Discrimination of Aspergillus lentulus from Aspergillus fumigatus by Raman spectroscopy and MALDI-TOF MS.

In 2005, a new sibling species of Aspergillus fumigatus was discovered: Aspergillus lentulus. Both species can cause invasive fungal disease in immune-compromised patients. The species are morphologically very similar. Current techniques for identification are PCR-based or morphology-based. These techniques are labour-intense and not sufficiently discriminatory. Since A. lentulus is less susceptible to several antifungal agents, it is important to correctly identify the causative infectious agent in order to optimize antifungal therapy. In this study we determined whether Raman spectroscopy and/or MALDI-TOF MS were able to differentiate between A. lentulus and A. fumigatus. For 16 isolates of A. lentulus and 16 isolates of A. fumigatus, Raman spectra and peptide profiles were obtained using the Spectracell and MALDI-TOF MS (VITEK MS RUO, bioMérieux) respectively. In order to obtain reliable Raman spectra for A. fumigatus and A. lentulus, the culture medium needed to be adjusted to obtain colourless conidia. Only Raman spectra obtained from colourless conidia were reproducible and correctly identified 25 out of 32 (78 %) of the Aspergillus strains. For VITEK MS RUO, no medium adjustments were necessary. Pigmented conidia resulted in reproducible peptide profiles as well in this case. VITEK MS RUO correctly identified 100 % of the Aspergillus isolates, within a timeframe of approximately 54 h including culture. Of the two techniques studied here, VITEK MS RUO was superior to Raman spectroscopy in the discrimination of A. lentulus from A. fumigatus. VITEK MS RUO seems to be a successful technique in the daily identification of Aspergillus spp. within a limited timeframe.

Isavuconazole activity against Aspergillus lentulus, Neosartorya udagawae, and Cryptococcus gattii, emerging fungal pathogens with reduced azole susceptibility.

Isavuconazole is an extended-spectrum triazole with in vitro activity against a wide variety of fungal pathogens. Clinical isolates of molds Aspergillus lentulus and Neosartorya udagawae and yeast Cryptococcus gattii VGII (implicated in the outbreak in the Pacific Northwest, North America) exhibit reduced susceptibilities to several azoles but higher susceptibilities to isavuconazole.

Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1.

The pathogenesis of candidiasis involves invasion of host tissues by filamentous forms of the opportunistic yeast Candida albicans. Morphology-specific gene products may confer proinvasive properties. A hypha-specific surface protein, Hwp1, with similarities to mammalian small proline-rich proteins was shown to serve as a substrate for mammalian transglutaminases. Candida albicans strains lacking Hwp1 were unable to form stable attachments to human buccal epithelial cells and had a reduced capacity to cause systemic candidiasis in mice. This represents a paradigm for microbial adhesion that implicates essential host enzymes.

Genetic organization and sequence analysis of the hypha-specific cell wall protein gene HWP1 of Candida albicans.

A previously isolated partial cDNA encoding a cell wall protein antigen found on hyphal surfaces of the opportunistic fungal pathogen, Candida albicans (Staab et al., 1996) was used to clone the complete hyphal wall protein 1 gene (HWP1). Hyphal forms of C. albicans invade mucosal surfaces of immunocompromised patients such as those with AIDS. HWP1 consisted of an open reading frame predicting an acidic protein (pI of 3.37) with a calculated molecular size of 61,122. The antigenic domain was located in the N-terminal third of the protein. The remainder of the protein contained abundant hydroxy amino acids, and terminated with a string of 15 amino acids typical of sequences specifying post-translational modification with glycosylphosphatidylinositol (6PI). The analyses suggested that Hwp1 is a glucan-linked protein with serine/threonine-rich regions that are predicted to function in extending a ligand-binding domain into the extracellular space.

Developmental expression of a tandemly repeated, proline-and glutamine-rich amino acid motif on hyphal surfaces on Candida albicans.

cDNA sequences encoding a cell wall protein have been isolated from the opportunistic pathogen, Candida albicans, an organism that can cause serious disease in immunocompromised patients such as those with AIDS. The cDNA encodes a peptide that is largely composed of an acidic, repeated motif 10 amino acids in length that is rich in proline and glutamine residues. The cDNA gene product was found to be present on hyphal surfaces by immunofluorescence assays using monospecific antisera raised to the recombinant protein produced in Pichia pastoris. The hyphae-specific surface location was also seen on organisms colonizing the gastrointestinal mucosa of mice, indicating that the antigen is produced and developmentally regulated during growth in host tissues. The cDNA clone hybridized to an abundant messenger RNA 2.3 kilobases in size that was present in hyphal but not yeast forms. These studies demonstrate that the bud-hypha transition is accompanied by the de novo synthesis of proteins that are targeted to hyphal surfaces. The primary sequence of the unique amino acid motif shares features with surface proteins of other lower eukaryotic microorganisms and with host acidic salivary proline-rich proteins.

A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.

Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.

EntG activity of Escherichia coli enterobactin synthetase.

The last steps in the biosynthesis of the Escherichia coli siderophore enterobactin (Ent) are carried out by Ent synthetase, a multienzyme complex believed to be composed of the entD, -E, -F, and -G products (EntD to -G). However, sequencing data showed that there is no separate entG gene and, unlike EntD to -F, no distinct EntG polypeptide has been identified. In this study, genetic, biochemical, and immunological approaches were used to study the anomalies associated with EntG activity. Two plasmids, pJS43 and pJS100, were isolated that had mutations resulting in truncated EntB proteins; both had the phenotype EntB+ EntG-. PJS43 had a Tn5 inserted 198 bp from the entB termination codon, and pJS100 had the last 25 codons of entB deleted. Plasmids isolated with Tn5 insertions in the 5' half of entB had the phenotype EntB- EntG+. These latter Tn5 mutations were EntB- EntG- when moved to the bacterial chromosome. Polyclonal antiserum was prepared and shown to react only with intact EntB in Western immunoblots. Addition of anti-EntB antiserum to Ent synthetase assays resulted in complete inhibition of enzyme activity, whereas preimmune serum had no effect. Lastly, AN462, the type strain for entG which was derived by Mu insertion and which has the phenotype EntB-G-A-, was characterized. Southern blot data showed a Mu insertion, presumably with polar effects, in the vicinity of the 5' end of entB. In summary, EntG activity was found to be encoded by the entB 3' terminus. The evidence, while not rigorously eliminating the possibility that a separate EntG polypeptide exists, strongly supports the idea that EntB is a bifunctional protein.

Nucleotide sequence of the Escherichia coli entE gene.

The Escherichia coli entE gene encodes a polypeptide necessary in the latter stages of biosynthesis of the siderophore enterobactin. The entE gene and adjacent DNA were sequenced. The predicted EntE polypeptide consists of 536 amino acids and has a Mr of 58,299 and a net charge of -7.33. Genetic evidence combined with this and previous sequencing data indicate that the genes entCEB(G)A are transcribed as unit from a promoter upstream of entC.