Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded characterization. Polysaccharides remain attached to the cell as capsular polysaccharide (CPS) or are shed into the surroundings in the form of exopolysaccharide (EPS). While a great deal of study has been done examining the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and removed capsular material. Sonication and glucanex digestion yield soluble CPS preparations, while French Press and modified glucanex digestion plus vortexing remove the capsule and cell wall producing polysaccharide aggregates that we call capsule ghosts. The existence of capsule ghosts implies an inherent organization that allows it to exist independent of the cell wall surface. As sonication and glucanex digestion were noncytotoxic, it was possible to observe the cryptococcal cells rebuilding their capsule, revealing new insights into capsule architecture and synthesis consistent with a model in which the capsule is assembled from smaller polymers, which are then assemble into larger ones.

Cryptococcus neoformans: Diagnostic Dilemmas, Electron Microscopy and Capsular Variants

Two cases of cryptococcal meningitis went undetected by a cryptococcal antigen (CrAg) lateral flow assay on blood in a reflex CrAg screen-and-treat programme in South Africa, although Cryptococcus neoformans was identified by culturing the cerebrospinal fluid specimens. Further investigations into these discordant diagnostic results included multilocus sequence typing (which showed no mutations in the CAP59 gene) and transmission electron microscopy using a capsule-staining protocol (which revealed a >50% reduction in capsular material in both cases, relative to a control culture). A multi-disciplinary approach for resolving discordant diagnostic test results is recommended.

Mycobacterium tuberculosis Cpn60.2 and DnaK Are Located on the Bacterial Surface, Where Cpn60.2 Facilitates Efficient Bacterial Association with Macrophages

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis, initially contacts host cells with elements of its outer cell wall, or capsule. We have shown that capsular material from the surface of M. tuberculosis competitively inhibits the nonopsonic binding of whole M. tuberculosis bacilli to macrophages in a dose-dependent manner that is not acting through a global inhibition of macrophage binding. We have further demonstrated that isolated M. tuberculosis capsular proteins mediate a major part of this inhibition. Two-dimensional polyacrylamide gel electrophoresis analysis of the capsular proteins showed the presence of a wide variety of protein species, including proportionately high levels of the Cpn60.2 (Hsp65, GroEL2) and DnaK (Hsp70) molecular chaperones. Both of these proteins were subsequently detected on the bacterial surface. To determine whether these molecular chaperones play a role in bacterial binding, recombinant Cpn60.2 and DnaK were tested for their ability to inhibit the association of M. tuberculosis bacilli with macrophages. We found that recombinant Cpn60.2 can inhibit ∼57% of bacterial association with macrophages, while DnaK was not inhibitory at comparable concentrations. Additionally, when polyclonal F(ab′)2 fragments of anti-Cpn60.2 and anti-DnaK were used to mask the surface presentation of these molecular chaperones, a binding reduction of ∼34% was seen for anti-Cpn60.2 F(ab′)2, while anti-DnaK F(ab′)2 did not significantly reduce bacterial association with macrophages. Thus, our findings suggest that while M. tuberculosis displays both surface-associated Cpn60.2 and DnaK, only Cpn60.2 demonstrates adhesin functionality with regard to macrophage interaction.

Granulicoccus phenolivorans gen. nov., sp. nov., a Gram-positive, phenol-degrading coccus isolated from phenol-degrading aerobic granules

A Gram-positive bacterium, designated strain PG-02T, was isolated by serial dilution from aerobic granules obtained from a laboratory-scale sequencing batch reactor for bioremediation of phenolic wastewater. Strain PG-02T grew axenically as cocci and is an oxidase-negative and catalase-positive, non-motile facultative anaerobe. It does not reduce nitrate and grows between 15 and 37 °C, with an optimum temperature of 30 °C. The pH range for growth is between 5.0 and 8.5, with an optimum pH of 7.0. Strain PG-02T contains type A3γ peptidoglycan (ll-A2pm←Gly with alanine at position 1 of the peptide subunit). The G+C content of the DNA is 69 mol%. Menaquinone MK-9(H4) was the major isoprenoid quinone. The polar lipids included diphosphatidylglycerol and phosphatidylglycerol, while 13-methyltetradecanoic acid (i-C15 : 0) and 1,1-dimethoxy-iso-pentadecane (i-C15 : 0 DMA) were the major components in whole-cell methanolysates. PG-02T stained positively for intracellular polyphosphate granules but not poly-β-hydroxyalkanoates. It produces capsular material and possesses an autoaggregation capability. Phenotypic and 16S rRNA gene sequence analyses showed that PG-02T differed from its closest phylogenetic relatives, namely members of the suborder Propionibacterineae, which includes the genera Tessaracoccus, Microlunatus, Luteococcus, Micropruina, Propionibacterium, Propioniferax, Nocardioides, Friedmanniella and Aeromicrobium, and that it should be placed in a new genus and species as Granulicoccus phenolivorans gen. nov., sp. nov. The type strain of Granulicoccus phenolivorans is PG-02T (=ATCC BAA-1292T=DSM 17626T).