Comparison of Raman spectroscopy and two molecular diagnostic methods for Burkholderia cepacia complex species identification.

OBJECTIVES: Burkholderia cepacia complex (Bcc) and Pseudomonas aeruginosa strains, colonize the respiratory tract of cyctic fibrosis patients. These strains are phenotypically difficult to discriminate, but differ greatly in their pathogenic potential and species identification is relevant. Here, three methods were compared for their diagnostic capacity. METHODS: A Bcc collection was analyzed with Raman spectroscopy, AFLP and rep-PCR analysis. RESULTS: Raman spectroscopy of 40 strains revealed high similarity. Rep-PCR and AFLP of respectively 96 and 112 strains revealed that Bcc strains could be distinguished from Pseudomonas strains. Both molecular methods allowed the identification of most Bcc species according to previous phenotypic and molecular characterization. CONCLUSION: Both AFLP and rep-PCR method data correspond with the previously reported species identification. However, Raman spectroscopy does not discriminate among P. aeruginosa and Bcc species and is therefore not useful as a diagnostic tool.

Detection of colon flora in peritoneal drain fluid after colorectal surgery: can RT-PCR play a role in diagnosing anastomotic leakage?

A semi-quantitative Real-Time PCR strategy was developed to identify potential indicator organisms for anastomotic leakage in peritoneal drainage fluid, Escherichia coli and Enterococcus faecalis. The analytical performance of the amplification method was validated with 10 culture-positive and 7 culture-negative peritoneal drain fluid samples, obtained from 9 different patients with a colorectal anastomosis. Real-Time PCR results were fully concordant with the microbiological culture results. However, among the culture-negative samples, four false-positive RT-PCR results were found. All false-positives originated from a single patient with a surgical site infection. This may indicate an elevated sensitivity of the RT-PCR method. The results showed that the semi-quantitative RT-PCR method has a clear potential to be useful as a powerful tool in early detection of anastomotic leakage.

Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein.

The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses. The phage display method allows for selection of human antibody fragments with potential use in clinical applications. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human Fab fragments recognising major vault protein we used a large non-immunized human Fab fragment phage library. Phages displaying major vault protein-reactive Fabs were obtained through several rounds of selection on major vault protein-coated immunotubes and subsequent amplification in TG1 E coli bacteria. Eventually, one major vault protein-reactive clone was selected and further examined. The anti-major vault protein Fab was found suitable for immunohistochemical and Western blot analysis of tumour cell lines and human tissues. BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs. The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies. Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms.

Guided selection of a pan carcinoma specific antibody reveals similar binding characteristics yet structural divergence between the original murine antibody and its human equivalent.

Antibody engineering provides an excellent tool for the generation of human immunotherapeutics for the targeted treatment of solid tumours. We have engineered and selected a completely human antibody to epithelial glycoprotein-2 (EGP-2), a transmembrane glycoprotein present on virtually all human simple epithelia and abundantly expressed on a variety of human carcinomas. We chose to use the procedure of "guided selection" to rebuild a high-affinity murine antibody into a human antibody, using two consecutive rounds of variable domain shuffling and phage library selection. As a starting antibody, the murine antibody MOC-31 was used. After the first round of guided selection, where the V(H) of MOC-31 was combined in Fab format with a human V(L)C(L) library, a small panel of human light chains was identified, originating from a segment of the VkappaIII family, whereas the MOC-31 V(L) is more homologous to the VkappaII family. Nevertheless, one of the chimaeric Fabs, C3, displayed an off-rate similar to MOC-31 scFv. Combining the V(L) of C3 with a human V(H) library, while retaining the V(H) CDR3 of MOC-31, clones were selected using human V(H) genes originating from the rarely used V(H)7 family. The best clone, 9E, shows over 13 amino acid mutations from the germline sequence, has an off-rate comparable to the original antibody and specifically binds to the "MOC-31"-epitope on EGP-2 in specificity and competition ELISA, FACS analysis and immunohistochemistry. In both V(L) and V(H) of antibody 9E, three germline mutations were found creating the MOC-31 homologue residue. Structural modelling of both murine and human antibodies reveals that one of the germline mutations, 53Y in V(H) CDR2, is likely to be involved in antigen binding. We conclude that, although they may bind the same epitope and have similar binding affinity to the antigen as the original murine antibody, human antibodies derived by guided selection unlike CDR-grafted antibodies, may retain only some of the original key elements of the binding site chemistry. The selected human anti-EGP-2 antibody will be a suitable reagent for tumour targeting.

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K.

The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 10(5). Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 10(2). With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol.

An affinity column for phospholipase A2 based on immobilised acylaminophospholipid analogues.

A synthetic route was developed to prepare 2-acylamino phospholipid analogues suitable for immobilisation. The inhibitors, synthesised in either the (R)- and (S)-configuration, carried an omega-carboxyl group in one acyl chain for immobilisation to the matrix. As a matrix Sepharose 6B, derivatised with a polar, non-charged 16 atom spacer was used. Low-molecular weight phospholipase A2 binds in a calcium-dependent way to the immobilised (S)-inhibitor and not to the immobilised (R)-inhibitor which shows that binding involves specific active site interactions rather than hydrophobic chromatography. The specificity was further demonstrated by the fact that the immobilised (S)-inhibitor binds porcine pancreatic and snake venom phospholipases A2, but not the porcine pancreatic zymogen. Moreover, a mutant porcine pancreatic phospholipase A2 in which the active side residue His48 has been replaced by Gln, was not bound by the column. This column material might be applicable for affinity purification of phospholipase A2 and for screening of phage display libraries.

Incorporation of an unnatural amino acid in the active site of porcine pancreatic phospholipase A2. Substitution of histidine by 1,2,4-triazole-3-alanine yields an enzyme with high activity at acidic pH.

The effect of the substitution of the active site histidine 48 by the unnatural 1,2,4-triazole-3-alanine (TAA) amino acid analogue in porcine pancreas phospholipase A2 (PLA2) was studied. TAA was introduced biosynthetically using a his-auxotrophic Escherichia coli strain. To study solely the effect of the substitution of the active site histidine, two nonessential histidines (i.e. His17 and His115) were replaced by asparagines, resulting in a fully active mutant enzyme (His-PLA2). In this His-PLA2 the single histidine as position 48 was substituted by TAA with an incorporation efficiency of about 90%, giving a mixture of His-PLA2 and TAA-PLA2. Based on the charge difference at acidic pH, both forms could be separated by FPLC, allowing for the purification of TAA-PLA2 free from His-PLA2. At pH 6, TAA-PLA2 has a fivefold reduced activity compared with His-Pla2. This reduced activity paralells a reduced rate of covalent modification with p-nitrophenacyl bromide of TAA-PLA2 compared with His-PLA2. Competitive inhibition gave comparable IC50 values for WT-PLA2, His-PLA2 and TAA-PLA2. These results indicate that the reduction in activity is not caused by a different affinity for the substrate, but more likely results from a reduced kcat value in TAA-PLA2. The enzymatic activities for native and mutant PLA2s were measured at different pH values. For WT-PLA2 and His-PLA2 the activity is optimal at pH 6 and is strongly deminished at acidic pH, with no observable activity at pH 3. In contrast, TAA-PLA2 is as active at pH 3 as at pH 6. Most likely, the decrease in activity observed for WT-PLA2 and His-PLA2 is caused by the protonation of the active site His48, which is the general base involved in the activation of the nucleophilic water molecule. In TAA-PLA2, however, the active site residue TAA48 is unprotonated at both pH 3 and 6 as a result of the low pKa of TAA compared with histidine.

An extended binding pocket determines the polar head group specificity of porcine pancreatic phospholipase A2.

Porcine pancreatic phospholipase A2 (PLA2) was studied by site-directed mutagenesis. Arg53 and/or Lys56 were replaced by a methionine (R53M or K56M, respectively) in combination with the Tyr69-->Phe (Y69F) substitution. These substitutions improved the activity on micellar and monomeric zwitterionic substrates and reduced the activity on negatively charged substrates compared to the Y69F mutant. With the neutral substrate 1,2-didodecanoyl-sn-glycerol-3-dimethyl phosphate (Lau2GroMe2P) a 20-fold increase of activity was observed for the 69F53M56M mutant, whereas this mutant showed a lower activity than native PLA2 on zwitterionic substrates. Thus the ratio Lau2GroMe2P/Lau2GroPCho has become 65 times higher for 69F53M56M compared to native phospholipase A2, illustrating that the substrate specificity has changed enormously. The methionine substitutions were also prepared in a 69F mutant in which a part of the surface loop (residues 62-66) was deleted. Also in this deletion mutant these substitutions showed a similar effect as the substitutions in the native 69F mutant. Furthermore it was shown that deletion of the loop increases the activity on micellar lecithins and negatively charged micellar substrates, but reduces the activity on Lau2GroMe2P. Therefore it can be concluded that the loop is important for the recognition of substrates. We also show that the loop plays a role in the dimerization of these proteins. Dimerization may account for the high activities observed for some mutants acting on monomeric substrate.

Structural elements of secretory phospholipases A2 involved in the binding to M-type receptors.

Specific membrane receptors for secretory phospholipases A2 (sPLA2s) have been initially identified with novel snake venom sPLA2s called OS1 and OS2. One of these sPLA2 receptors (muscle (M)-type, 180 kDa) has a very high affinity for OS1 and OS2 and a high affinity for pancreatic and inflammatory-type mammalian sPLA2s, which might be the natural endogenous ligands of PLA2 receptors. Primary structures of OS1 and OS2 were determined and compared with sequences of other sPLA2s that bind less tightly or do not bind to the M-type receptor. In addition, the binding properties of pancreatic sPLA2 mutants to the M-type receptor have been analyzed. Residues within or close to the Ca(2+)-binding loop of pancreatic sPLA2 are crucially involved in the binding step, although the presence of Ca2+ that is essential for the enzymatic activity is not required for binding to the receptor. These residues include Gly-30 and Asp-49, which are conserved in all sPLA2s. Leu-31 is also essential for binding of pancreatic sPLA2 to its receptor. Many other mutations have been considered. Those occurring in the N-terminal alpha helices and the pancreatic loop do not change binding to the M-type receptor. Conversion of pancreatic prophospholipase to phospholipase is essential for the acquisition of binding properties to the M-type receptor.