Visualizing Biomaterial Degradation by Candida albicans Using Embedded Luminescent Molecules To Report on Substrate Digestion and Cellular Uptake of Hydrolysate.

Microbial pathogens use hydrolases as a virulence strategy to spread disease through tissues and colonize medical device surfaces; however, visualizing this process is a technically challenging problem. To better understand the role of secreted fungal hydrolases and their role in Candida albicans virulence, we developed an in situ model system using luminescent Re(I) and Ir(III) containing probe molecules embedded in a biodegradable (poly(lactic-co-glycolic acid), PLGA) polymer and tracked their uptake using epifluorescent imaging. We found that secretion of esterases can explain how physically embedded probes are acquired by fungal cells through the degradation of PLGA since embedded probes could not be liberated from nonbiodegradable polystyrene (PS). It was important to verify that epifluorescent imaging captured the fate of probe molecules rather than naturally occurring fungal autofluorescence. For this, we exploited the intense luminescent signals and long spectral relaxation times of the Re and Ir containing probe molecules, resolved in time using a gated imaging system. Results provide a visual demonstration of a key virulence trait of C. albicans: the use of hydrolases as a means to degrade materials and acquire hydrolysis products during fungal growth and hyphal development. These results help to explain the role of nonspecific hydrolases using a degradable material that is relevant to the study of fungal pathogenesis on biotic (tissues) surfaces. Additionally, understanding how fungal pathogens condition surfaces by using nonspecific hydrolases is important to the study of fungal attachment on abiotic surfaces, the first step in biofilm formation on medical devices.

Detection of Wzy/Wzz interaction in Shigella flexneri.

The O antigen (Oag) component of Shigella flexneri lipopolysaccharide (LPS) is important for virulence and a protective antigen. It is synthesized by the Wzy-dependent mechanism. S. flexneri Wzy has 12 transmembrane segments and two large periplasmic loops. The modal chain length of the Oag is determined by Wzz. Experimental evidence supports multi-protein interactions in the Wzy-dependent pathway. However, evidence for direct interaction of Wzy with the other proteins of the Wzy-dependent pathway is limited. Initially, we purified Wzy-GFP-His8 and detected the presence of a dimeric form. In vivo cross-linking was then performed in an S. flexneri wzy mutant strain carrying plasmids encoding Wzy-GFP-His8 and untagged Wzz. Following solubilization with n-dodecyl-ß-D-maltopyranoside (DDM) and affinity purification of Wzy-GFP-His8, Western immunoblotting with Wzz antibody detected co-purification of Wzz; this was supported by MS analysis. To the best of our knowledge, this is the first reported isolation of a complex between Wzy and Wzz. Wzy mutants (WzyR164A, WzyV92M, WzyY137H, and WzyR250K) whose properties are affected by Wzz were able to form complexes with Wzz. Their mutational alterations did not affect the interaction of Wzy with Wzz. Thus, the interaction may involve many regions of Wzy.

Mutational analysis of the major periplasmic loops of Shigella flexneri Wzy: identification of the residues affecting O antigen modal chain length control, and Wzz-dependent polymerization activity.

The O antigen (Oag) component of LPS is a major Shigella flexneri virulence determinant. Oag is polymerized by WzySf, and its modal chain length is determined by WzzSf and WzzpHS2. Site-directed mutagenesis was performed on wzySf in pWaldo-wzySf-TEV-GFP to alter Arg residues in WzySf's two large periplasmic loops (PLs) (PL3 and PL5). Analysis of the LPS profiles conferred by mutated WzySf proteins in the wzySf deficient (Δwzy) strain identified residues that affect WzySf activity. The importance of the guanidium group of the Arg residues was investigated by altering the Arg residues to Lys and Glu, which generated WzySf mutants conferring altered LPS Oag modal chain lengths. The dependence of these WzySf mutants on WzzSf was investigated by expressing them in a wzySf and wzzSf deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles identified a role for the Arg residues in the association of WzySf and WzzSf during Oag polymerization. Colicin E2 and bacteriophage Sf6c susceptibility supported this conclusion. Comparison of the expression levels of different mutant WzySf-GFPs with the wild-type WzySf-GFP showed that certain Arg residues affected production levels of WzySf in a WzzSf-dependent manner. To our knowledge, this is the first report of S. flexneri WzySf mutants having an effect on LPS Oag modal chain length, and identified functionally significant Arg residues in WzySf.

Mutational analysis of the Shigella flexneri O-antigen polymerase Wzy: identification of Wzz-dependent Wzy mutants.

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySf and WzzSf was investigated by transforming these mutated wzySf plasmids into a wzySf- and wzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified WzySf mutants whose polymerization activities were WzzSf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for WzzSf in WzySf stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also affects WzySf activity and stability.