Niclosamide-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The Hyr1 protein from the fungus Candida albicans is a cross kingdom immunotherapeutic target for Acinetobacter bacterial infection.

Different pathogens share similar medical settings and rely on similar virulence strategies to cause infections. We have previously applied 3-D computational modeling and bioinformatics to discover novel antigens that target more than one human pathogen. Active and passive immunization with the recombinant N-terminus of Candida albicans Hyr1 (rHyr1p-N) protect mice against lethal candidemia. Here we determine that Hyr1p shares homology with cell surface proteins of the multidrug resistant Gram negative bacterium, Acinetobacter baumannii including hemagglutinin (FhaB) and outer membrane protein A (OmpA). The A. baumannii OmpA binds to C. albicans Hyr1p, leading to a mixed species biofilm. Deletion of HYR1, or blocking of Hyr1p using polyclonal antibodies, significantly reduce A. baumannii binding to C. albicans hyphae. Furthermore, active vaccination with rHyr1p-N or passive immunization with polyclonal antibodies raised against specific peptide motifs of rHyr1p-N markedly improve survival of diabetic or neutropenic mice infected with A. baumannii bacteremia or pneumonia. Antibody raised against one particular peptide of the rHyr1p-N sequence (peptide 5) confers majority of the protection through blocking A. baumannii invasion of host cells and inducing death of the bacterium by a putative iron starvation mechanism. Anti-Hyr1 peptide 5 antibodies also mitigate A. baumannii /C. albicans mixed biofilm formation in vitro. Consistent with our bioinformatic analysis and structural modeling of Hyr1p, anti-Hyr1p peptide 5 antibodies bound to A. baumannii FhaB, OmpA, and an outer membrane siderophore binding protein. Our studies highlight the concept of cross-kingdom vaccine protection against high priority human pathogens such as A. baumannii and C. albicans that share similar ecological niches in immunocompromised patients

The Hyr1 protein from the fungus <i>Candida albicans</i> is a cross kingdom immunotherapeutic target for <i>Acinetobacter</i> bacterial infection

<div><p>Different pathogens share similar medical settings and rely on similar virulence strategies to cause infections. We have previously applied 3-D computational modeling and bioinformatics to discover novel antigens that target more than one human pathogen. Active and passive immunization with the recombinant N-terminus of <i>Candida albicans</i> Hyr1 (rHyr1p-N) protect mice against lethal candidemia. Here we determine that Hyr1p shares homology with cell surface proteins of the multidrug resistant Gram negative bacterium, <i>Acinetobacter baumannii</i> including hemagglutinin (FhaB) and outer membrane protein A (OmpA). The <i>A</i>. <i>baumannii</i> OmpA binds to <i>C</i>. <i>albicans</i> Hyr1p, leading to a mixed species biofilm. Deletion of <i>HYR1</i>, or blocking of Hyr1p using polyclonal antibodies, significantly reduce <i>A</i>. <i>baumannii</i> binding to <i>C</i>. <i>albicans</i> hyphae. Furthermore, active vaccination with rHyr1p-N or passive immunization with polyclonal antibodies raised against specific peptide motifs of rHyr1p-N markedly improve survival of diabetic or neutropenic mice infected with <i>A</i>. <i>baumannii</i> bacteremia or pneumonia. Antibody raised against one particular peptide of the rHyr1p-N sequence (peptide 5) confers majority of the protection through blocking <i>A</i>. <i>baumannii</i> invasion of host cells and inducing death of the bacterium by a putative iron starvation mechanism. Anti-Hyr1 peptide 5 antibodies also mitigate <i>A</i>. <i>baumannii /C</i>. <i>albicans</i> mixed biofilm formation <i>in vitro</i>. Consistent with our bioinformatic analysis and structural modeling of Hyr1p, anti-Hyr1p peptide 5 antibodies bound to <i>A</i>. <i>baumannii</i> FhaB, OmpA, and an outer membrane siderophore binding protein. Our studies highlight the concept of cross-kingdom vaccine protection against high priority human pathogens such as <i>A</i>. <i>baumannii</i> and <i>C</i>. <i>albicans</i> that share similar ecological niches in immunocompromised patients.</p></div

Anti-Hyr1p peptide antibodies specifically binds <i>A</i>. <i>baumannii</i> but not <i>P</i>. <i>aeruginosa</i>.

<p>Flow cytometry analysis of pooled sera collected from rabbits separately immunized with 8 peptides of Hyr1p showing binding of these antibodies to <i>A</i>. <i>baumannii</i> but not to <i>P</i>. <i>aeruginosa</i> (bacterial cells at 10⁷ cells). Pooled sera from the same rabbits prior to immunization was used as a control (Control-Abs). The high and the low doses of the Abs were 30 and 3 μg/ml, respectively. <i>AB = A</i>. <i>baumannii</i>, <i>PA = P</i>. <i>aeruginosa</i>.</p

<i>Acinetobacter</i> OmpA is the ligand to <i>Candida</i> Hyr1p.

<p><i>Acinetobacter</i> biotin-labeled cell wall proteins that bound to <i>C</i>. <i>albicans</i> wild-type, <i>hyr1/hyr1</i>, <i>hyr1/hyr1+HYR1</i>, or <i>als3/als3</i> hyphae were separated on SDS-PAGE, probed with anti-biotin antibody, and the hybridized bands were identified by Mass spectrometry. <i>C</i>. <i>albicans</i> wild-type or <i>hyr1/hyr1+HYR1</i> complemented hyphae bound a major band at 38 kDa which was identified as OmpA by MS/MS analysis (A). In contrast, the <i>hyr1/hyr1</i> mutant had severe reduction in its ability to bind OmpA. <i>C</i>. <i>albicans</i> Als3p was not found to be a receptor as proteins equally bound to <i>als3/als3</i> mutant strain (A). To confirm the identity of the 38 kDa band, blots were probed with an anti-OmpA antibody, which confirmed that the 38 kDa proteins were indeed OmpA (B).</p

Active or passive immunization targeting Hyr1p protect diabetic and neutropenic mice from <i>A</i>. <i>baumannii</i> infections.

<p>Survival of diabetic mice (n = 10 for alum control or 9 for rHyr1p-N + Alum) were vaccinated and boosted with 30 μg dose mixed with 0.1% aluminum hydroxide then infected with <i>A</i>. <i>baumannii</i> HUMC1 (confirmed inoculum of 2.5 x 10⁷) via the tail vein. *<i>P</i> = 0.005 vs. alum control (A). Tissue bacterial burden of target organs harvested from diabetic mice (n = 10 per arm) vaccinated with alum or rHyr1p + alum three days after infecting with <i>A</i>. <i>baumannii</i> (5 x 10⁶). **<i>P</i> <0.02 vs. alum vaccinated mice (B). Survival of diabetic mice that were given an intraperitoneal (i.p.) injection of 1 mg of pooled anti-Hyr1p IgG raised against 8 individual peptides of Hyr1p (n = 20 from 2 independent experiments with similar results for <i>A</i>. <i>baumannii</i> [AB] infection and 10 for <i>P</i>. <i>aeruginosa</i> [P<i>A</i>]) or control IgG (n = 18 for AB and 10 for PA) 2 hours prior to infecting them intravenously (i.v.) with AB or PA (confirmed inocula of 3.4 x10⁷ cells for AB and 1.1 x 10⁸ for PA). ^¥<i>P</i> = 0.00001 vs. Control IgG of AB ^‡<i>P</i> = 0.1 vs. Control IgG of PA (C). Survival of diabetic mice (n = 8 per group) that were given an i.p. injection of 1 mg of each anti-Hyr1p IgG raised against individual peptides of Hyr1p 2 hours prior to infecting them i.v. with <i>A</i>. <i>baumannii</i> (confirmed inoculum of 6.2 x 10⁷). ^§<i>P <</i>0.05 vs. all arms except peptide, 2, 7, or 8 (D). Survival of neutropenic mice (n = 10 per group) infected with <i>A</i>. <i>baumannii</i> via inhalation (inhaled inoculum of 2.2 x 10⁷) and 24 h later treated i.p. with either isotype matching control IgG (100 μg), or with anti-peptide 5 IgG (at 30 or 100 μg). A repeat dose of the antibody was given on Day +8. ***<i>P</i> <0.002 vs. Control IgG arm (E).</p

Striking 3 D structural homology between <i>C</i>. <i>albicans</i> Hyr1p and <i>A</i>. <i>baumannii</i> FhaB.

<p>Ribbon diagram depicting the full scale model for <i>C</i>. <i>albicans</i> Hyr1 as computed by the iTasser server (A). The region of Hyr1 corresponding to homologous domains in the viral tailspike, <i>BP</i> FhaB and <i>HI</i> HMW1 proteins is indicated in blue. Note the striking similarity in 3D structure of Hyr1p and FhaBp (B). Hyr1 models showing the sequence and location of the eight peptides used as antigens in the study in red as van der Waals space-filling spheres (C). Note the similar location of peptide 5 on Fhab (B) and on Hyr1p (C) (red regions). Models for three putative cross-reactive <i>A</i>. <i>baumannii</i> targets (FhaBp, OmpA, BIg) are shown with domains homologous to peptide 5 in red (D). In addition, the <i>A</i>. <i>baumannii</i> BIg protein has many additional near matches (6 identical residues) which are shown on the full model as orange. Sequence alignments between <i>C</i>. <i>albicans</i> (CA) peptide 5 and putative cross-reactive <i>A</i>. <i>baumannii</i> (AB) targets with identical residues are depicted in boxes. Higher resolution views of identified domains showing identical and physicochemically conserved residues are visualized as space-filling spheres. Coloration is a modified RasMol schema (Gly, Ala—cream; Asn, Gln—light brown; Thr—orange; Val, Ile—green; Trp—olive green; Asp—red; His—sky blue; Pro—chartreuse).</p