Evolution of metabolic divergence in <i>Pseudomonas aeruginosa</i> during long-term infection facilitates a proto-cooperative interspecies interaction

The effect of polymicrobial interactions on pathogen physiology and how it can act either to limit pathogen colonization or to potentiate pathogen expansion and virulence are not well understood. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens commonly found together in polymicrobial human infections. However, we have previously shown that the interactions between these two bacterial species are strain dependent. Whereas P. aeruginosa PAO1, a commonly used laboratory strain, effectively suppressed S. aureus growth, we observed a commensal-like interaction between the human host-adapted strain, DK2-P2M24-2003, and S. aureus. In this study, characterization by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) imaging mass spectrometry (IMS) and mass spectral (MS) molecular networking revealed a significant metabolic divergence between P. aeruginosa PAO1 and DK2-P2M24-2003, which comprised several virulence factors and signaling 4-hydroxy-2-alkylquinoline (HAQ) molecules. Strikingly, a further modulation of the HAQ profile was observed in DK2-P2M24-2003 during interaction with S. aureus, resulting in an area with thickened colony morphology at the P. aeruginosa–S. aureus interface. In addition, we found an HAQ-mediated protection of S. aureus by DK2-P2M24-2003 from the killing effect of tobramycin. Our findings suggest a model where the metabolic divergence manifested in human host-adapted P. aeruginosa is further modulated during interaction with S. aureus and facilitate a proto-cooperative P. aeruginosa–S. aureus relationship

Importance of the evolutionarily conserved glycine residue in the N-terminal region of cystatin C (Gly-11) for cysteine endopeptidase inhibition

Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by site-directed mutagenesis and expression in Escherichia coli. The five variants were isolated and structurally verified. Their inhibitory properties were compared with those of wild-type recombinant cystatin C by determination of the equilibrium constants for dissociation (Ki) of their complexes with the cysteine endopeptidases papain and human cathepsin B and with the cysteine exopeptidase dipeptidyl peptidase I. The Ser-11 and Ala-11 cystatin C variants displayed Ki values for the two endopeptidases that were approx. 20-fold higher than those of wild-type cystatin C, while the corresponding values for the Trp-11. Arg-11 and Glu-11 variants were increased by a factor of about 2000. In contrast, the Ki values for the interactions of all five variants with the exopeptidase differed from that of wild-type cystatin C by a factor of less than 10. Wild-type cystatin C and the Ser-11, Ala-11 and Glu-11 variants were incubated with neutrophil elastase, which in all cases resulted in the rapid hydrolysis of a single peptide bond, between amino acid residues 10 and 11. The Ki values for the interactions with papain of these three N-terminal-decapeptide-lacking cystatin C variants were 20-50 nM, just one order of magnitude higher than the value for N-terminally truncated wild-type cystatin C, which in turn was similar to the corresponding values for the full-length Glu-11, Arg-11 and Trp-11 variants. These data indicate that the crucial feature of the conserved Gly residue in position 11 of wild-type cystatin C is that this residue, devoid of a side-chain, will allow the N-terminal segment of cystatin C to adopt a conformation suitable for interaction with the substrate-binding pockets of cysteine endopeptidases, resulting in high-affinity binding and efficient inhibition. The functional properties of the remaining part of the proteinase contact area, which is built from more C-terminal inhibitor segments, are not significantly affected even when amino acids with bulky or charged side-chains replace the Gly-11 residue of the N-terminal segment

High-level expression of active human cystatin C in Escherichia coli

Expression of the human cysteine proteinase inhibitor, cystatin C (CysC) in the cytoplasm of Escherichia coli was studied using a cDNA fragment encoding the cysteine proteinase inhibitor controlled by the phage λ pImage /cI857 system. The yield of CysC was low, probably due to proteolytic degradation. By fusing the cysC cDNA to a DNA fragment encoding the signal peptide of the E. coli outer membrane protein A, it was possible to produce a substantial amount of CysC in the periplasm. The processing of the signal peptide was shown to be quantitative and to result in CysC with the correct N-terminal amino acid. Yields higher than 1000 μg CysC/ml can be obtained by initiating the product formation at a moderate temperature (40 °C) late in an optimized fermentation process. A method that gives selective extraction of the periplasmic proteins and at the same time stabilizes CysC has been used

Human cystatin D: cDNA cloning, characterization of the E. coli expressed inhibitor, and identification of the native protein in saliva

A cDNA coding for cystatin D, a human member of the cystatin protein family, has been cloned after specific amplification of reverse- transcribed parotid gland RNA. After replacing the segment encoding the putative 20-residue signal peptide with one encoding the Escherichia coli OmpA leader sequence, the cDNA was expressed in E. coli. The isolated recombinant protein exhibited Ki values of 1.2 nM and > 1 microM for papain and cathepsin B, respectively. An antiserum raised against recombinant cystatin D recognized a protein in human saliva with electrophoretical mobility identical to that of the recombinant protein. Immunoenzymatic analysis revealed that this cysteine proteinase inhibitor is present in human saliva and tears at concentrations of 3.8 and 0.5 mg/liter, respectively, while it was not detected in seminal plasma, blood plasma, milk, or cerebrospinal fluid. Cystatin D purified from human saliva by immunosorption displayed a heterogeneous N-terminal end, with sequences starting at residues 5, 7, 9, and 11 of the predicted N-terminal portion of the mature protein. On the basis of structural and functional properties, cystatin D represents a novel cysteine proteinase inhibitor possibly playing a protective role against proteinases present in the oral cavity

An enzyme with rhamnogalacturonase activity

An enzyme exhibiting rhamnogalacturonase activity, which enzyme: a) is encoded by the DNA sequence shown in SEQ ID No. 1 or a sequence homologous thereto encoding a polypeptide with RGase activity, b) has the amino acid sequence shown in SEQ ID No. 2 or an analogue thereof, c) is reactive with an antibody raised against the enzyme encoded by the DNA sequence shown in SEQ ID No. 1, d) has a pH optimum above pH 5, and/or e) has a relative activity of at least 30% at a pH in the range of 5.5-6.5. The enzyme may be used for reducing the viscosity of a plant cell wall material and for extracting high molecular weight molecules from a plant cell wall material.<br/