe3+ must undergo reduction to Fe2+ to be translocated by HitABC transporter into cytosol.19, 30 However, it remains unknown how Ga3+ was transferred from Ga3+-HitA to HitB and then released into cytosol since Ga3+ is redox-inactive. One possible mechanism is that the



weaker binding affinity enables Ga3+ transfer and delivery through HitABC, for which further investigation is warranted.

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ACKNOWLEDGMENTS We thank the staff from the BL17U1 and BL19U1 beamlines of Shanghai Synchrotron Radiation Facility (SSRF) in China for assistance during data collection. This work was supported by the National Natural Science Foundation of China (21671203, 21877131), Science and Technology Program of Guangzhou, China (201707010038), RGC of Hong Kong (17305415, 17333616), the Ministry of Education of China (IRT-17R111), the Fundamental Research Funds for the Central Universities.

REFERENCE Scheme 1. Schematic representation of pyochelin-facilitated and ABC transporter pathways for Ga3+ and Fe3+ uptake in P. aeruginosa.

In conclusion, we have identified pyochelin-facilitated uptake and ABC transporter (HitABC) are two major pathways for Ga3+ internalization in P. aeruginosa (Scheme 1). Ga3+ has a lower binding affinity towards HitA compared to Fe3+, which may render its subsequent transfer to HitB. The X-ray crystallography studies demonstrate that both Ga3+-HitA and Fe3+-HitA share the same structures and coordination spheres, so that P. aeruginosa cannot distinguish Ga3+ from Fe3+ for metal ion internalization. Our studies decipher the molecular mechanism for Ga3+ internalization in P. aeruginosa, providing a basis for the development of new galliumbased anti-P. aeruginosa drugs.

ASSOCIATED CONTENT Supporting Information Experimental procedures; X-ray diffraction data collection and structure refinement statistics for HitA structures (Table S1); plasmids (Table S2), bacterial strains (Table S3) and primers (Table S4) used in the study. Accession Codes Atomic coordinates and structure factors for apo-HitA, Fe3+HitA and Ga3+-HitA have been deposited in the Protein Data Bank with accession codes 6IWF, 6IVY and 6J2S.

AUTHOR INFORMATION Corresponding Author * Email: [email protected] ORCID Wei Xia: 0000-0001-6480-3265

Notes The authors declare no competing financial interests.

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Identification and characterization of a metalloprotein involved in

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