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Calprotectin-mediated zinc chelation inhibits Pseudomonas aeruginosa protease activity in cystic fibrosis sputum

Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to control cultures, but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc-starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to post-translationally inhibit zinc metalloprotease activity and thereby impact the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment. Importance The factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc-starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.


Introduction
sputum sample in our study that elicited the strongest zinc-starvation response had a zinc 148 concentration of ~2 µg/g (~2000 µg/L, ~31 µM) (Fig. 2a). Given the concomitant high zinc 149 concentration in our CF sputum samples and the elevated zinc starvation response in P. 150 aeruginosa grown in these CF sputum samples, it is likely that the zinc in our CF sputum samples 151 is bound by zinc-sequestering proteins. CP is one such host zinc-sequestering protein that is 152 found in high concentrations in the sputum of CF patients (2,22). CP has also been shown to 153 induce expression of Zur-regulated genes in P. aeruginosa strain PA14 (51). Therefore, we 154 hypothesized that CP binds zinc to induce a zinc starvation response in P. aeruginosa grown in 155 CF sputum. To test this, we first expressed and purified recombinant human CP as previously 156 described (52) and as illustrated in Fig. S2. The ability of our recombinant CP to induce a zinc-9 concentrations in the lung can reach 1 mg/ml (~40 µM) (29), therefore, 1 mg/ml CP was used for The addition of excess zinc (1 mM) in the presence of CP prevented promoter activation (Fig.   163   3a). These results confirm that our purified recombinant human CP can induce a zinc-starvation 164 response in P. aeruginosa which is quenched with the addition of exogenous zinc.

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Despite the reportedly high concentrations of CP in the serum, sputum, and BALF of CF 166 patients (2, 21-24), P. aeruginosa appears to be able to access enough zinc to persist. Various 167 environmental factors may influence CP zinc binding such as calcium concentrations (53), pH 168 (54), or the presence of oxidants (55,56). Additionally, while CP in its tetrameric state is resistant 169 to proteolytic degradation, CP is susceptible to oxidation which in turn makes it susceptible to 170 proteolytic degradation by both host and bacterial proteases (55,56). Because it was unclear if 171 CP in sputum would remain intact and/or active to bind zinc, we tested the ability of recombinant 172 human CP to bind zinc and thereby induce a zinc-starvation response in P. aeruginosa grown in 173 CF sputum. P. aeruginosa strain PAO1 att::PPA3600-lacZ was grown in unamended CF sputum, 174 sputum supplemented with 1 mM zinc, and sputum supplemented with both 1 mM zinc and 1 175 mg/ml (~40 µM) CP (Fig. 3b). The addition of zinc lowered PA3600 promoter activity in sputum 176 ( Fig. 3b), supporting our NanoString data (Fig. 1c), while addition of CP to zinc-amended sputum 177 significantly prevented reduction of promoter activity (Fig. 3b). These data confirm that 178 recombinant CP added to CF sputum remains intact to bind zinc, which induces a zinc starvation 179 response in colonizing P. aeruginosa.

180
While recombinant CP added to zinc-amended sputum increased P. aeruginosa PA3600 181 promoter activity on average compared to zinc-amended sputum controls, the CF sputum 182 samples tested varied in their responses (Fig. 3b, Fig. 1b). The inverse correlation between 183 sputum zinc concentrations and induction of the PA3600 promoter suggests that sputum samples 184 that result in high promoter activity have lower concentrations of zinc than samples that induce 10 low promoter activity, comparatively (Fig. 2). The high promoter activity by P. aeruginosa was 186 readily quenched by the addition of zinc but remained high when CP was also added ( Fig. 3b; 187 green, lavender, lilac). Conversely, the low promoter activity by P. aeruginosa grown in sputum 188 samples with presumably high zinc is not affected greatly by the addition of zinc nor CP ( Fig. 3b; 189 pink, light pink, gray). Overall, these data show that addition of recombinant CP to zinc-amended 190 sputum can induce a zinc-starvation response dependent on sputum zinc concentration.

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Since both TPEN and CP were confirmed to bind zinc and induce a zinc-starvation 194 response in P. aeruginosa in culture medium (Fig. 1a, Fig. 3a), we wanted to further measure the 195 effects of TPEN-and CP-mediated zinc sequestration on P. aeruginosa growth. Addition of TPEN 196 or CP to cultures grown in LB decreased the final OD600 of P. aeruginosa compared to control 197 conditions (Fig. 3d), but neither inhibited earlier growth stages (Fig. 3c). These data show that P.

198
aeruginosa grows in the presence of CP under the conditions tested.

199
While CP does not prevent the growth of P. aeruginosa, little is known about how CP-200 mediated zinc starvation affects P. aeruginosa physiology. Unlike the chemical chelator TPEN,

201
CP is not membrane permeable and instead exerts its effects on pathogens by binding metals in 202 the extracellular environment. CF sputum has been reported to contain high concentrations of 203 both CP (2, 22, 23) and secreted P. aeruginosa proteases including zinc metalloproteases (57). studies and curated databases suggest that CP and P. aeruginosa-secreted zinc 217 metalloproteases are abundant in the extracellular milieu of the CF mucus environment.

220
Given the importance of zinc to the activity of zinc metalloenzymes, we hypothesized that 221 zinc chelation by TPEN and CP would inhibit the activity of secreted zinc metalloproteases. Our 222 initial studies suggested that LasB and LasA accounted for the majority of proteolytic activity by 223 P. aeruginosa strain PAO1 (WT) because filtered supernatants from ΔlasAB cultures spotted onto 224 milk plates cleared the milk plates substantially less than filtered WT supernatants (Fig. 4a, inset 225 i-ii). As a result, this study focuses on the effect of zinc chelation on LasB and LasA activity.

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To test the above hypothesis, LasB activity was determined quantitatively using azocasein 227 as a substrate. The azocasein degradation assay was previously described to measure total 228 proteolytic activity (14). However, by comparing the ability of P. aeruginosa WT, ΔlasA, and

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ΔlasAB supernatants to degrade azocasein, we found that azocasein degradation was LasB-230 dependent under the conditions tested (Fig. 4a). As a result, we tested the effect of TPEN and 231 CP on LasB activity using the azocasein degradation assay. P. aeruginosa supernatants were 232 filtered and then left untreated, treated with TPEN or CP, or treated with both TPEN or CP and 233 zinc. Treatment with TPEN or CP inhibited LasB enzymatic activity while addition of excess zinc

234
(1 mM) in the presence of TPEN or CP restored LasB activity (Fig. 4b, Fig. 4c). Furthermore, 235 treatment of ΔlasAB supernatants with TPEN ( Fig. S3a) or CP (Fig. S3b)  245 aureus under the conditions tested ( Fig. 5a-b). This assay was then used to measure LasA 246 activity in P. aeruginosa cell-free supernatants left untreated, treated with TPEN or CP, or treated 247 with both TPEN or CP and zinc. Treatment of supernatants with TPEN or CP inhibited LasA 248 activity while treatment with TPEN or CP in the presence of excess zinc (500 µM and 160 µM, 249 respectively) restored LasA activity ( Fig. 5c-f). Furthermore, treatment of ΔlasA supernatants with 250 zinc, TPEN, or CP had no effect on lysis of S. aureus, confirming that treatment of supernatants 251 did not have LasA-independent cytotoxic effects on S. aureus (Fig. S5). Therefore, treatment of 252 P. aeruginosa cell-free supernatants with zinc chelators TPEN and CP inhibits LasA-mediated 253 lysis of S. aureus.

256
Here we show that P. aeruginosa strain PAO1 grown in aliquots of expectorated CF 257 sputum exhibits a zinc-starvation response despite relatively high concentrations of zinc in the 258 sputum samples. Treatment with recombinant host CP was sufficient to induce a zinc-starvation 259 response in P. aeruginosa grown in zinc-amended CF sputum samples from different subjects, 260 demonstrating that CP retains its function in sputum. Furthermore, treatment of P. aeruginosa 261 supernatants with CP inhibited the activity of secreted, extracellular zinc metalloproteases LasB 262 and LasA. The data presented in this study support a model in which CP released from recruited 13 neutrophils sequesters zinc from the environment to induce a zinc-starvation response in P.

267
A variety of strategies have been used to learn about the environment that P. aeruginosa 268 encounters in the CF lung including analysis of bacteria grown in buffered media supplemented 269 with CF sputum compared to bacteria grown in laboratory media (8,9), and direct analysis of sputum from a variety of CF patients. Our study also found that P. aeruginosa activates its zinc-275 starvation response in CF sputum on average but revealed differences across samples from 276 different CF donors ( Fig. 1b-c, Fig. 3b). These findings taken together underscore the fact that

282
CP concentrations during infections can reach 1 mg/ml or ~40 µM which is often posited 283 to be higher than or in excess of the bioavailable zinc concentration in most environments (29).

292
There is mounting evidence that divalent-metal sequestration by CP affects P. aeruginosa.  59) showed that treatment with CP significantly reduced 299 intracellular iron and manganese in P. aeruginosa, but did not significantly affect intracellular zinc, 300 though intracellular zinc trended downward (59). Our data show that CP induces a Zur-regulated 301 zinc-starvation response in vitro and in expectorated CF sputum which is repressed upon the 302 addition of excess zinc ( Fig. 3a-b). We also observed CP-mediated growth defects in vitro (

307
Additionally, while Filkins et al. (60) showed that in vitro co-culture of P. aeruginosa and 308 S. aureus on CF bronchial epithelial cells reduced the viability of S. aureus, Wakeman et al. (51) 309 showed that zinc chelation by CP promotes P. aeruginosa and S. aureus co-culture in in vitro, in 310 vivo, and ex vivo models, in part, by downregulating genes encoding anti-staphylococcal factors 311 such as pyocyanin, hydrogen cyanide, and PQS/HQNO. Interestingly, treatment of P. aeruginosa 312 with CP did not reduce the expression of lasA though the functionality of LasA was not tested 313 (51). In this study, we show that CP-mediated zinc chelation inhibits LasA-mediated lysis of S. via zinc sequestration. Furthermore, colonization of the CF airways is usually described as a 317 pattern of succession where S. aureus is the predominant colonizer early on in younger patients 318 before being outcompeted by P. aeruginosa in older patients (60). However,Fischer et al. (61) 319 recently showed that P. aeruginosa and S. aureus chronically co-colonize the CF lung. Wakeman 320 et al. also showed that P. aeruginosa, S. aureus, and CP colocalize in CF lung explants (51).

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Further studies are required to determine if CP modulates protease-dependent and/or protease-322 independent co-colonization of P. aeruginosa and S. aureus in the CF lung.

336
LasB is an abundant protease with broad substrate specificity that is implicated in amino 337 acid liberation and consumption (70). In addition to nutrient acquisition, LasB also plays a role in 338 the ability of P. aeruginosa to invade host epithelial cells (39) and to evade host immune 339 responses via processes such as degrading cytokines (40). Interestingly, degradation of pro-formation (73) and in this study we show that CP-mediated zinc chelation inhibits the activity of 345 secreted LasB (Fig. 4c). Taken together, there appears to be a complex interplay between LasB, 346 neutrophils, and CP during the course of infection which may contribute to exacerbations in CF.

347
Furthermore, recent work suggests that secreted LasB activates Protease IV which then 348 predominantly processes and activates LasA (36,37). Therefore, CP-mediated inhibition of 349 secreted LasB activity may have downstream effects on the processing and activity of other 350 secreted zinc metalloproteases.

351
In conclusion, the results of our study show that CP can induce a zinc-starvation response 352 in P. aeruginosa in CF sputum as well as chelate zinc to inhibit the activity of virulence-associated 353 zinc metalloproteases. Future studies will focus on how competition for zinc in a zinc-limited or

425
Plasmid S100A8/A9 was obtained from Futami et al. (52) and recombinant CP was expressed 426 and purified as previously described with minor modification. Plasmid S100A8/A9 was first

455
A superloop was assembled with the appropriate volume for sample application. Sample was then 456 loaded in the column using the superloop at 2.5 ml/min. The column was then washed with 10 457 CV of SP Sepharose HP buffer A at about 5 ml/min. The column was then washed with a step 458 gradient of SP Sepharose HP buffer B: 5 CV of 5% SP Sepharose HP buffer B, 10 CV at 30% SP

22
Caseinolytic activity was determined qualitatively by spotting P. aeruginosa supernatants onto 1% 486 milk plates or quantitatively using azocasein as a substrate as previously described (14) with of CP buffer without DTT, and/or 1 mM ZnSO4 • 7 H2O or an equivalent volume of diH2O.

498
Staphylolytic activity was determined by monitoring the decrease in absorbance at 595 nm of a 499 heat-killed S. aureus suspension as previously described (14) with modification. S. aureus strain 500 SH1000 (77) was cultured in TSB overnight (16 h) at 37°C with rolling. Cultures were centrifuged 501 at 4,500 x g for 10 min, resuspended in 20 mM Tris-HCl, pH 8.8 to a final OD600 of 1.0, and then 502 killed by heating at 100°C for 30 min. Heat-killed S. aureus suspensions were cooled to room 503 temperature before use. P. aeruginosa culture supernatants were treated overnight (16 h) with 50 504 μM TPEN or an equivalent volume of 100% EtOH, 1 mg/ml (~40 µM) CP or an equivalent volume 505 of CP buffer without DTT, and/or 160-500 µM ZnSO4 • 7 H2O or an equivalent volume of diH2O.

506
Because increasing concentrations of zinc were previously reported to inhibit LasA activity (46), 507 an appropriate concentration of zinc to use in add-back experiments was determined 508 experimentally. For undiluted WT supernatants, the addition of 500 µM zinc had no effect on LasA 509 activity, while increasing concentrations of zinc inhibited LasA-mediated lysis of S. aureus (Fig.  23 supernatants, the addition of 50 µM zinc had no effect on LasA activity, while increasing 512 concentrations of zinc inhibited LasA-mediated lysis of S. aureus (Fig. S4c-d). However, a 513 tetramer of CP can potentially bind up to four zinc ions. Therefore, to ensure that zinc would be 514 in excess in CP-based experiments, we used 160 µM zinc which was four times the concentration 515 of CP but still less than 250 µM zinc which was the concentration tested that started to inhibit 516 LasA activity independent of CP. P. aeruginosa supernatants (20 µl) were added to 180 µl of heat-               LasA. LasB is a protease that can degrade host proteins, such as elastin, as well as peptides.

850
These degraded proteins/peptides can then be taken up and utilized as nutrients by P.    . 2 Activation of the PA3600 promoter in CF sputum by P. aeruginosa is inversely correlated with total sputum zinc concentration. (a) P. aeruginosa strain PAO1 P PA3600 -lacZ was inoculated into 8 different CF sputum samples. Zinc concentration of the same 8 CF sputum samples was determined by ICP-MS. Β-Gal activity on the left y-axis (Miller Units; gray bars) was then compared to sputum zinc concentration on the right y-axis (µg/g; red dots), (b) Pearson correlation matrix comparing Β-Gal activity (Miller units), sputum zinc concentration, sputum iron concentration, and sputum manganese concentration. *p<0.05, **p<0.01