Catalase Activity is Critical for Proteus mirabilis Biofilm Development, EPS Composition, and Dissemination During Catheter-Associated Urinary Tract Infection

Proteus mirabilis is a leading uropathogen of catheter-associated urinary tract infections (CAUTIs), which are among the most common healthcare-associated infections worldwide. A key factor that contributes to P. mirabilis pathogenesis and persistence during CAUTI is the formation of catheter biofilms, which provide increased resistance to antibiotic treatment and host defense mechanisms. Another factor that is important for bacterial persistence during CAUTI is the ability to resist reactive oxygen species (ROS), such as through the action of the catalase enzyme. Potent catalase activity is one of the defining biochemical characteristics of P. mirabilis, and its single catalase gene (katA) was recently identified as a candidate fitness factor for UTI, CAUTI, and bacteremia. Here we show that disruption of katA results in increased ROS levels, increased sensitivity to peroxide, and decreased biofilm biomass. The biomass defect was due to a decrease in extracellular polymeric substances (EPS) production by the ΔkatA mutant, and specifically due to reduced carbohydrate content. Importantly, the biofilm defect resulted in decreased antibiotic resistance in vitro and a colonization defect during experimental CAUTI. The ΔkatA mutant also exhibited decreased fitness in a bacteremia model, supporting a dual role for catalase in P. mirabilis biofilm development and immune evasion.

Proteus mirabilis is a leading uropathogen of catheter-associated urinary tract infections 23 (CAUTIs), which are among the most common healthcare-associated infections 24 worldwide. A key factor that contributes to P. mirabilis pathogenesis and persistence 25 during CAUTI is the formation of catheter biofilms, which provide increased resistance 26 to antibiotic treatment and host defense mechanisms. Another factor that is important 27 for bacterial persistence during CAUTI is the ability to resist reactive oxygen species 28 (ROS), such as through the action of the catalase enzyme. Potent catalase activity is 29 one of the defining biochemical characteristics of P. mirabilis, and its single catalase 30 gene (katA) was recently identified as a candidate fitness factor for UTI, CAUTI, and 31 bacteremia. Here we show that disruption of katA results in increased ROS levels, 32 increased sensitivity to peroxide, and decreased biofilm biomass. The biomass defect 33 was due to a decrease in extracellular polymeric substances (EPS) production by the 34 ∆katA mutant, and specifically due to reduced carbohydrate content. Importantly, the 35 biofilm defect resulted in decreased antibiotic resistance in vitro and a colonization 36 defect during experimental CAUTI. The ∆katA mutant also exhibited decreased fitness

Introduction
The absence of catalase severely alters the composition of the EPS matrix, resulting in 109 increased antibiotic susceptibility of biofilm-associated bacteria. Additionally, we 110 demonstrate that catalase contributes to the pathogenesis of P. mirabilis during CAUTI 111 and fitness during bacteremia.

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Generation and characterization of a P. mirabilis ∆katA mutant 114 In order to investigate the importance of catalase activity to P. mirabilis growth, 115 biofilm formation, and pathogenesis, a mutant was generated in P. mirabilis strain 116 HI4320 to disrupt the catalase gene (katA) by insertion of a kanamycin resistance 117 cassette. Disruption of the katA gene and resulting catalase activity were confirmed by 118 PCR and a catalase foam height assay (Fig. 1A). As expected, the ∆katA mutant was 119 catalase-negative when exposed to hydrogen peroxide, and providing the katA gene on 120 a plasmid restored catalase activity. The complemented activity was more potent than 121 that of the parental strain, which is likely due to plasmid copy number. The ∆katA 122 mutant grew similarly to wild-type P. mirabilis in LB broth and did not exhibit any defects 123 in swimming motility, swarming motility, or urease activity ( Fig. 1B-E), indicating that 124 disruption of the katA gene does not affect standard laboratory behaviors of P. mirabilis. 125 Disruption of katA also had no impact on expression of either adjacent gene by qRT-126 PCR (Fig. S1). Taken together, these data indicate that targeted disruption of katA 127 abrogates catalase activity without obvious polar effects. decrease in total biomass that was detectable at 4 hours and remained evident for the 159 rest of the time course (Fig. 3A). Importantly, assessment of viable bacteria within the 160 biofilms at each time point revealed that the defect was not due to a decrease in 161 bacterial CFUs, and therefore likely represents a defect in biofilm architecture (Fig. S2). 162 These results were also recapitulated for biofilms established in pooled human urine, as 163 a physiologically-relevant growth medium for CAUTI (Fig. 3B). 164 To define the specific contribution of peroxide detoxification to P. mirabilis biofilm 165 formation, wild-type P. mirabilis and the ΔkatA mutant were supplemented with an 166 amount of bovine catalase that results in the equivalent foam height produced by 1x10 8 167 CFU/ml of wild-type P. mirabilis (~0.7 mg , ~40,000 units). The addition of bovine 168 catalase had no impact on biofilm development by wild-type P. mirabilis in LB, but fully 169 restored biofilm biomass for the ΔkatA mutant in both LB and human urine, albeit not 170 until the 20 hour time point (Fig. 3A, B). These data suggest that catalase is important 171 for later stages of biofilm development and maturation rather than the initial stages of 172 attachment and microcolony formation. Importantly, the contribution of bovine catalase 173 was confirmed to specifically derive from enzymatic activity, as both 10 kDa filtration 174 and heat inactivation of the catalase solution abrogated complementation of the ΔkatA 175 mutant biofilm defect (Fig. S3). 176 To verify that the contribution of catalase to P. mirabilis biofilm development 177 pertains specifically to peroxide detoxification, we next sought to determine if the 178 addition of a non-lethal concentration of peroxide (~109 mM, as seen in Fig. 2D) could 179 impair biofilm formation by wild-type P. mirabilis. The addition of peroxide at the time of 180 inoculation significantly decreased the P. mirabilis biofilm biomass for the first 6 hours of 181 biofilm development, after which time enough of the peroxide was likely broken down to 182 restore full biofilm development (Fig. 3C). Similar results were also recapitulated in 183 pooled human urine (Fig. 3D). Taken together, these data confirm that the biofilm defect 184 of the ΔkatA mutant is specifically due to the loss of catalase activity and peroxide 185 detoxification, and likely pertains to biofilm maturation. 186 We next sought to determine if the biofilm defect of the ΔkatA mutant may be the 4B). Importantly, the addition of bovine catalase at the time of inoculation slightly 219 enhanced EPS production by wild-type P. mirabilis ( Fig 4C) and fully restored EPS 220 production by the ∆katA mutant ( Fig 4D). These data indicate that the biomass defect of 221 the ∆katA mutant is likely due to a decrease in the production of EPS.

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The exact composition of P. mirabilis HI4320 biofilm EPS has not been fully 223 elucidated in the literature; however, the EPS of bacterial biofilms is generally 224 comprised of proteins, carbohydrates, and extracellular DNA (eDNA). To assess biofilm 225 EPS composition, wild-type P. mirabilis and the ∆katA mutant were incubated for 20 226 hours in LB broth with aeration (planktonic suspension, PS) or stationary in wells of a 24-well plate from which a total biofilm sample was collected (biofilm suspension, BS).

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The remaining biofilm material was then treated with formaldehyde to fix the bacterial 229 cells and prevent lysis during subsequent steps of EPS extraction, and NaOH to 230 promote dissociation of the EPS from the biofilm and increase its solubility. Following 231 these treatments, a cellular fraction (CF) and dialyzed EPS fraction (EF) were collected.

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No differences in eDNA concentration were observed between strains in any of the 233 tested fractions (Fig. 4E), and total protein measurement revealed only a slight 234 decrease in the ∆katA biofilm suspension compared to wild-type (Fig. 4F). However, 235 total carbohydrate measurement revealed a substantial decrease in the biofilm 236 suspension and EPS fraction of the ∆katA mutant compared to wild-type P. mirabilis 237 (Fig. 4G). Thus, the biofilm biomass defect of the ∆katA mutant derives from a decrease 238 in EPS carbohydrate content.  complemented by the addition of bovine catalase at the time of inoculation (Fig. 6A, B). 274 Thus, the ∆katA biofilm biomass defect is present on a physiologically-relevant biofilm 275 substrate for CAUTI. 278 Considering the important contribution of bacterial biofilms to CAUTI in humans, 279 we next sought to determine the contribution of P. mirabilis catalase to colonization and and Table 1). Thus, while the ∆katA mutant exhibited reduced spleen CFUs at 24 hpi, 297 pathogenesis was equivalent to wild-type by 96 hpi.

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A caveat to the CAUTI model is that the bacterial inoculum is introduced as a 299 suspension, and biofilm formation occurs concurrently with defense against the host 300 immune response. In order to isolate the contribution of the ∆katA biofilm defect to P. 301 mirabilis pathogenesis during CAUTI, we next inoculated mice with catheter segments 302 that had been pre-colonized for 12 hours by either wild-type P. mirabilis or the ΔkatA 303 mutant to establish catheter biofilms. Importantly, pre-colonizing the catheter segments  (Table 1). Infection with the ∆katA mutant also displayed greater uniformity in urine and 312 bladder CFUs in the pre-colonized model compared to the traditional CAUTI model; 313 however, in comparison to wild-type P. mirabilis, the ∆katA mutant exhibited a profound 314 decrease in kidney colonization and a significantly lower incidence of hematuria, 315 bladder hematoma, and kidney stones, as well as a trend towards reduced bacterial 316 burden in the urine and spleen ( Fig. 7C and Table 1). Based on these data, wild-type 317 displays greater infection severity and the ∆katA mutant displays a more pronounced 318 defect when the infection is seeded from a pre-colonized catheter, which underscores the importance of P. mirabilis biofilm development and EPS composition for CAUTI 320 pathogenesis.

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While the pre-colonized catheter model clearly demonstrates a pathogenesis 322 contribution for catalase stemming from its role in biofilm development, these studies oxidative burst represents a potent source of hydrogen peroxide during CAUTI, we next 341 investigated the contribution of P. mirabilis catalase to defense against opsonophagocytic killing by neutrophils. Interestingly, the ∆katA mutant was no more 343 susceptible to opsonophagocytic killing than wild-type P. mirabilis (Fig. S6). These data 344 indicate that the contribution of catalase to fitness within the bloodstream is likely due to 345 factors other than defense against neutrophil oxidative burst.

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The high degree of resistance against antimicrobials and host defenses that is 348 conferred by bacterial biofilms represents a substantial challenge for the effective infection-specific fitness factor for survival in the bloodstream (44). We therefore 368 endeavored to delineate the contribution of catalase to several stages of P. mirabilis pathogenesis. Here, we show that disruption of catalase results in a slight increase in 370 ROS levels during growth in broth culture, which does not appear to induce oxidative 371 stress in P. mirabilis but does increase sensitivity to additional peroxide insult. We also 372 uncovered a critical role for peroxide detoxification via catalase activity in the production 373 of a mature biofilm by P. mirabilis. Specifically, loss of catalase activity decreased 374 biofilm biomass due to a severe alteration of EPS composition, particularly the 375 carbohydrate fraction. Importantly, this EPS defect rendered the ∆katA biofilms more 376 susceptible to antibiotics and also decreased colonization and dissemination in a mouse 377 model of CAUTI.

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The exact sugar moieties that comprise the EPS of P. mirabilis strain HI4320 379 have yet to be fully elucidated; however, hexose moieties previously identified in the contribution of catalase to biofilm architecture (66). In Mycoplasma pneumoniae (an organism that lacks superoxide dismutase and catalase), treatment with catalase 393 enhanced biofilm formation and altered biofilm structure, such that fewer and smaller 394 tower structures were produced and the resulting biofilms were smoother and more 395 homogenous (3). While treatment of wild-type P. mirabilis with bovine catalase did not 396 impact total biofilm biomass, our electron microscopy images suggest that it may 397 increase EPS production and biofilm smoothness. Full determination of P. mirabilis 398 HI4320 EPS carbohydrate composition will be necessary to reveal which components 399 are altered when catalase is disrupted, and which (if any) are increased in the wild-type 400 strain upon supplementation with excess catalase. 401 We also revealed that disruption of P. mirabilis catalase results in a biofilm-  routinely cultured at 37°C with aeration in 5 mL of LB broth (10 g/L tryptone, 5 g/L yeast 460 extract, 0.5 g/L NaCl) or on Low salt LB agar (10 g/L tryptone, 5 g/L yeast extract, 0.1 g/L NaCl, 15 g/L agar). Biofilm assays were performed using LB broth (10 g/L tryptone, 462 5 g/L yeast extract, 0.5 g/L NaCl) or filter-sterilized human urine (Cone Bioproducts, 463 Sequin, TX).   Mouse model of CAUTI. CAUTI studies were carried out as previously described (43). 609 Briefly, the inoculum was prepared by washing overnight cultures of wild-type P.       , and swarming motility (E) in comparison to wild-type P. mirabilis. (A) An SDS/peroxide foam height assay was conducted to provide phenotypic validation of successful generation of a P. mirabilis ∆katA mutant and plasmid generated ∆katA+ complemented strain. (B) Bacterial growth in LB at 37°C was assessed by measurement of OD 600 at hourly intervals for 18 hours. Error bars represent mean ± standard deviation (SD) of 6 independent experiments with 6 technical replicates each (representative graph shown). No significant differences in growth were determined by two-way ANOVA with Dunnett's test for multiple comparisons. (C) Urease activity in human urine was assessed by measurement of phenol red color change (OD 562 ) at 5-minute intervals for 80 minutes, and a P. mirabilis ∆ureF mutant was included as negative control. Error bars represent mean ± SD of 2 independent experiments with 6 technical replicates each. No significant differences between WT and ∆katA were identified by two-way ANOVA. (D and E) Bacteria were cultured in LB broth overnight and stab inoculated into motility agar (D) or inoculated onto the surface of swarm agar plates (E). Motility diameter was measured in millimeters after 16 hours of incubation at 30°C (D) or 37°C (E). R1-3 indicate individual swarm ring diameters. Error bars represent mean ± SD for 3 independent experiments with at least 2 replicates each. No significant differences in motility diameter were determined by Student's t test (D) or two-way ANOVA (E). (E) A competitive index (CI) was calculated for the ∆katA mutant on a per-mouse basis for the spleen, liver, and kidneys from the ratio of mutant to wild-type recovered from the organ divided by the ratio of mutant to wild-type in the inoculum. Each data point represents the Log10 CI from an individual mouse. Solid lines represent the median, dashed line indicates a competitive index of 1, or a 1:1 ratio of mutant to wild-type. *P < 0.05 by Wilcoxon signed rank test.

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