A Compensatory RNase E Variation Increases Iron Piracy and Virulence in Multidrug-Resistant Pseudomonas aeruginosa during Macrophage Infection

During chronic cystic fibrosis (CF) infections, evolved Pseudomonas aeruginosa antibiotic resistance is linked to increased pulmonary exacerbations, decreased lung function, and hospitalizations. However, the virulence mechanisms underlying worse outcomes caused by antibiotic resistant infections are poorly understood. Here, we investigated evolved aztreonam resistant P. aeruginosa virulence mechanisms. Using a macrophage infection model combined with genomic and transcriptomic analyses, we show that a compensatory mutation in the rne gene, encoding RNase E, increased siderophore gene expression, causing macrophage ferroptosis and lysis. Macrophage killing could be eliminated by treatment with the iron mimetic gallium. RNase E variants were abundant in clinical isolates, and CF sputum gene expression data show that clinical isolates phenocopied RNase E variant functions during macrophage infection. Together these data show how P. aeruginosa RNase E variants can cause host damage via increased siderophore production and host cell ferroptosis but may also be targets for gallium precision therapy.

232 Prussian blue staining ( Figure 5A, 5D). The iron content was significantly greater in BMDM 233 infected with AzEvC10 compared to those infected with the WT strain at both 3 hpi (p<0.01) and 234 6 hpi (p<0.05) ( Figure 5A). As the iron content in BMDM infected with WT PAO1 remained the 235 same between 3 hpi and 6 hpi, it significantly increased between 3 hpi and 6 hpi in BMDM infected 236 with the AzEvC10 mutant (p<0.05).
237 Iron overload predisposes cells to oxidative stress through the Fenton reaction 30-32 . Reactive 238 oxygen species (ROS) production is a defensive mechanism used by host cells to kill intracellular 239 pathogens. However, too much ROS can cause host damage and lead to cell death. Therefore, 240 we quantified general ROS production in BMDM during bacterial infection ( Figure 5B). As 241 expected, both WT and AzEvC10 infected BMDM increased their production of ROS at 6 hpi 242 (p<0. 01 and p<0.0001 compared to 3 hpi respectively). However, BMDM infected with the 243 AzEvC10 mutant had significantly greater ROS production at 6 hpi compared to macrophages 244 infected with WT PAO1 (p<0.01).
245 We then assessed if this degree of ROS stimulated by infection with the AzEvC10 mutant was 246 sufficient to induce cell damage. One of the consequences of iron-induced ROS toxicity in cells 247 is lipid peroxidation, in which free radicals attack double bonds of fatty acids leading to oxidative 248 damage of polyunsaturated fatty acids and cell structure damage 30 . We used a ratiometric sensor 249 to measure lipid peroxidation in BMDM and found that lipid peroxidation was significantly greater 250 in AzEvC10 infected BMDM compared to those infected with WT PAO1 (p<0.05) ( Figure 5C). Iron 251 overload, ROS production, and lipid peroxidation can lead to ferroptosis, a form of iron-mediated 252 programmed cell death. Consistent with this, we found that BMDM infected with AzEvC10 253 exhibited high levels of annexin V and propidium iodide staining (Figure 5E). The increase of 254 these programmed cell death markers matched the increases in AzEvC10-mediated macrophage 255 death shown in Figure 1B. It is possible that other programmed or non-programmed cell death 256 pathways (pyroptosis, necroptosis, necrosis, etc.) are also involved in BMDM death, although 257 together our results suggest that the AzEvC10 mutant impairs iron regulation in macrophages 258 and induces excessive ROS and lipid peroxidation leading to ferroptosis of macrophages.
259 Gallium treatment efficiently kills multidrug-resistant and hypervirulent P. aeruginosa 260 Gallium is an ion with similar properties to iron and has been proposed as an antimicrobial agent 261 against P. aeruginosa 33 . Because virulence of the AzEvC10 mutant included a higher capacity to 262 acquire iron and induce toxicity with siderophores, we sought to test if gallium was efficient against 263 this multidrug-resistant mutant during BMDM infection. We first confirmed inhibition of bacterial 296 in vitro ( Figure 7B, Table S2). We then compared the genes that were differentially expressed 297 between the sputum samples and in vitro grown P. aeruginosa with the DEGs of our WT PAO1 298 or AzEvC10 strains between macrophage infection and LB growth. As expected, common 299 upregulated genes between AzEvC10 mutant 3 hpi and sputum included pyoverdine and 300 pyochelin biosynthesis enzymes and transport receptors ( Figure 7C, Table S2). Other common 301 genes were involved in the response to oxidative stress (ahpC), T1SS (aprA, aprFED), and 302 regulation of the T3SS (rsmZ and pcrG, respectively). Interestingly, the alkaline protease (aprA) 303 and its secretory apparatus (aprFED) expressions are regulated by iron levels and facilitate iron 304 acquisition by the proteolytic cleavage of transferrins 35,36 . This highlights the importance of iron 305 acquisition during infection and a role for rne mutants in iron-mediated bacterial virulence. A 306 subset of commonly upregulated genes shared by the AzEvC10 mutant 6 hpi and sputum P. 307 aeruginosa are known to be expressed under anaerobic conditions, including the nir and nor 308 operons, cytochrome C oxidase subunits (ccoP2 and ccoQ2), arginine deaminase (arcA), and 309 malate/L-lactate dehydrogenase (dpkA) ( Figure 7D, Table S2). This switch to anaerobic 310 metabolism contributes to increased fitness of P. aeruginosa under hypoxic conditions. The open 311 reading frame PA3271 was also overexpressed in both AzEvC10 at 6 hpi and sputum and 312 encodes a probable two-component sensor. Because more than 50% of two-component systems   Figure 7A). We found that all isolates (n=45) had multiple non-synonymous mutations 324 in the rne gene ( Figure 7E). All non-synonymous mutations except for two were found in the 331 Further investigation will be needed to determine which variants affect RNase E functions and 332 bacterial phenotypes during infection.

333
334 Discussion 335 P. aeruginosa is present in more than 50% of adults with CF and is thought to adapt to the CF 336 lung environment by decreasing production of virulence factors and acquiring antibiotic resistance 337 features. In this study, we challenge this concept of an inverse correlation between acquisition of 338 antibiotic resistance and virulence factor production. In a previous study, we showed that 346 We showed that although aztreonam resistance was due to a loss-of-function nalD mutation, the 347 AzEvC10 hypervirulent phenotype resulted from a rne gene variant ( Figure 2, Figure S2). The 348 evolution of increased virulence towards macrophages was surprising, given that this phenotype 349 evolved only in response to aztreonam, suggesting that antibiotic selection alone can lead to 350 hypervirulent phenotypes.
351 RNase E is an endonuclease with two main domains: a catalytic amino-terminus, and a carboxy-352 terminus which serves as a scaffold for the degradosome complex 48,49 . In E. coli the last residues 353 of C-terminus contain the binding site for the exoribonuclease Polynucleotide phosphorylase 354 (PNPase) 49 . Thus, a 17-residue deletion in that region as seen in our AzEvC10 could prevent or 355 impair PNPase binding and overall degradosome activity. Although the absence of the 356 degradosome complex enhanced RNA half-lives in E. coli, some studies suggested that PNPase 357 was unnecessary for RNA degradation and was rather a scavenger of RNA intermediates 37,50-52 .
358 However, mutants lacking PNPase or the PNPase-binding site in RNase E were sufficient to 359 increase RNA half-lives of specific mRNAs, suggesting that the presence of PNPase in the 360 degradosome complex is necessary for degradation of specific mRNAs 53 . In our study, a 17-361 residue deletion in the PNPase-binding site of RNase E was sufficient to create a global 362 transcriptomic shift in the AzEvC10 mutant during macrophage infection. We found a striking 363 upregulation of pyoverdine and pyochelin secretion by AzEvC10, rne 50bp , and rne::Tn strains 364 ( Figure 3, Table S1). Interestingly, heme sensor genes of the Has system were also upregulated, 365 but not those involved in the Phu system ( Figure 3, Table S1). This suggests that the mutation in 366 AzEvC10 RNase E impacts specific substrates as opposed to the whole mRNA pool. E. coli 367 RNase E was also shown to interact with RNA-binding proteins such as Hfq in a degradosome-368 independent manner 51,54,55 . However, the literature differs on the necessity of this interaction for 369 RNA cleavage 51,54,56 . The RNase E carboxy-terminal region was also shown to be essential for 370 binding and localization of the degradosome to the bacterial membrane, which could affect its 371 activity 57,58 . In P. aeruginosa in general, and in our study, it is unknown whether the RNase E-372 Hfq interaction or cellular localization are impaired in the AzEvC10 mutant. However, due to the 373 location of the AzEvC10 RNase E mutation, we believe this to be unlikely. Ultimately, future work 374 will be necessary to determine the precise effects of the RNase E C-terminal variation on its 375 biochemical functions.  (Table S1).
383 The T3SS can also induce macrophage lysis 9,10 and was previously shown to be positively 384 regulated by RNase E in P. aeruginosa 62 . Although T3SS genes were upregulated at 3 hpi by the 385 AzEvC10 mutant, their negative regulators, the rsmY and rsmZ small non-coding RNAs were 386 upregulated as well (Figure 4, Table S1). Therefore, it is not clear whether the T3SS is active or 387 inactive in the AzEvC10 strain. Future studies will examine the role of this virulence system in the  431 Since siderophores were upregulated in the AzEvC10 mutant and induced toxicity during 432 macrophage infection, we sought to target and inhibit these molecules using gallium nitrate.
433 Previous work suggested gallium as a novel therapy for its antimicrobial properties against P. 434 aeruginosa and Klebsiella pneumoniae 33,75-80 . In the present study, gallium completely abolished 435 siderophore-mediated cytotoxicity in BMDM ( Figure 6). Moreover, gallium treatment for 24 h 436 decreased bacterial growth by a factor of 100 to 1000-fold. This is important since AzEvC10 437 mutant P. aeruginosa is resistant to multiple antibiotics currently used in CF patients 26  469 Some limitations exist in our study. We speculated that the degradosome assembly and/or 470 function was impaired in the AzEvC10 mutant, explaining its increased virulence by siderophore 471 overexpression. However, have not directly tested the effects of the rne mutation on degradosome 472 function, nor the interaction of RNase E with PNPase. The mutation could also impair RNase E 473 interaction with other proteins, like RNA-binding proteins, rather than PNPase. These aspects will 474 need to be elucidated in further studies. Finally, we used healthy instead of CF macrophages for      533 aeruginosa in red, and DAPI in blue. n = 3-6 independent replicates for each experiment. *p<0.05, 534 **p<0.01, ***p<0.001, ****p<0.0001. See Table S5 for statistical tests used and exact p-values.  cytotoxicity was assessed by LDH assay at 6 hpi with indicated bacteria. n=5-8 independent 548 replicates for each experiment *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Table S5 Table S5 for statistical tests used and exact p-values.