Glycosaminoglycan Metabolism by Commensal and Pathogenic Bacteria of the Urinary Tract

Abstract

Urinary tract infections (UTIs) are among the most common adult bacterial infections. When a patient suffers ≥3 UTIs per year, it is defined as recurrent UTI (rUTI). Current rUTI therapies rely on antimicrobials to achieve sterility in the urinary tract (UT). However, research has shown that urine is not sterile in healthy individuals and that the urinary microbiota has not been fully characterized. A constant flux of urine containing electrolytes, osmolytes, amino acids, and carbohydrates supply nutrients to support urinary microbiota. An overlooked carbon source in the UT is the glycosaminoglycan (GAG) layer lining the urothelium. GAGs are linear heteropolysaccharides containing repeating disaccharide units composed of uronic acid (or galactose) and amino sugars. Previous studies have determined that species belonging to the genera Lactobacillus, Bifidobacterium, and Bacteroides derived from the human gut express enzymes that degrade GAGs into metabolizable disaccharides. However, the ability of the urinary microbiota and invading uropathogens to metabolize GAGs has not been assessed. To fill this knowledge gap, we screened a library of urinary bacteria clinically isolated from women with and without rUTI for the ability to degrade and utilize GAGs such as chondroitin sulfate (CS), heparan sulfate (HS), and hyaluronic acid (HA). To test this, we developed a plate-based, semi-quantitative GAG degradation and growth assay. Briefly, isolates were inoculated in modified MRS lacking carbohydrates and supplemented with CS, HS, HA, glycogen, glucose, or maltose. The resuspension was added to a microtiter plate and incubated at 35°C for 72 hours in microaerophilic conditions. Optical density at 600nm (OD600) was measured to record growth. Plates were centrifuged and the cell-free supernatant was added to 1% bovine serum albumin (BSA), which complexes with GAGs to form a white precipitate in the presence of acetic acid. To calculate percent GAG degradation, OD600 was recorded after addition of acetic acid and compared to bacteria-free controls. Using this method, we screened 16 representative isolates of four Lactobacillus species (L. crispatus, L. gasseri, L. delbrueckii, L. vaginalis), Streptococcus agalactiae, and Escherichia coli. Preliminary results show that all urinary Lactobacilli and E. coli exhibited no GAG degradation. However, all 4 Lactobacillus species were able to utilize maltose as a sole carbon source. Two S. agalactiae strains were able to degrade HA, but no other GAGs. Although urinary Lactobacilli were unable to degrade GAGs in vitro, these findings suggest that urinary Lactobacilli may rely on a bacterial consortia or host enzymes to degrade and utilize GAGs. Future work will focus on screening more common urinary microbiota species and analyzing the ability of urinary Lactobacilli to utilize GAGs in the presence of host GAG degradative enzymes and in co-culture with S. agalactiae. Upon completion, this work will define the metabolic requirements of key urinary microbiota and elucidate metabolic interactions between the urinary microbiota, host environment, and invading uropathogens.