Identification and characterization of human archaemetzincin-1 and -2, two novel members of a family of metalloproteases widely distributed in Archaea

Systematic analysis of degradomes, the complete protease repertoires of organisms, has demonstrated the large and growing complexity of proteolytic systems operating in all cells and tissues. We report here the identification of two new human metalloproteases that have been called archaemetzincin-1 (AMZ1) and archaemetzincin-2 (AMZ2) to emphasize their close relationship to putative proteases predicted by bioinformatic analysis of archaeal genomes. Both human proteins contain a catalytic domain with a core motif (HEXXHXXGX3CX4CXMX17CXXC) that includes an archetypal zinc-binding site, the methionine residue characteristic of metzincins, and four conserved cysteine residues that are not present at the equivalent positions of other human metalloproteases. Analysis of genome sequence databases revealed that AMZs are widely distributed in Archaea and vertebrates and contribute to the defining of a new metalloprotease family that has been called archaemetzincin. However, AMZ-like sequences are absent in a number of model organisms from bacteria to nematodes. Phylogenetic analysis showed that these enzymes have undergone a complex evolutionary process involving a series of lateral gene transfer, gene loss, and genetic duplication events that have shaped this novel family of metalloproteases. Northern blot analysis showed that AMZ1 and AMZ2 exhibit distinct expression patterns in human tissues. AMZ1 is mainly detected in liver and heart whereas AMZ2 is predominantly expressed in testis and heart, although both are also detectable at lower levels in other tissues. Both human enzymes were produced in Escherichia coli, and the purified recombinant proteins hydrolyzed synthetic substrates and bioactive peptides, demonstrating that they are functional proteases. Finally, these activities were abolished by inhibitors of metalloproteases, providing further evidence that AMZs belong to this catalytic class of proteolytic enzymes.     

Bacterial phospholipases C.

A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.     

Bacterial phospholipases C.

A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.     

Analysis of a new family of widely distributed metal-independent alpha-mannosidases provides unique insight into the processing of N-linked glycans

The modification of N-glycans by α-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for α1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-α1,6-mannosidase activity consistent with specificity for N-linked glycans having their α1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substrate-bound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (α/α)(6)-fold, recognize and hydrolyze the α1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-α1,6-mannosidases.     

The use of clostridial spores for cancer treatment

Hypoxic/necrotic regions, absent in normal tissues, can be exploited to target tumours in cancer therapy using nonpathogenic strains of the bacterial genus Clostridium. Following administration of Clostridium spores to tumour-bearing organisms, these spores can only germinate within the hypoxic/necrotic regions of solid tumours, proving their exquisite selectivity. Low oxygen tension is a common feature of solid tumours, which may arise from the unique physiological environment, generated to a large extent by the abnormal tumour vasculature, and provides as such a niche for anaerobic bacteria. Some clostridia tested clearly showed innate oncolytic activity, but they could not completely eradicate the tumour. Recombinant clostridia producing prodrug-converting enzymes or cytokines resulted in the production of such proteins solely within the tumour, and where applicable, could convert the prodrug in a toxic compound. Moreover, in some cases, tumour eradication or tumour control could be observed. This review brings an overview of the relative successes and failures of the Clostridium-directed tumour therapy with both wild-type strains and strains producing proteins useful in antitumour therapy.     

Homology Modeling of Hemagglutinin/Protease [HA/P (vibriolysin)] from <Emphasis Type="Italic">Vibrio Cholerae</Emphasis>: Sequence Comparision,Residue Interactions and Molecular Mechanism

Vibrio cholerae produces a zinc-containing and calcium-stabilized soluble hemagglutinin/protease, which has been earlier shown to have the ability to cleave several physiologically important substrates including mucin, fibronectin and lactoferin. This study presents homology modeling of hemagglutinin/protease (vibriolysin) from Vibrio cholerae in the presence of inhibitor HPI [N-(1-carboxy-3-phenylpropyl)-phenylalanyl-alpha-aspargine]. The 3D structure was predicted based on its sequence homology with Pseudomonas aeruginosa elastase (PAE). Comparison of the 3D structures of PAE and HA/P reveals a remarkable similarity having a conserved α + β domain. The inhibitor shows similar binding features as seen in other metalloproteases of M4 peptidase family. The study also highlights the key catalytic residues as well as the residues at the S₁ and binding sub-sites. The similarities between the two proteins provide support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action. The fact that both enzymes are secreted as zinc-containing proteases, led us to further hypothesize that they may play similar role in pathogenesis.     

Site-2 proteases in prokaryotes: regulated intramembrane proteolysis expands to microbial pathogenesis

Regulated intramembrane proteolysis (RIP) is a widely distributed mechanism of signal transduction in which membrane-bound proteases cleave transmembrane domains of substrate proteins. The site-2 protease (S2P) class of RIP metalloproteases is present in most bacterial genomes but is generally of unknown function except for the well-characterized proteases RseP and SpoIVFB. In this review we will discuss the biochemical functions and physiologic roles of S2P proteases in bacteria and highlight recent data implicating S2P family members in host-pathogen interactions.     

Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in two processive endocellulases, Cel48A and Cel9B, both of which possess a novel modular architecture

The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B(2) (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.     

Protease inhibitors: synthesis of matrix metalloproteinase and bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions

Matrix metalloproteinase (MMP)/bacterial collagenase inhibitors incorporating 5-amino-2-mercapto-1,3,4-thiadiazole zinc binding functions are reported. A series of compounds was prepared by reaction of arylsulfonyl isocyanates or arylsulfonyl halides with phenylalanyl-alanine, followed by coupling with 5-amino-2-mercapto-1,3,4-thiadiazole in the presence of carbodiimides. These new compounds were assayed as inhibitors of human MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from the anaerobe Clostridium histolyticum (ChC). The new derivatives proved to be powerful inhibitors of these metalloproteases, with activities in the low micromolar range for some of the target enzymes, depending on the substitution pattern at the arylsulfonyl(ureido) moieties.     

Unique features of the sodC-encoded superoxide dismutase from Mycobacterium tuberculosis, a fully functional copper-containing enzyme lacking zinc in the active site

The sodC-encoded Mycobacterium tuberculosis superoxide dismutase (SOD) shows high sequence homology to other members of the copper/zinc-containing SOD family. Its three-dimensional structure is reported here, solved by x-ray crystallography at 1.63-A resolution. Metal analyses of the recombinant protein indicate that the native form of the enzyme lacks the zinc ion, which has a very important structural and functional role in all other known enzymes of this class. The absence of zinc within the active site is due to significant rearrangements in the zinc subloop, including deletion or mutation of the metal ligands His115 and His123. Nonetheless, the enzyme has a catalytic rate close to the diffusion limit; and unlike all other copper/zinc-containing SODs devoid of zinc, the geometry of the copper site is pH-independent. The protein shows a novel dimer interface characterized by a long and rigid loop, which confers structural stability to the enzyme. As the survival of bacterial pathogens within their host critically depends on their ability to recruit zinc in highly competitive environments, we propose that the observed structural rearrangements are required to build up a zinc-independent but fully active and stable copper-containing SOD.     

Thermophilic archaeal enzymes and applications in biocatalysis

Thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. They can be used as biocatalysts in the application of 'green chemistry'. The thermophilic archaea contain enzymes that have already been used in commercial applications such as the L-aminoacylase from Thermococcus litoralis for the resolution of amino acids and amino acid analogues. This enzyme differs from bacterial L-aminoacylases and has similarities to carboxypeptidases from other archaeal species. An amidase/γ-lactamase from Sulfolobus solfataricus has been used for the production of optically pure γ-lactam, the building block for antiviral carbocyclic nucleotides. This enzyme has similarities to the bacterial signature amidase family. An alcohol dehydrogenase from Aeropyrum pernix has been used for the production of optically pure alcohols and is related to the zinc-containing eukaryotic alcohol dehydrogenases. A transaminase and a dehalogenase from Sulfolobus species have also been studied. The archaeal transaminase is found in a pathway for serine synthesis which is found only in eukaryotes and not in bacteria. It can be used for the asymmetric synthesis of homochiral amines of high enantioselective purity. The L-2-haloacid dehalogenase has applications both in biocatalysis and in bioremediation. All of these enzymes have increased thermostability over their mesophilic counterparts.     

Thiosulfate, polythionates and elemental sulfur assimilation and reduction in the bacterial world

Among sulfur compounds, thiosulfate and polythionates are present at least transiently in many environments. These compounds have a similar chemical structure and their metabolism appears closely related. They are commonly used as energy sources for photoautotrophic or chemolithotrophic microorganisms, but their assimilation has been seldom studied and their importance in bacterial physiology is not well understood. Almost all bacterial strains are able to cleave these compounds since they possess thiosulfate sulfur transferase, thiosulfate reductase or S-sulfocysteine synthase activities. However, the role of these enzymes in the assimilation of thiosulfate or polythionates has not always been clearly established. Elemental sulfur is, on the contrary, very common in the environment. It is an energy source for sulfur-reducing eubacteria and archaebacteria and many sulfur-oxidizing archaebacteria. A phenomenon still not well understood is the 'excessive assimilatory sulfur metabolism' as observed in methanogens which perform a sulfur reduction which exceeds their anabolic needs without any apparent benefit. In heterotrophs, assimilation of elemental sulfur is seldom described and it is uncertain whether this process actually has a physiological significance. Thus, reduction of thiosulfate and elemental sulfur is a common but incompletely understood feature among bacteria. These activities could give bacteria a selective advantage, but further investigations are needed to clarify this possibility. Presence of thiosulfate, polythionates and sulfur reductase activities does not imply obligatorily that these activities play a role in thiosulfate, polythionates or sulfur assimilation as these compounds could be merely intermediates in bacterial metabolism. The possibility also exists that the assimilation of these sulfur compounds is just a side effect of an enzymatic activity with a completely different function. As long as these questions remain unanswered, our understanding of sulfur and thiosulfate metabolism will remain incomplete.     

PcrA/UvrD/Rep DNA helicases in bacterial genomes

Helicases, which utilize the energy liberated by the hydrolysis of nucleotides to unwind nucleic acids, are involved in many aspects of nucleic acid metabolism. Various DNA helicases from the PcrA/UvrD/Rep subfamily are essential for the survival of different pathogenic bacteria and we have recently shown that they can be inhibited with small synthetic molecules. Altogether this suggests that these enzymes are potential new drug targets. Since little is known about the presence of these enzymes in bacterial genomes, 99 bacterial genomes were analyzed in the present study. This analysis reveals which and how many of these enzymes are found in bacteria, but more important, it identifies several of these enzymes as potential drug target candidates. In addition, this work identifies several proteins, called here PURL, that have a high homology with the PcrA/UvrD/Rep proteins and that may form an additional group in this helicase subfamily.     

How microbes utilize host ubiquitination

Activity, abundance and localization of eukaryotic proteins can be regulated through covalent attachment of ubiquitin and ubiquitin-like moieties. Ubiquitination is important in various aspects of immunity. Pathogens utilize host ubiquitination for the suppression of immune signalling and reprogramming host processes to promote microbial life. They deliver so-called effector molecules into host cells, which functionally or structurally resemble components of the host ubiquitination machinery utilizing this enzymatic process or they secrete molecules to inhibit ubiquitin-mediated degradation. Since prokaryotic pathogens lack a classical ubiquitination system, effector mimicry of components of the ubiquitin machinery could be achieved through gene flow. Horizontal gene transfer allows pathogenic bacteria to access ubiquitination enzymes from a potential host, while lateral gene transfer recruits components from another pathogen providing spread within the microbial community. Additionally, convergent evolution can shape bacterial proteins to acquire ubiquitination functions.     

Assessment of the bacterial diversity of breast milk of healthy women by quantitative real-time PCR

Aims:  Breast milk has been described as a source of bacteria influencing the development of the infant gut microbiota. Up to the present, few studies have been focused on the application of culture-independent techniques to study bacterial diversity in breast milk. In this context, the aim of this study was to characterize the breast milk microbiota of healthy women by applying the quantitative real-time PCR technique (qRTi-PCR).
Methods and Results:  A total of 50 breast milk samples were analysed by qPCR to assess the presence of different bacterial genera or clusters, including the Bifidobacterium , Lactobacillus , Staphylococcus , Bacteroides , Enterococcus , Streptococcus , Clostridium cluster IV and Clostridium cluster XIVa–XIVb groups. Staphylococcus , Streptococcus , Bifidobacterium and Lactobacillus were the predominant groups and were detected in all the samples. Clostridium XIVa–XIVb and Enterococcus were detected in most of the samples in contrast to the Bacteroides and Clostridium cluster IV groups.
Conclusions:  Our results confirm the abundance of bacterial DNA in breast milk samples and suggest that the qRTi-PCR technique has a huge potential in the microbiological analysis of human milk.
Significance and Impact of the study:  qRTi-PCR allowed the detection of bacterial DNA of streptococci, staphylococci, lactic acid bacteria and bifidobacteria in the samples of human milk, which confirms that breast milk can be an important source of bacteria and bacterial DNA to the infant gut.     

Multilocus enzyme analysis in aerobic and anaerobic bacteria using gel electrophoresis-nitrocellulose blotting

An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide-agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5-15 min each) from a single polyacrylamide gel could be achieved for most enzymes studied, and allowed an increased definition of enzyme bands on nitrocellulose as compared to migration gels. Four enzymes, which could not be blotted in such conditions, could still be stained in gels after blotting. Thus, the method allowed the combined analysis of several enzymes after a single gel electrophoresis separation. The analysis of enzyme polymorphism in the various species studied raised the interest of polymorphic loci such as esterase or glutamic-oxaloacetic transaminase for epidemiologic studies. The method characterized a genetic diversity of enzyme loci of S. pneumoniae higher than previously reported, and is thus convenient for the analysis of genetic relationships between related isolates. Since the present method reduces the tediousness of multilocus enzyme electrophoresis and requires experimental conditions that are not specific for the bacterial population studied, it may be proposed for rapid population genetics analysis of a wide variety of bacteria.     

Bacterial CMP-sialic acid synthetases: production,properties, and applications

Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell–cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5′-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC 2.7.7.43), the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.     

细菌中基因组岛的转移与调控机制研究进展

基因水平转移可导致细菌不同种属间个体DNA的交换,从而使细菌对环境的适应性增强,是细菌进化的重要途径之一。基因组岛是基因水平转移的重要载体,可移动的基因组岛能够整合到宿主的染色体上,并在特定的条件下切除,进而通过转化、接合或转导等方式转移到新的宿主中。基因组岛具有多种生物学功能,如抗生素抗性、致病性、异源物质降解、重金属抗性等。基因组岛的转移造成可变基因在不同种属细菌间的广泛传播,例如毒力和耐药基因的传播导致了多重耐药细菌的产生,威胁人类健康。基因组岛由整合酶介导转移,同时在转移的过程受到多种不同转录因子的调控。本文对细菌中基因组岛的结构特点、转移和调控机制以及预测等方面进行了综述,并最终阐明基因组岛的转移及其调控机制是遏制基因组岛传播的重要策略。