Denis Tielker

Structural Basis of Carbohydrate Recognition by the Lectin LecB from Pseudomonas aeruginosa

The crystal structure of Pseudomonas aeruginosa fucose-specific lectin LecB was determined in its metal-bound and metal-free state as well as in complex with fucose, mannose and fructopyranose. All three monosaccharides bind isosterically via direct interactions with two calcium ions as well as direct hydrogen bonds with several side-chains. The higher affinity for fucose is explained by the details of the binding site around C6 and O1 of fucose. In the mannose and fructose complexes, a carboxylate oxygen atom and one or two hydroxyl groups are partly shielded from solvent upon sugar binding, preventing them from completely fulfilling their hydrogen bonding potential. In the fucose complex, no such defects are observed. Instead, C6 makes favourable interactions with a small hydrophobic patch. Upon demetallization, the C terminus as well as the otherwise rigid metal-binding loop become more mobile and adopt multiple conformations.

Responses to hypoxia in fungal pathogens

Hypoxia is typical for most battlefields of host–pathogen interactions in the human host. While adaptation of human cells to low levels of oxygen has been well established, little information exists on mechanisms of hypoxic adaptation in microbial pathogens. Importantly, the impact of hypoxia on microbial infection, virulence and pathogenesis is rarely investigated. Recent results on the human pathogens Candida albicans and Cryptococcus neoformans indicate that these fungi adapt to hypoxia specifically by altering several morphological phenotypes, metabolic and transcriptomal activities, as well as virulence traits. In this review, novel components and mechanisms involved in hypoxic adaptation of human fungals pathogens are summarized and discussed.

Msb2 Shedding Protects Candida albicans against Antimicrobial Peptides

Msb2 is a sensor protein in the plasma membrane of fungi. In the human fungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinase pathway to maintain cell wall integrity and allow filamentous growth. Msb2 doubly epitope-tagged in its large extracellular and small cytoplasmic domain was efficiently cleaved during liquid and surface growth and the extracellular domain was almost quantitatively released into the growth medium. Msb2 cleavage was independent of proteases Sap9, Sap10 and Kex2. Secreted Msb2 was highly O-glycosylated by protein mannosyltransferases including Pmt1 resulting in an apparent molecular mass of >400 kDa. Deletion analyses revealed that the transmembrane region is required for Msb2 function, while the large N-terminal and the small cytoplasmic region function to downregulate Msb2 signaling or, respectively, allow its induction by tunicamycin. Purified extracellular Msb2 domain protected fungal and bacterial cells effectively from antimicrobial peptides (AMPs) histatin-5 and LL-37. AMP inactivation was not due to degradation but depended on the quantity and length of the Msb2 glycofragment. C. albicans msb2 mutants were supersensitive to LL-37 but not histatin-5, suggesting that secreted rather than cell-associated Msb2 determines AMP protection. Thus, in addition to its sensor function Msb2 has a second activity because shedding of its glycofragment generates AMP quorum resistance.

Pseudomonas aeruginosa lectins I and II and their interaction with human airway cilia

The bacterium Pseudomonas aeruginosa (PA) produces two carbohydrate binding lectins, designated PA lectin-I and lectin-II (PA-IL, PA-IIL). Both lectins are used by the bacterium to adhere to the glycocalyx of mammalian cells. In addition, the lectins immobilize ciliary beat. The kinetics of ciliary beat inhibition by each individual lectin have been analysed; however, their joint action on cilia has not been reported. Here we demonstrate that PA-IL and PA-IIL inhibit ciliary beat in a similar time-dependent manner. If applied simultaneously, ciliary beat inhibition after five hours of incubation was weaker than if each lectin was applied separately. Thus it can be hypothesized that the lectins compete for the same binding site(s) of the glycocalyx. Sugar inhibition experiments demonstrate that D-galactose and L-fucose inhibit both lectins, although clear preferences of D-galactose for PA-IL and of L-fucose for PA-IIL exist. These interactions have to be kept in mind when designing sugar-based therapies.

Growth-dependent secretome of Candida utilis

Recently, the food yeast Candida utilis has emerged as an excellent host for production of heterologous proteins. Since secretion of the recombinant product is advantageous for its purification, we characterized the secreted proteome of C. utilis. Cells were cultivated to the exponential or stationary growth phase, and the proteins in the medium were identified by MS. In parallel, a draft genome sequence of C. utilis strain DSM 2361 was determined by massively parallel sequencing. Comparisons of protein and coding sequences established that C. utilis is not a member of the CUG clade of Candida species. In total, we identified 37 proteins in the culture solution, 17 of which were exclusively present in the stationary phase, whereas three proteins were specific to the exponential growth phase. Identified proteins represented mostly carbohydrate-active enzymes associated with cell wall organization, while no proteolytic enzymes and only a few cytoplasmic proteins were detected. Remarkably, cultivation in xylose-based medium generated a protein pattern that diverged significantly from glucose-grown cells, containing the invertase Inv1 as the major extracellular protein, particularly in its highly glycosylated S-form (slow-migrating). Furthermore, cultivation without ammonium sulfate induced the secretion of the asparaginase Asp3. Comparisons of the secretome of C. utilis with those of Kluyveromyces lactis and Pichia pastoris, as well as with those of the human fungal pathogens Candida albicans and Candida glabrata, revealed a conserved set of 10 and six secretory proteins, respectively.

Lipase LipC affects motility, biofilm formation and rhamnolipid production in Pseudomonas aeruginosa

Pseudomonas aeruginosa produces and secretes several lipolytic enzymes, among them the lipases LipA and LipC. LipA is encoded within the lipA/lipH operon, together with its cognate foldase LipH, which was also found to be required for the functional expression of LipC. At present, the physiological function of LipC is unknown. We have cloned a synthetic operon consisting of the lipC structural gene and the foldase gene lipH obtained from the lipA/lipH operon and have constructed, in parallel, a lipC-deficient P. aeruginosa mutant. Inactivation of the lipC gene significantly impaired type IV pilus-dependent twitching and swarming motility, but also the flagella-mediated swimming motility of P. aeruginosa. Moreover, for the lipC mutant, we observed a significant decrease in the amount of extracellular rhamnolipids. Also, the P. aeruginosa lipC mutant showed a significantly altered biofilm architecture. Proteome analysis revealed the accumulation of the response regulator protein PhoP in the lipC mutant.

Lectin-based affinity tag for one-step protein purification

The production of pure protein is indispensable for many applications in life sciences, however protein purification protocols are difficult to establish, and the experimental procedures are usually tedious and time-consuming. Therefore, a number of tags were developed to which proteins of interest can be fused and subsequently purified by affinity chromatography. We report here on a novel lectin-based affinity tag using the D-mannose-specific lectin LecB from Pseudomonas aeruginosa. A fusion protein was constructed consisting of yellow fluorescent protein and LecB separated by an enterokinase cleavage site. This protein was overexpressed in Escherichia coli Tuner (DE3), and the cell extract was loaded onto a column containing a mannose agarose matrix. Electrophoretically pure fusion protein at a yield of 24 mg/L culture was eluted with a D-mannose containing buffer The determination of equilibrium adsorption isotherms revealed an association constant of the lectin to the mannose agarose matrix of Ka = 3.26 x 10(5)/M. Enterokinase treatment of the purified fusion protein resulted in the complete removal of the LecB-tag. In conclusion, our results indicate that the lectin LecB of P. aeruginosa can be used as a tag for the high-yield one-step purification of recombinant proteins.

Heterologous protein secretion by Candida utilis

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.

A temperature-induced narrow DNA curvature range sustains the maximum activity of a bacterial promoter in vitro.

Among the molecular strategies bacteria have set up to quickly match their transcriptional program to new environments, changes in sequence-mediated DNA curvature play a crucial role. Bacterial promoters, especially those of mesophilic bacteria, are in general preceded by a curved region. The marked thermosensitivity of curved DNA stretches allows bacteria to rapidly sense outer temperature variations and affects transcription by favoring the binding of activators or repressors. Curved DNA is also able to influence the transcriptional activity of a bacterial promoter directly, without the involvement of trans-acting regulators. This study attempts to quantitatively analyze the role of DNA curvature in thermoregulated gene expression using a real-time in vitro transcription model system based on a specific fluorescence molecular beacon. By analyzing the temperature-dependent expression of a reporter gene in a construct carrying a progressively decreasing bent sequence upstream from the promoter, we show that with a decrease in temperature a narrow curvature range accounts for a significant enhancement of promoter activity. This strengthens the view that DNA curvature-mediated regulation of gene expression is likely a strategy offering fine-tuning control possibilities and that, considering the widespread presence of curved sequences upstream from bacterial promoters, it may represent one of the most primitive forms of gene regulation.

Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications

The yeast Candida utilis is used as a food additive and as a host for heterologous gene expression to produce various metabolites and proteins. Reliable protocols for intracellular production of recombinant proteins are available for C. utilis and have now been expanded to secrete proteins into the growth medium or to achieve surface display by linkage to a cell wall protein. A recombinant C. utilis strain was recently shown to induce oral tolerance in a mouse model of multiple sclerosis suggesting future applications in autoimmune therapy. Whole genome sequencing of C. utilis and its presumed parent Cyberlindnera (Pichia) jadinii demonstrated different ploidy but high sequence identity, consistent with identical recombinant technologies for both yeasts. C. jadinii was recently described as an antagonist to the important human fungal pathogen Candida albicans suggesting its use as a probiotic agent. The review summarizes the status of recombinant protein production in C. utilis, as well as current and future biotechnological and medical applications of C. utilis and C. jadinii.