微生物学

The most important virulence factor in the Cryptococcus genus is the polysaccharide capsule. This genus includes several species that cause life-threatening invasive disease. An increase in capsule thickness is important during fungal infection. The capsule is usually imaged using India ink, and crucial insights on the dynamics of its growth have been obtained using capsule-binding proteins such as specific antibodies or complement. We have developed an alternative method that allows both static and time-lapse imaging of the capsule using Percoll^®, a suspension of nanometric spheres that do not penetrate the capsule. Given that these particles have a higher refractive index than the capsule, the latter can be imaged by differential interference contrast (DIC) microscopy. Static observation of the capsule with DIC and Percoll^® results in capsule thickness measurements that match those made with India ink. Using capsule-inducing media, a glass-bottom incubation chamber and a live-imaging system equipped for DIC microscopy, this method allows time-lapse imaging of capsule growth. In contrast with India ink staining, DIC imaging of Percoll^® exclusion halos result in crisp images. The greatest advantage of this method, though, is that unlike India ink, the Percoll^® particles are non-toxic and unlike opsonins they do not bind the capsule, resulting in observations of capsule growth that are free from interference of bound proteins on capsule physiology.

The peptidoglycan sacculus, or cell wall, is what defines bacterial cell shape. Cell wall composition can be best characterized at the molecular level by digesting the peptidoglycan murein polymer into its muropeptide subunits and quantifying the abundance of muropeptides using high-pressure liquid chromatography. Certain features of the cell wall including muropeptide composition, glycan strand length, degree of crosslinking, type of crosslinking and other peptidoglycan modifications can be quantified using this approach. Well-established protocols provide us with highly-resolved and quantitatively reproducible chromatographic data, which can be used to investigate bacterial cell wall composition under a variety of environmental or genetic perturbations. The method described here enables the purification of muropeptide samples, their quantification by HPLC, and fraction collection for peak identification by mass spectrometry. Although the methods for peptidoglycan purification and HPLC analysis have been previously published, our method includes important details on how to re-equilibrate the column between runs to allow for automated analysis of multiple samples.

Teichoic acids (TA) are anionic polymers comprised of polyol phosphate repeat units that are abundant in the cell wall of Gram-positive bacteria. Both wall teichoic acid (WTA) and lipoteichoic acid (LTA) play important roles in regulating cell wall remodeling as well as conferring antibiotic resistance. To analyze TA, we describe a polyacrylamide gel electrophoresis (PAGE) method for both WTA and LTA. To extract crude WTA, the peptidoglycan sacculus is first isolated and WTA is then liberated by hydrolysis. LTA is extracted by 1-butanol and pre-treated with lipase to prevent aggregation and improve single-band resolution by PAGE. Crude extracts of both TAs are then subjected to PAGE followed by Alcian blue and silver staining. These protocols are easily adoptable by laboratories interested in rapidly analyzing TAs and can be used determine the relative abundance, relative polymer length and whether TAs are glycosylated. More detailed TA structural and compositional information can be obtained using the described purification protocols by nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) analysis.