Cloning and characterization of M.LmoA118I, a novel DNA:m4C methyltransferase from the Listeria monocytogenes phage A118, a close homolog of M.NgoMXV.

A homolog of M.NgoMXV DNA:m4C methyltransferase has been identified among the open reading frames deduced from the genomic sequence of Listeria monocytogenes phage A118 [Loessner et al., 2000]. The gene coding for this putative protein has been cloned in Escherichia coli and its enzymatic activity in vivo in this host have been analyzed. Remarkably, despite M.NgoMXV and M.LmoA118I exhibit high sequence similarity (58% identical and 19% conservatively substituted residues), their target preferences differ: both proteins exhibit "relaxed" sequence specificity, but while M.LmoA118I more efficiently methylates GGCC sites, it seems to target only a subset of CCWGG sites methylated by M.NgoMXV.     

Structure and dynamics of the alpha-lactalbumin molten globule: fluorescence studies using proteins containing a single tryptophan residue

The fluorescence properties of three variants of alpha-lactalbumin (alpha-LA) containing a single tryptophan residue were investigated under native, molten globule, and unfolded conditions. These proteins have levels of secondary structure and stability similar to those of the wild type. The fluorescence signal in the native state is dominated by that of W104, with the signal of W60 and W118 significantly quenched by the disulfide bonds in their vicinity. In the molten globule state, the magnitude of the fluorescence signal of W60 and W118 increases, due to the loss of rigid, specific side chain packing. In contrast, the magnitude of the signal of W104 decreases in the molten globule state, perhaps due to the protonation of H107 or quenching by D102 or K108. The solvent accessibilities of individual tryptophan residues were investigated by their fluorescence emission maximum and by acrylamide quenching studies. In the native state, the order of solvent accessibility is as follows: W118 > W60 > W104. This order changes to W60 > W104 > W118 in the molten globule state. Remarkably, the solvent accessibility of W118 in the alpha-LA molten globule is lower than that in the native state. The dynamic properties of the three tryptophan residues were examined by time-resolved fluorescence anisotropy decay studies. The overall rotation of the molecule can be observed in both the native and molten globule states. In the molten globule state, there is an increase in the extent of local backbone fluctuations with respect to the native state. However, the fluctuation is not sufficient to result in complete motional averaging. The three tryptophan residues in the native and molten globule states have different degrees of motional freedom, reflecting the folding pattern and dynamic heterogeneity of these states. Taken together, these studies provide new insight into the structure and dynamics of the alpha-LA molten globule, which serves as a prototype for partially folded proteins.     

C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates

Listeria monocytogenes phage endolysins Ply118 and Ply500 share a unique enzymatic activity and specifically hydrolyse Listeria cells at the completion of virus multiplication in order to release progeny phage. With the aim of determining the molecular basis for the lytic specificity of these enzymes, we have elucidated their domain structure and examined the function of their unrelated and unique C-terminal cell wall binding domains (CBDs). Analysis of deletion mutants showed that both domains are needed for lytic activity. Fusions of CBDs with green fluorescent protein (GFP) demonstrated that the C-terminal 140 amino acids of Ply500 and the C-terminal 182 residues of Ply118 are necessary and sufficient to direct the murein hydrolases to the bacterial cell wall. CBD500 was able to target GFP to the surface of Listeria cells belonging to serovar groups 4, 5 and 6, resulting in an even staining of the entire cell surface. In contrast, the CBD118 hybrid bound to a ligand predominantly present at septal regions and cell poles, but only on cells of serovars 1/2, 3 and 7. Non-covalent binding to surface carbohydrate ligands occurred in a rapid, saturation-dependent manner. We measured 4 x 104 and 8 x 104 binding sites for CBD118 and CBD500 respectively. Surface plasmon resonance analysis revealed unexpected high molecular affinity constants for the CBD-ligand interactions, corresponding to nanomolar affinities. In conclusion, we show that the CBDs are responsible for targeting the phage endolysins to their substrates and function to confer recognition specificity on the proteins. As the CBD sequences contain no repeats and lack all known sequence motifs for anchoring of proteins to the bacterial cell, we conclude that they use unique structural motifs for specific association with the surface of Gram-positive bacteria.     

Modified Listeria bacteriophage lysin genes (ply) allow efficient overexpression and one-step purification of biochemically active fusion proteins.

Listeria bacteriophage lytic enzymes are useful for in vitro applications such as rapid, gentle cell disruption, and they provide new approaches as selective antimicrobial agents for destruction of Listeria monocytogenes in contaminated foods. We describe here the amino-terminal modification of three cloned Listeria phage lysin genes (ply), resulting in fusion proteins with a 12-amino-acid leader containing six consecutive histidine residues. The recombinant enzymes retain their native specific activity and can be efficiently overproduced in Escherichia coli. By one-step metal chelate affinity chromatography, active lysins could be purified to more than 90% homogeneity.     

Biochemical characterization of the component parts of intestinal mucin from patients with cystic fibrosis.

Previous studies have shown that human small-intestinal mucin consists of high-Mr glycoproteins and a smaller S-S-bonded protein of 118 kDa. The major antigenic determinants of the mucin were associated with the large glycoproteins, but depended for stability on intact disulphide bonds, and were destroyed by digestion with Pronase. In the present study we isolated and analysed the component parts of mucin from patients with cystic fibrosis with special attention being paid to the peptide constituents. After reduction with 0.2 M-beta-mercaptoethanol [5 min, 100 degrees C in 1% SDS (sodium dodecyl sulphate)], the large glycoproteins and smaller peptide with an apparent molecular size of 118 kDa were separated by equilibrium density-gradient centrifugation in CsCl, Sepharose 4B chromatography or preparative SDS/polyacrylamide-gel electrophoresis. The large glycoproteins contained about 70% of the protein of the native mucin. Digestion with Pronase resulted in a further loss of 'naked' protein (10% of the native mucin protein) from the C-terminal end of the glycoprotein peptide core, and left behind highly glycosylated proteins comprised mainly (70 mol%) of threonine, serine and proline. The 118 kDa component, which contained about 30% of the native mucin protein, consisted mainly of aspartic acid, serine, glutamic acid and glycine (40 mol%), plus threonine, proline, alanine, valine and leucine (35 mol%). Together with the 'naked' protein segment, the 118 kDa component contained most of the cysteine residues of the native mucin. Surprisingly, the peptide also contained carbohydrate (less than or equal to 5% of the native mucin carbohydrate but 50% by weight of the 118 kDa component), which included 9 mol% mannose, suggesting the presence of N-linked oligosaccharides. The peptide exhibited strong non-covalent interactions with the high-Mr glycoproteins and a tendency to self-aggregate in the absence of dissociating agents. Our findings therefore suggest that native mucin consists of large glycoproteins capable of forming disulphide bridges from their C-terminal 'naked' (antigenic) regions to a smaller glycopeptide having an Mr of 118 000.     

RPK118, a PX domain-containing protein, interacts with peroxiredoxin-3 through pseudo-kinase domains

RPK118 is a sphingosine kinase-1-binding protein that has been implicated in sphingosine 1 phosphate-mediated signaling. It contains a PX (phox homology) domain and two pseudo-kinase domains, and co-localizes with sphingosine kinase-1 on early endosomes. In this study we identified a novel RPK118-binding protein, PRDX3 (peroxiredoxin-3), by yeast two-hybrid screening. The interaction between these proteins was confirmed by pull-down assays and co-immunoprecipitation experiments. Deletion studies showed that RPK118 interacted with PRDX3 through its pseudokinase domains, and with early endosomes through its PX domain. Double immunofluorescence experiments demonstrated that PRDX3 co-localized with RPK118 on early endosomes in COS7 cells. PRDX3 is a member of the antioxidant family of proteins synthesized in the cytoplasm and functioning in mitochondria. Our findings indicate that RPK118 is a PRDX3-binding protein that may be involved in transporting PRDX3 from the cytoplasm to its mitochondrial site of function or to other membrane structures via endosome trafficking.     

Functional regulation of the Listeria monocytogenes bacteriophage A118 holin by an intragenic inhibitor lacking the first transmembrane domain

We have dissected the functional properties of the holin encoded by Listeria monocytogenes bacteriophage A118. Native hol118 was cloned into lambdaDeltaSthf, devoid of the S holin, and tested in an E. coli background. Surprisingly, it caused very late cell lysis, beginning at 80 min after induction. Immunological analyses demonstrated that Hol118 appears in the cytoplasmic membrane shortly after infection. The hol118 gene features a dual start motif similar to lambda S. Therefore, different N-terminally modified Hol118 variants were tested. However, in contrast to lambda S, inactivation of AUG-1 or AUG-2 showed no significant influence on lysis timing. In addition, Hol118-mediated lysis could not be triggered by energy poisons, indicating a functional regulation different from that of S. Toeprinting assays on hol118 mRNA revealed an unexpected translational start codon (AUG-3) at nucleotide position 40. We demonstrated by in vitro and in vivo approaches that the predicted Hol118(83) product is actually produced together with the full-length polypeptide. However, although the truncated holin lacking its first transmembrane domain appeared in the cytoplasmic membrane, it was shown to be functionally deficient and unable to support lambda R-mediated lysis. In contrast, specific mutations introduced to abolish translation initiation at AUG-3 drastically accelerated lysis, pointing to an inhibitor function of Hol118(83). This hypothesis was supported by the observation that hol118(83) inhibited holin function when expressed in trans. A deviation from the lambda S paradigm is proposed, which represents a new model of holin functional regulation: the intragenic, in frame translated Hol118(83) product, which is devoid of its first transmembrane domain, acts as a functional inhibitor and constitutes a key part of the lysis clock of A118. Presence of the dominant inhibitor function also explains the long latent period of A118, where the onset of lysis takes about 70 min, more than twice the time needed by lambda.     

Transposon-induced mutations in two loci of Listeria monocytogenes serotype 1/2a result in phage resistance and lack of N-acetylglucosamine in the teichoic acid of the cell wall.

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916DeltaE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins.

All species of the genus Listeria secrete a major extracellular protein called p60. A comparison of the deduced amino acid sequences of all listerial p60 proteins previously indicated there were only a few regions which were unique to the pathogenic, food-borne species Listeria monocytogenes. Two of these p60 regions were chosen for the development of antibodies specific for the facultative intracellular species L. monocytogenes. Initially, these regions were characterized via epitope mapping, and this led to the development of two different synthetic peptides. Rabbits immunized with these synthetic peptides generated polyclonal antibodies that were then used in Western blot (immunoblot) analyses. Antiserum against peptide A (PepA) recognized the p60 protein in the supernatants collected from most L. monocytogenes serotypes except for several strains belonging to serotypes 4a and 4c. No p60-related protein was detected in the supernatants from other Listeria species with this anti-PepA antiserum. Antibodies raised against peptide D (PepD) reacted with p60 from all L. monocytogenes serotypes, including all 4a and 4c strains that were tested, and also showed no cross-reactivity with supernatant proteins from other Listeria species. Both antisera also detected p60 in supernatants of a large number of environmental isolates of L. monocytogenes. Besides Western blot analyses, these antisera to PepA and PepD reacted with secreted p60 in an enzyme-linked immunosorbent assay, indicating recognition of the native antigen in addition to the denatured form. These data suggest that synthetic peptides derived from the variable region of the L. monocytogenes p60 protein may be useful for the development of an immunological diagnostic assay.     

In silico analysis of structural and functional consequences in p16INK4A by deleterious nsSNPs associated CDKN2A gene in malignant melanoma

In this study, we identified the most deleterious nsSNP in CDKN2A gene through structural and functional properties of its protein (p16INK4A) and investigated its binding affinity with cdk6. Out of 118 SNPs, 14 are nsSNPs in the coding region and 17 SNPs were found in the untranslated region (UTR). FastSNP suggested that 7 SNPs in the 5' UTR might change the protein expression levels. Sixty-four percent of nsSNPs are found to be damaged in PolyPhen server among the 14 nsSNPs investigated. With this effort, we modeled the mutant p16INK4A proteins based on these deleterious nsSNPs, out of which three nsSNPs associated p16INK4A had RMSD values of greater than 3.00 A with native protein. From a comparison of total energy of these three mutant proteins, we identified that the major mutation is from Aspartic acid to Tyrosine at the residue position of 84 of p16INK4A. Further, we compared the binding efficiency of both native and mutant p16INK4A with cdk6. We found that mutant p16INK4A has less binding affinity with cdk6 compared to native type. This is due to ten hydrogen bonds and eight salt bridges which exist between the native type and cdk6, whereas the mutant type makes only nine hydrogen bonds and five salt bridges with cdk6. Based on our investigation, we propose that the SNP with the ID rs11552822 could be the most deleterious nsSNP in CDKN2A gene, causing malignant melanoma, as it was well correlated with experimental studies carried out elsewhere.     

Characterization of MyD118, Gadd45, and proliferating cell nuclear antigen (PCNA) interacting domains. PCNA impedes MyD118 AND Gadd45-mediated negative growth control

MyD118 and Gadd45 are related genes encoding for proteins that play important roles in negative growth control, including growth suppression and apoptosis. MyD118 and Gadd45 are related proteins that previously were shown to interact with proliferating cell nuclear antigen (PCNA), implicated in DNA replication, DNA repair, and cell cycle progression. To establish the role of MyD118 and Gadd45 interactions with PCNA, in this work we sought to identify the interacting domains and analyze the significance of this interaction in negative growth control. Using complementary in vivo and in vitro interaction assays the N-terminal (1-46) and middle (100-127) regions of PCNA were identified as harboring MyD118- and Gadd45 interacting domains, whereas PCNA interacting domains within MyD118 and Gadd45 were localized to the C termini of these proteins (amino acids 114-156 and 137-165, respectively). These findings provide first evidence that similar domains within MyD118 and Gadd45 mediate interactions with PCNA. Importantly, ectopic expression of MyD118 or Gadd45 N-terminal peptides, lacking the PCNA interacting domain, was found to suppress colony formation or induce apoptosis more efficiently than the full-length proteins. These findings suggest that interaction of MyD118 or Gadd45 with PCNA, in essence, serves to impede negative growth control.     

Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell to cell

Cell-to-cell spread is a fundamental step in the infection cycle of Listeria monocytogenes that strictly depends on the formation of bacteria-induced protrusions. Since Listeria actin tails in the protrusions are tightly associated with the plasma membrane, we hypothesised that membrane-cytoskeleton linkers would be required for initiating and sustaining their formation and the subsequent cell-to-cell spread. We have found that ezrin, a member of the ezrin, radixin and moesin (ERM) family that functions as a key membrane-cytoskeleton linker, accumulates at Listeria protrusions. The ability of Listeria to induce protrusions and effectively spread between adjacent cells depends on the interaction of ERM proteins with both a membrane component such as CD44 and actin filaments. Interfering with either of these interactions or with ERM proteins phosphorylation not only reduces the number of protrusions but also alters their morphology, resulting in the formation of short and collapsed protrusions. As a consequence, Listeria cell-to-cell spread is severely impaired. Thus, ERM proteins are exploited by Listeria to escape the host immune response and to succeed in the development of the infection.     

Listeria monocytogenes, a unique model in infection biology: an overview

This review rather than covering the whole field intends to highlight recent findings on the Listeria monocytogenes infectious process or some Listeria specific traits, place them within the framework of well-established data, and demonstrate how this Gram-positive bacterium has, in two decades, emerged as a multifaceted paradigm. Indeed, the cell biology of the infectious process has been deciphered in great detail and provided insights in both the way bacterial pathogen manipulate the host and unsuspected functions of well-known cellular proteins. The intra- and intercellular motility has in particular been instrumental in understanding actin-based motility in general. The analysis of the two main Listeria invasion proteins and that of their host specificities have illustrated how in vitro studies can help generating or choosing relevant animal models for in vivo studies. Listeria post-genomics studies have highlighted the power of comparative genomics in virulence studies. Together, Listeria, after being recognized as a powerful tool in immunology, now appears as one of the most insightful models in infection biology.     

Extremely fast hydrogen exchange of ribonuclease-(1-118) as compared with native RNase A and its implication for the conformational energy state

RNase-(1-118) containing native disulfide bonds is similar in fold to native RNase A but not of lowest Gibbs energy as compared with the isomers containing non-native disulfide bonds. The present n.m.r. studies have indicated a dramatic increase in the exchange rate of all of the 'protected' amide protons of RNase-(1-118) over RNase A. A calculation shows a large increase in the rate of 'opening' of the structure. The exchange rate of the protected amide protons of RNase-(1-120) is slower than RNase-(1-118) but much faster than RNase A. Binding with a synthetic complementing fragment (114-124) markedly reduces the exchange rate of 20 to 25 amide protons of RNase-(1-118). It has previously been shown that binding with a complementing fragment of RNase-(1-118) generates a lowest Gibbs energy state. Thus, using available thermodynamic information for interpretation, we suggest that a) removal of six carboxy terminal residues of RNase A would disrupt coupling between these residues and those distant in the structure (loss of extra stabilizing energy), b) this would, in turn, alter the enthalpy-entropy compensation in such a way that the magnitude of Gibbs energy change favoring folding is significantly reduced without a large change of fold and c) in this activated state the molecule would be highly motile.     

The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity.

Expression of the iap gene of Listeria monocytogenes in the L. monocytogenes rough mutant RIII and in Bacillus subtilis DB104 caused the disruption of the cell chains which these two strains normally form under exponential growth conditions. The p60 protein produced by L. monocytogenes and B. subtilis DB104 also exhibited bacteriolytic activity detected in denaturing polyacrylamide gels containing heat-killed Micrococcus lysodeikticus. Purification of the p60 protein led to aggregation of p60 and loss of the cell chain disruption and bacteriolytic activities. A cysteine residue in the C-terminal part of p60 which is conserved in all p60-like proteins from the other Listeria species seems to be essential for both activities. The iap gene could not be inactivated without a loss of cell viability, indicating that p60 is an essential housekeeping protein for L. monocytogenes and probably also for other Listeria species. These data suggest that p60 possesses a murein hydrolase activity required for a late step in cell division.     

Detection and prevention of protein aggregation before,during, and after purification

The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins.     

Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.