Rat Indwelling Urinary Catheter Model of Candida albicans Biofilm Infection 2 3 4

ABSTRACT 24 Indwelling urinary catheters are commonly used in the management of 25 hospitalized patients. Candida can adhere to the device surface and propagate 26 as a biofilm. These communities differ from free-floating Candida, exhibiting high 27 tolerance to antifungal therapy. The significance of catheter-associated 28 candiduria is often unclear and treatment may be problematic considering the 29 biofilm drug resistant phenotype. Here we describe a rodent model for study of 30 urinary catheter-associated Candida albicans biofilm infection that mimics this 31 common process in patients. In the setting of a functioning, indwelling urinary 32 catheter in a rat, Candida proliferated as a biofilm on the device surface

The Extracellular Matrix of <i>Candida albicans</i> Biofilms Impairs Formation of Neutrophil Extracellular Traps

<div><p>Neutrophils release extracellular traps (NETs) in response to planktonic <i>C</i>. <i>albicans</i>. These complexes composed of DNA, histones, and proteins inhibit <i>Candida</i> growth and dissemination. Considering the resilience of <i>Candida</i> biofilms to host defenses, we examined the neutrophil response to <i>C</i>. <i>albicans</i> during biofilm growth. In contrast to planktonic <i>C</i>. <i>albicans</i>, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The <i>C</i>. <i>albicans pmr1Δ/Δ</i>, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that <i>C</i>. <i>albicans</i> biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.</p></div

<i>C</i>. <i>albicans</i> biofilms resist killing by neutrophils and impair release of NETs.

<p>(A) Planktonic and biofilm <i>C</i>. <i>albicans</i> were co-cultured with human neutrophils at an effector:target ratio of 1:1 and fungal inhibition was estimated by an XTT assay after neutrophil lysis. Neutrophils have very little activity against biofilms, but strongly inhibit planktonic <i>Candida</i>, <i>n = 11</i>. (B) The rate of NET release was estimated by Sytox Green detection of free DNA. Planktonic <i>Candida</i> generated high fluorescence, representing NET release, comparable to the levels induced by PMA. In contrast, biofilms did not produce fluorescence, similar to the neutrophil only control, <i>n = 3</i>. (C) Following co-culture with neutrophils for 4 h, the neutrophil response to planktonic and biofilm <i>C</i>. <i>albicans</i> was visualized by immunofluorescence using an anti-citrullinated H4 antibody. NETs were observed in response to planktonic <i>Candida</i>, but were rarely produced in response to biofilm. (D) By scanning electron microscopy, thread-like NETs covered planktonic cells after a 4 h co-culture with neutrophils. In contrast, neutrophils exposed to biofilm appeared rounded, with few NETs released. Measurement bars represent 20 μm and 2 μm for 2,000x and 10,000x images, respectively. *<i>P<0</i>.<i>05</i> Error bars represent SEM.</p

Neutrophils adhere to <i>C</i>. <i>albicans</i> biofilm.

<p>(A) Fluorescently-labeled neutrophils were added to microfluidic channels with <i>C</i>. <i>albicans</i> biofilms, which had been propagated on the sidewall, or to planktonic <i>C</i>. <i>albicans</i>. Over the course of 90 min, neutrophils migrated to the biofilm, adhered, and extended over the surface of the hyphae. In contrast, neutrophils engulfed planktonic <i>C</i>. <i>albicans</i> and did not appear to elongate. (B) Neutrophil interactions with <i>C</i>. <i>albicans</i> were examined by scanning electron microscopy. In response to biofilms, neutrophils initially adhered to hyphae, then elongated with extended filopodia, and ultimately appeared rounded. Upon co-culture with planktonic cells, NETs developed over this 4 h time period. Measurement bars represent 2 μm for 10,000x images. Representative data are shown for experiments performed with neutrophils from at least 3 different donors on different days.</p

C. <i>albicans</i> biofilm extracellular matrix inhibits neutrophil production of ROS and protects against neutrophil activity.

<p>(A-D) Neutrophils were pre-labeled with CM-H2DCFDA and exposed to <i>C</i>. <i>albicans</i> biofilms in the presence or absence of PMA and ROS was measured by fluorescence. (A) Neutrophils produced higher levels of ROS in response to planktonic cells over the course of 3 h, <i>n = 4</i>, <i>representative data with SD shown</i>. (B) Dispersion of biofilms increased neutrophil production of ROS <i>n = 5</i>, <i>SEM shown</i>. (C) Neutrophils generated increased ROS in response to the <i>C</i>. <i>albicans pmr1Δ/Δ</i> mutant biofilm, several fold above the reference strain, <i>n = 9</i>, <i>SEM shown</i>. (D) <i>C</i>. <i>albicans</i> biofilms inhibited ROS production in response to PMA stimulation <i>n = 11</i>, <i>SEM shown</i>. (E-F) <i>C</i>. <i>albicans</i> biofilms and planktonic cells were co-cultured with human neutrophils and fungal killing was estimated by an XTT assay after neutrophil lysis. Neutrophils exhibited increased activity against the <i>pmr1Δ/Δ</i> mutant biofilm compared to the reference strain (E) but no difference was observed for planktonic cells (F), <i>n = 6 and 4</i>, <i>SEM shown</i>. *<i>P<0</i>.<i>05</i> compared to reference.</p

<i>C</i>. <i>albicans</i> biofilm mannosylation impairs NET release and contributes to virulence in vivo.

<p>(A) <i>C</i>. <i>albicans</i> biofilms were formed in rat venous catheters in vivo and imaged by scanning electron microscopy. While both the reference strain and the <i>pmr1</i>Δ/Δ mutant formed biofilms in vivo, the <i>pmr1</i>Δ/Δ mutant biofilm was associated with increased host cells and overlying fibrillary material. On higher magnification, the web of fibrils appeared to extrude from host cells. (B) The contribution of mannosylation by <i>PMR1</i> to biofilm immune protection was assessed by rat catheter viable burden. Disruption of <i>PMR1</i> was associated with a 70% decrease in burden compared to the reference strain. *<i>P<0</i>.<i>05</i>.</p

NET inhibition by <i>C</i>. <i>albicans</i> is dependent on intact biofilm architecture and is not due to a soluble factor.

<p>(A) Planktonic, biofilm, and partially dispersed biofilms were co-cultured with human neutrophils for 4 h and NET release was estimated by Sytox Green detection of free DNA. Dispersed biofilms induced NETs, marked by elevated Sytox Green, <i>n = 6</i>. (B) By scanning electron microscopy, NETs were triggered in response to disrupted biofilm, indicating intact structure is necessary for NET inhibition. The measurement bars represent 20 μm and 2 μm for the 2,000x and 10,000x images, respectively. (C) <i>C</i>. <i>albicans</i> biofilms and PMA (an inducer of NETs), alone and in combination were incubated with neutrophils for 4 h and NET release was estimated by Sytox Green. While PMA alone generated a robust NET response, the combination of PMA and biofilm did not trigger DNA release. (D) Supernatants from biofilms (24 h) were collected at 2 h and added to Sytox Green assays in combination with PMA. Biofilm supernatants alone did not block PMA induction of NETs, <i>n = 5</i>. (E) Neutrophils were pre-stimulated (pre-stim) with PMA to induce NETs for 90 min prior to addition to biofilms. While pre-stimulation increased NET release, biofilms still inhibited NETs compared to the PMA only control, <i>n = 4</i>. (F) The activity of pre-stimulated neutrophils against biofilms was estimated by XTT assay following lysis of neutrophils. Neutrophils pre-stimulated to induce NETs inhibited biofilms <i>n = 4</i>. <i>*P<0</i>.<i>05</i> compared to reference, <i>**P<0</i>.<i>05</i> compared to *. Error bars represent SEM.</p

Biofilm inhibition of NETs is dependent on <i>C</i>. <i>albicans</i> mannosylation.

<p>(A) NET release was estimated by Sytox Green after neutrophils were exposed to <i>C</i>. <i>albicans</i> biofilms for 4 h. Five mutants with disruption of mannan pathways triggered NET release in response to neutrophils from multiple donors, <i>n = 4</i>. (B) By scanning electron microscopy, neutrophils exposed to the reference strain biofilm appeared rounded, while the <i>pmr1Δ/Δ</i> biofilm elicited NETs. Measurement bars represent 2 μm for 10,000x images. (C) Complementation of the <i>pmr1Δ/Δ</i> mutant mostly restored the NET inhibition phenotype <i>n = 4</i>. (D) NET release was measured in response to planktonic <i>C</i>. <i>albicans</i>. In contrast to the response to biofilms, no significant difference was observed between <i>pmr1Δ/Δ</i> and the reference strain, <i>n = 6</i>. <i>*P<0</i>.<i>05</i> compared to reference. Error bars represent SEM.</p