Enhancing Candida auris Surveillance in High-Risk Settings by Implementing a High-Throughput Molecular Assay on the Hologic Fusion Open Access Platform.

Candida auris, a resilient pathogenic yeast with frequent multidrug resistance, presents a persistent challenge in healthcare settings. The timely identification of C. auris is crucial for infection control and prevention, especially in facilities facing unique hurdles, such as our institution, which serves four major hospitals and approximately 80% of the Texas inmate population. Understaffing, communal living, and financial constraints exacerbate infection control issues. To address common staff shortages, streamline testing services, and enhance testing efficiency, there was a pressing need for rapid and high-throughput detection of C. auris. This study presents the validation and utility of an assay implemented on the Hologic Fusion Open Access platform using samples collected from high-risk patients' axilla and groin areas, as well as environmental swab samples from patient rooms. Our assay complemented efforts to control C. auris outbreaks within our healthcare system, providing valuable insights into its presence within surveillance samples. This assay demonstrated the value of high-throughput molecular detection platforms in challenging healthcare environments by aiding infection preventionists in containing the spread of C. auris and preventing nosocomial infections. Our research contributes essential data on the suitability and performance of the Hologic Fusion Open Access platform for C. auris detection. These findings hold significant implications for enhancing surveillance and control measures in high-risk settings, making a significant impact on the field of infection control and prevention.

The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch.

Resistant starch is a prebiotic accessed by gut bacteria with specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses Sas6 (Starch Adherence System member 6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here, we present the crystal structures of Sas6 and of RbCBM74 bound with a double helical dimer of maltodecaose. The RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. Isothermal titration calorimetry and native mass spectrometry demonstrate that RbCBM74 recognizes longer single and double helical α-glucans, while RbCBM26 binds short maltooligosaccharides. Bioinformatic analysis supports the conservation of the amylopectin-targeting platform in CBM74s from resistant-starch degrading bacteria. Our results suggest that RbCBM74 and RbCBM26 within Sas6 recognize discrete aspects of the starch granule, providing molecular insight into how this structure is accommodated by gut bacteria.

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome.

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure-function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal ß-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (Kd ≤ 20 µM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

Starch Digestion by Gut Bacteria: Crowdsourcing for Carbs.

Starch is a polymer of glucose and is one of the most abundant carbohydrates in a Western diet. Resistant starch escapes digestion by host small intestinal glucoamylases and transits the colon where it is degraded by the combined efforts of many gut bacteria. Bacterial metabolism and fermentation of resistant starch leads to increases in short-chain fatty acids, including the clinically beneficial butyrate. Here, we review the molecular machinery that gut bacteria use to degrade starch and how these functions may intersect to facilitate complete starch digestion. While the protein complexes that gut bacteria use to degrade starch differ across phyla, some molecular details converge to promote the optimal positioning of enzymes and substrate for starch degradation.