Microbial adhesion to medical devices is common for hospital-acquired infections, particularly for urinary catheters. If not properly treated these infections cause complications and exacerbate antimicrobial resistance. Catheter use elicits bladder inflammation, releasing host serum-proteins, including fibrinogen, into the bladder, which deposit on the urinary catheter. Enterococcus faecalis uses fibrinogen as a scaffold to bind and persist in the bladder despite antibiotic treatments. Inhibition of fibrinogen-pathogen interaction significantly reduces infection. Here, we show deposited fibrinogen is advantageous for uropathogens E. faecalis, E. coli, P. aeruginosa, K. pneumoniae, A. baumannii and C. albicans, suggesting that targeting catheter protein deposition may reduce colonization creating an effective intervention for catheter-associated urinary tract infections. In a mouse model of CAUTI, host-protein deposition was reduced, using liquid-infused silicone catheters, resulting in decreased colonization on catheters, in bladders, and dissemination in vivo. Furthermore, proteomics revealed a significant decrease in deposition of host-secreted proteins on liquid-infused catheter surfaces. Our findings suggest targeting microbial binding scaffolds may be an effective antibiotic-sparing intervention for use against catheter-associated urinary tract infections and other medical device infections.

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans, which employs a guide RNA to target an activator complex to the promoter region of a gene of interest, thus driving transcriptional expression of that gene. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust and constitutive overexpression. We further demonstrate the specificity of the system via RNA sequencing. We highlight the application of CRISPRa to overexpress genes involved in pathogenesis and drug susceptibility and contribute towards the identification of novel phenotypes. Consequently, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.

Catheter-associated urinary tract infections (CAUTIs) account for 40% of hospital-acquired infections (HAIs). As 20 to 50% of hospitalized patients receive catheters, CAUTIs are one of the most common HAIs, resulting in increased morbidity, mortality, and health care costs. Candida albicans is the second most common CAUTI uropathogen, yet relative to its bacterial counterparts, little is known about how fungal CAUTIs are established. Here, we show that the catheterized bladder environment induces Efg1- and fibrinogen (Fg)–dependent biofilm formation that results in CAUTI. In addition, we identify the adhesin Als1 as the critical fungal factor for C. albicans Fg-urine biofilm formation. Furthermore, we show that in the catheterized bladder, a dynamic and open system, both filamentation and attachment are required, but each by themselves are not sufficient for infection. Our study unveils the mechanisms required for fungal CAUTI establishment, which may aid in the development of future therapies to prevent these infections.

Catheter-associated urinary tract infections (CAUTIs) are a serious public health problem and account for approximately 40% of hospital-acquired infections worldwide. Candida spp are a major causative agent of CAUTI (17.8%) – specifically Candida albicans – that has steadily increased to become the second most common CAUTI uropathogen 1 . Yet, there is poor understanding of the molecular details of how C. albicans attaches, grows in the bladder, forms biofilms, survives, and persists during CAUTI 2 . Understanding of the mechanisms that contribute to CAUTI and invasive fungal infection will give insights into the development of more effective therapies, which are needed due to the spread of antifungal resistance and complex management of CAUTI in patients that require a urinary catheter 3 . Here, we characterize the ability of five Candida albicans clinical and laboratory strains to colonize the urinary catheter, grow and form biofilm in urine, and their ability to cause CAUTIs using our mouse model. Analysis of C. albicans strains revealed that growth in urine promotes morphological transition from yeast to hyphae, which is important for invasive infection. Additionally, we found that biofilm formation was dependent on the presence of fibrinogen, a protein released on the bladder to promote bladder healing 4,5 . Furthermore, deletion of hyphae regulatory genes resulted in defective bladder and catheter colonization and abolished dissemination. These results indicate that novel antifungal therapies preventing the morphological transition of C. albicans from yeast to hyphae have considerable promise for the treatment of fungal CAUTIs.

For the fungal pathogen Candida albicans, genetic overexpression readily occurs via a diversity of genomic alterations, such as aneuploidy and gain-of-function mutations, with important consequences for host adaptation, virulence, and evolution of antifungal drug resistance. Given the important role of overexpression on C. albicans biology, it is critical to develop and harness genetic tools that enable the analysis of genes expressed at high levels in the fungal cell. Here, we describe the development, optimization, and application of a novel, single-plasmid-based CRISPR activation (CRISPRa) platform for targeted genetic overexpression in C. albicans. Our CRISPRa system exploits a nuclease-dead dCas9 fused to the tripartite activator complex VP64-p65-Rta (VPR), and a Golden Gate site for efficient guide RNA cloning to overexpress genes of interest. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in C. albicans, and we assess optimal guide RNA targeting for robust overexpression. We further demonstrate the application of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these traits. Together, this tool will facilitate a broad range of applications for the study of C. albicans genetic overexpression.