Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization.

Candida albicans colonization is required for invasive disease. Unlike humans, adult mice with mature intact gut microbiota are resistant to C. albicans gastrointestinal (GI) colonization, but the factors that promote C. albicans colonization resistance are unknown. Here we demonstrate that commensal anaerobic bacteria-specifically clostridial Firmicutes (clusters IV and XIVa) and Bacteroidetes-are critical for maintaining C. albicans colonization resistance in mice. Using Bacteroides thetaiotamicron as a model organism, we find that hypoxia-inducible factor-1α (HIF-1α), a transcription factor important for activating innate immune effectors, and the antimicrobial peptide LL-37 (CRAMP in mice) are key determinants of C. albicans colonization resistance. Although antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease. In the setting of antibiotics, Hif1a and Camp (which encodes CRAMP) are required for B. thetaiotamicron-induced protection against C. albicans colonization of the gut. Thus, modulating C. albicans GI colonization by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

RNA isolation of Pseudomonas aeruginosa colonizing the murine gastrointestinal tract.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice in order to identify changes in bacterial gene expression during alterations to the host's immune status. As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.