Catheterized-bladder environment induces hyphal Candida albicans formation , promoting fungal 1 colonization and persistence

27 Catheter-associated urinary tract infections (CAUTIs) are a serious public health problem and 28 account for approximately 40% of hospital-acquired infections worldwide. Candida spp are a 29 major causative agent of CAUTI (17.8%) – specifically Candida albicans– that has steadily 30 increased to become the second most common CAUTI uropathogen1. Yet, there is poor 31 understanding of the molecular details of how C. albicans attaches, grows in the bladder, forms 32 biofilms, survives, and persists during CAUTI 2. Understanding of the mechanisms that contribute 33 to CAUTI and invasive fungal infection will give insights into the development of more effective 34 therapies, which are needed due to the spread of antifungal resistance and complex management 35 of CAUTI in patients that require a urinary catheter 3. Here, we characterize the ability of five 36 Candida albicans clinical and laboratory strains to colonize the urinary catheter, grow and form 37 biofilm in urine, and their ability to cause CAUTIs using our mouse model. Analysis of C. albicans 38 strains revealed that growth in urine promotes morphological transition from yeast to hyphae, 39 which is important for invasive infection. Additionally, we found that biofilm formation was 40 dependent on the presence of fibrinogen, a protein released on the bladder to promote bladder 41 healing4,5. Furthermore, deletion of hyphae regulatory genes resulted in defective bladder and 42 catheter colonization and abolished dissemination. These results indicate that novel antifungal 43 therapies preventing the morphological transition of C. albicans from yeast to hyphae have 44 considerable promise for the treatment of fungal CAUTIs. 45

INTRODUCTION static conditions; YPD is a standard C. albicans growth media and BHI was used since the clinical 142 strains were isolated on this media. Static growth was used to mimic the bladder environment and 143 shaking growth was used as a comparison with standard lab culture conditions. For restrictive 144 environment, we used human urine and to further mimic the plasma protein extravasation in the 145 catheterized bladder 37 , urine was supplemented with either 3% human serum or different nitrogen  (Fig. 1). The growth of the lab strains and Pt62 was not enhanced, or was inhibited, 156 with BSA or Aa supplementation when compared with urine alone (Fig. 1A, D-F). However, Fg 157 enhanced growth of all strains, at different magnitudes when compared with urine alone (Fig. 1), 158 except for PNCL1, which already exhibited good growth in urine alone (Fig. 1C).

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Urine conditions promotes C. albicans hyphal formation. 161 Environmental conditions induce C. albicans morphological changes that are associated with 162 virulence 10,19 . C. albicans can exhibit different morphologies 21 , but the main ones are vegetative 163 yeast, pseudohyphae, and hyphae 19 . Hyphae morphology leads to the spread of infection and 164 increased virulence of the pathogen 20,22 . Therefore, we wanted to determine how the bladder 165 environment affects C. albicans morphology. To assess the pathogen morphologies, C. albicans' 166 laboratory and clinical strains were grown in urine with 3% human serum to mimic plasma protein 167 extravasation in the catheterized bladder 37,38 . YPD alone was used as a negative control and when 168 supplemented with serum was used as a positive control for inducing hyphal morphology [44][45][46] . 169 Strains were incubated at 37ºC with 5% CO2 for 48 hrs and samples were collected at 0, 24, and 170 48 hours. C. albicans strains were stained with calcofluor white to assess morphology using 171 fluorescence microscopy (Zeiss Axio Observed inverted scope). The cell morphology was 172 analyzed automatically using CellProfiler software (available from the Broad Institute at 173 www.cellprofiler.org) 47 to quantify the percentages of yeast, pseudophyphal, or hyphal forms 174 based on the circularity value of each outlined cell (Fig. 2G, Table S1). All strains showed 175 predominantly yeast morphology in YPD media and YPD with serum induced pseudohyphal and 176 hyphal formation in all strains, except Pt65 and SC5314 efg-1Δcph-1Δ. Notably, our analysis 177 showed that urine conditions promote pseudohyphal and hyphal formation in all strains (Fig. 2,   178 Table S1) except SC5314 efg-1Δcph-1Δ (Fig. 2F Fg may be an important factor to promote C. albicans CAUTI pathogenesis. Based on these and 192 our previous findings (Fig. 2), we hypothesized that urine conditions induce factors responsible 193 for Fg-binding and biofilm formation. Thus, we assessed biofilm formation under rich (YPD and 194 BHI) and restrictive conditions (human urine) and compared between BSA-and Fg-coated 195 microplates as we have previously described 54 . At 48 hrs, immunostaining analyses were 196 performed to assess fungal biofilm formation by using anti-Candida antibodies and biofilm 197 biomass was quantified by fluorescence intensity 54 . We found that for the clinical strains, Fg 198 promoted biofilm formation in all conditions but Fg-dependent biofilm formation was further 199 enhanced in human urine condition ( Fig. 3A-C). For the laboratory strains, we found a similar 200 Fg-dependent biofilm formation in YPD, BHI, and urine, but Fg had no effect in DAY230 when 201 grown in BHI ( Fig. 3D and E). In contrast, SC5314 efg-1Δcph-1Δ was not able to form biofilms 202 in human urine regardless of the coated surface (Fig. 3F), highlighting the importance of 203 filamentation for biofilms under urine conditions. Notably, we observed that the hyphae-defective 204 mutant was able to form Fg-dependent biofilms in YPD and BHI (Fig. 3F), suggesting that the 205 mechanisms of biofilm formation are different. This difference could be related to adhesins that 206 are expressed during yeast form that may contribute to biofilm but not in urine conditions. 207 Therefore, using conditions that closely mimic the in vivo environment are important to identify 208 physiologically-relevant determinants for biofilm formation. Fibrinogen promotes C. albicans biofilm formation. 211 Furthermore, we analyzed the C. albicans strains biofilm by immunofluorescence (IF) microscopy.

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C. albicans strains' biofilms were grown for 48 hrs in human urine using glass-bottom petri dishes 213 coated with BSA or Fg. Biofilms on BSA were barely monolayers or small aggregates composed 214 of yeast, pseudohyphae, and hyphae ( Fig. 3G-K), except for SC5314 efg-1Δcph-1Δ, where all 215 cells were in yeast form (Fig. 3L). On the other hand, Fg promoted a robust biofilm in all strains 216 when compared with BSA-dependent biofilms (Fig. 3G-K); except for the hyphae-deficient were added and incubated for 48 hrs at 37°C in human urine. Visualization of the interaction was 225 done by confocal microscopy and 3D reconstruction at 10x and 40x. We found that most of the 226 cells had a pseudohyphal and hyphal morphology surrounding and going through the fibrin fibers 227 and nets (Fig. 4A-E), except for SC5314 efg-1Δcph-1Δ (Fig. 3F).  Pt65, PNCL1, DAY230 and SC5314 and the catheter and bladder were colonized to the same 241 extent (Fig. 5). Importantly, we found that the hyphae-deficient mutant, SC5314 efg-1Δcph-1Δ, 242 had a significantly defective bladder and catheter colonization when compared with the SC5314 243 wild-type (WT) strain (p-value < 0.005; Fig. 5E). Interestingly, in the absence of a catheter, the 244 hyphae-deficient mutant behaved and colonized to the same extent as the WT strain.

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In human infection, the incidence of candidemia and systemic dissemination arising from 246 Candida UTI or candiduria Candida in urine are relatively low (1-8% of all candidemia cases) 58 .

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However, the prevalence of candidemia due to candiduria increases in critically ill and 248 immunocompromised patients 59,60 , especially if the patients are undergoing urinary 249 catheterization 61 . Since our mouse model of CAUTI allows us to assess dissemination, we 250 analyzed fungal burden of kidneys, spleen, and heart after 24 hpi (Fig. 5). We found that urinary with Fg ( Fig. 6 and Fig. S1-S6). 278 Importantly, neutrophils were highly recruited into the bladder, specifically in the areas 279 with fungal colonization ( Fig. 6 and Fig. S1-S6). We found that C. albicans breached the  found that all strains form a robust biofilm on the implanted catheter ( Fig. 7), colonizing 59% to 295 79% of the surface of the catheter (Table S2). SC5314 WT showed 78.9 ± 14% colonization of 296 the catheters while SC5314 efg-1Δcph-1Δ catheters' colonization was 10.4 ± 7.5%, exhibiting a 297 significant defective colonization ( Fig. 7F-G, L; Table S2). Our IF analysis showed that C. 298 albicans strains were preferentially binding onto deposited Fg on the catheter (Fig. 7B-G). We 299 then further quantified the percentage of the catheter-colonizing fungal population that was 300 colocalizing with Fg. We found that 75% to 91% of the C. albicans strains' staining was 301 colocalized with deposited Fg (Fig. 7H-L). Moreover, in the SC5314 efg-1Δcph-1Δ strain, that 302 was only able to colonize 10% of the catheter (Fig. 7G, Table S2), 79% of it was colocalizing 303 with Fg (Fig. 7L). This result further corroborates that hyphal formation is important for a robust 304 biofilm formation during CAUTI and that Fg serves as a platform for catheter colonization in vivo. In this study, we have shown that hyphal morphology and fungal interactions with Fg and 309 fibrin are critical for establishment of CAUTI. We showed that C. albicans is able to survive and 310 grow in urine and supplementation with 3% serum and Fg promotes growth. Importantly, we found  Our results showed that hyphal formation was critical for Fg-dependent biofilm formation 317 in urine conditions but not in YPD and BHI media. This seemingly contradictory result is not with C. albicans biofilms in urinary catheters. retrieved from rats 24 . These results, taken together 324 with our previous data showing that Fg is accumulated in the bladder and deposited on the catheters 325 1,5,13 , and that Fg promotes fungal biofilm formation in urine conditions and during urinary 326 catheterization (Fig. 3, 5-6), suggest that expression of Fg binding proteins could mediate fungal 327 biofilm formation in CAUTI.

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It has been reported that C. albicans encodes a Fg-binding protein, Mp58, which is    i.e. 9 fields of view) were randomly acquired and at least three images were analyzed per condition.

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