Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes
TL;DR: The environmental parameters necessary for increased production of mature, 35‐kDa active protease in strains of L. monocytogenes are described and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease is described.
Abstract: Listeria monocytogenes, a facultative intracellular pathogen, synthesizes an extracellular protease which is responsible for the maturation of phosphatidylcholine phospholipase C (lecithinase), a virulence factor involved in cell-to-cell spread. This work describes the environmental parameters necessary for increased production of mature, 35-kDa active protease in strains of L. monocytogenes, and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease. High performance liquid affinity chromatography was exploited to isolate the biologically active form of the mature protease, which was then subjected to biochemical characterization using casein as a substrate. The protease is a zinc-dependent metalloprotease which degrades casein over a wide range of temperatures and pH values. It can also degrade actin, the most abundant protein in many eukaryotic cells. The Listeria protease was shown to exhibit a high thermal stability and a relatively narrow substrate specificity. A three-dimensional model built on the basis of the homology with thermolysin was used to understand the structural basis of these characteristics.
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TL;DR: What is currently known about animal and human listeriosis is summarized, the pathogen, host and environmental factors that contribute to pathogenesis are detailed, and the interactions among those factors that influence the occurrence of human infection are examined.
Abstract: Listeriosis is a severe human and animal disease caused by two species of pathogenic bacteria from the genus Listeria, L. monocytogenes and L. ivanovii. In humans, listeriosis is overwhelmingly a foodborne disease, yet much remains to be learned regarding the transmission dynamics of pathogenic Listeria from the environment, through food, to humans. Similarly, our understanding of the various host, pathogen and environmental factors that impact the pathogenesis of listeriosis at the cellular and molecular level is incomplete. This review will summarize what is currently known about animal and human listeriosis, detail the pathogen, host and environmental factors that contribute to pathogenesis and, finally, examine the interactions among those factors that influence the occurrence of human infection.
188 citations
TL;DR: The state of the art in the design of such enzyme inhibitors with potential therapeutic applications, as well as recent advances in the use of some of these proteases in therapy are presented.
Abstract: Serine-, cysteine-, and metalloproteases are widely spread in many pathogenic bacteria, where they play critical functions related to colonization and evasion of host immune defenses, acquisition of nutrients for growth and proliferation, facilitation of dissemination, or tissue damage during infection. Since all the antibiotics used clinically at the moment share a common mechanism of action, acting as inhibitors of the bacterial cell wall biosynthesis or affecting protein synthesis on ribosomes, resistance to these pharmacological agents represents a serious medical problem, which might be resolved by using new generation of antibiotics, possessing a different mechanism of action. Bacterial protease inhibitors constitute an interesting such possibility, due to the fact that many specific as well as ubiquitous proteases have recently been characterized in some detail in both gram-positive as well as gram-negative pathogens. Few potent, specific inhibitors for such bacterial proteases have been reported at this moment except for some signal peptidase, clostripain, Clostridium histolyticum collagenase, botulinum neurotoxin, and tetanus neurotoxin inhibitors. No inhibitors of the critically important and ubiquitous AAA proteases, degP or sortase have been reported, although such compounds would presumably constitute a new class of highly effective antibiotics. This review presents the state of the art in the design of such enzyme inhibitors with potential therapeutic applications, as well as recent advances in the use of some of these proteases in therapy.
165 citations
TL;DR: Evaluated enzymes of the thermolysin family have an innovative potential for biotechnological applications and are implicated as virulence factors of the microorganisms that produce them and are therefore potential drug targets.
Abstract: Zinc containing peptidases are widely distributed in nature and have important roles in many physiological processes. M4 family comprises numerous zinc-dependent metallopeptidases that hydrolyze peptide bonds. A large number of these enzymes are implicated as virulence factors of the microorganisms that produce them and are therefore potential drug targets. Some enzymes of the family are able to function at the extremes of temperatures, and some function in organic solvents. Thereby enzymes of the thermolysin family have an innovative potential for biotechnological applications.
135 citations
Cites background from "Characteristics of the biologically..."
...A metalloprotease from Listeria monocytogenes is active at temperatures up to 80 C, and with a T50 of 88 C it is the most heat-stable natural thermolysin-like peptidase known (95)....
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TL;DR: The nature and the expression patterns of proteins in the various secretomes suggest that distinct CO(2)-responsive chromosome- and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.
Abstract: Bacillus anthracis is a gram-positive spore-forming bacterium that is the etiological agent of anthrax, a lethal disease sporadically affecting humans and animals, in particular herbivores. In its most severe manifestation, B. anthracis infection is initiated by inhalation of spores, which are taken up by alveolar macrophages and germinate into fast-dividing vegetative cells which secrete toxins and virulence factors during growth (81, 99). If untreated by prompt antibiotic administration, the bacteria invade the bloodstream, resulting in massive bacteremia and consequently generalized systemic failure and death. B. anthracis is considered to represent a potential biothreat agent, owing to the severity of the anthrax disease, the ease of respiratory contamination, and the perpetual environmental stability of the infective spores. The recent deliberate dissemination of B. anthracis (15) accelerated the efforts to identify new B. anthracis virulence-related determinants for the design of novel diagnostic, preventive, and/or therapeutic strategies.
Fully virulent B. anthracis strains harbor two native plasmids, pXO1 and pXO2, which encode critical pathogenicity factors. The absence of either one of the two plasmids results in a pronounced attenuation of B. anthracis virulence. The pXO2 plasmid encodes proteins involved in the biosynthesis of the poly-d-glutamic acid capsule, which may inhibit phagocytosis of bacteria during infection; pXO1 encodes the three toxin components protective antigen (PA), lethal factor (LF) (a zinc-dependent metalloprotease which proteolytically inactivates protein kinase kinases 1 and 2), and edema factor (EF) (a calmodulin-dependent adenylate cyclase), which form two binary toxins, lethal toxin and edema toxin. PA, the common component of both toxins, is not toxic by itself, yet it plays the central role of binding a specific receptor on the host cells and translocating LF and EF into the cytosol of infected cells, where they exert their detrimental activities. Anthrax is acknowledged as a toxinogenic disease, owing to the lethality of pure toxin preparations (77); on the other hand, additional B. anthracis secreted proteins are most probably involved in the onset and course of the disease and in survival of the bacteria in the host.
The regulatory circuits governing the virulence of B. anthracis are still to be fully deciphered, yet certain observations suggest that the virulence of the bacteria entails cross talk mechanisms which link expression of plasmid-encoded and chromosomally encoded genes. The regulatory AtxA protein, encoded by pXO1, is essential for expression of the toxin and capsule synthesis genes in vivo (a situation which can be mimicked by growing the bacteria in minimal medium under high bicarbonate-CO2 conditions) (75). Two additional regulatory proteins, AcpA and AcpB, encoded by pXO2, were suggested to act downstream of AtxA and to affect capsule synthesis (35, 36). AtxA was found also to influence expression of chromosomal genes, either directly or via AcpA and AcpB. In addition, the protein AbrB, which is a chromosomally encoded transition state regulator, was suggested to negatively control the activity of the toxin gene promoters (123, 131) via AtxA.
Secreted proteins include factors involved in pathogenicity, in particular in gram-positive bacteria (79). Such proteins may serve as possible targets for diagnostic purposes and/or therapeutic intervention. Bacteria of the Bacillus cereus phylogenetic group (B. cereus and Bacillus thuringiensis), to which B. anthracis belongs, secrete a diversity of factors that are essential for virulence, including toxins, hemolysins, proteases, and lecithinases. Notably, in these bacteria the secretion of certain virulence factors is regulated by a pleiotropic regulator, PlcR (2, 82, 87, 110), which is inactive in B. anthracis (2). It has been suggested that the evolutionary inactivation of the PlcR regulon in B. anthracis was due to incompatibility with the AtxA-controlled regulon and reflects the fact that the PlcR target genes are not essential for anthrax pathogenicity (96). Several studies have postulated that secreted proteases, other than those belonging to the silenced PlcR regulon, are responsible for some clinical manifestations of anthrax (1, 9, 117, 140). Such proteases could damage host tissues, interfere with immune effectors of the host, and/or provide nutrients for bacterial survival (103). Some chromosomally encoded B. anthracis extracellular proteases were suggested to be controlled by Cot43, a novel regulatory gene encoded by pXO1 (9).
The availability of the B. anthracis genomic DNA sequence (109, 121) paved the way for high-throughput genomic, transcriptomic, and proteomic analyses of B. anthracis (6, 7, 8, 17, 25, 41, 50, 64, 78, 85, 121, 144) in an effort to elucidate pathogenicity mechanisms by identification of novel virulence factors or in search for specific therapeutic and/or diagnostic targets. Indeed, bioinformatic surveys of the B. anthracis genome (7, 8, 121) suggested that proteins other than PA, LF, and EF, may participate in anthrax pathogenesis. Furthermore, many B. anthracis open reading frames (ORFs) encode potentially secreted or membrane-bound proteins exhibiting homology to known virulence factors from other bacteria (7, 8).
A preliminary proteomic study carried out in our laboratory examined membrane proteins prepared from a nonvirulent B. anthracis strain and led to the recognition of a number of immunodominant exposed proteins (8, 25). Here, we document an extended proteomic study, focusing on B. anthracis secreted proteins, which expands the data set of expressed B. anthracis proteins from both virulent and nonvirulent strains (6, 25, 41, 50, 64, 78, 85, 144). Based on identification of more than 400 two-dimensional electrophoresis (2-DE)-separated protein spots, we report the expression of 64 proteins which represent the most abundant B. anthracis secreted proteins, many of which resemble factors involved in the virulence of other pathogens. Comparison of the relative abundances of proteins in pXO1- and pXO2-containing and plasmid-cured strains reveals about 30 ORFs which are either preferentially expressed or repressed in the virulent B. anthracis strain under conditions which are considered to simulate those encountered within the mammalian host. The pattern of expression of these specific proteins demonstrates that B. anthracis possesses distinct regulatory pathways which involve plasmid- or chromosome-encoded CO2-inducible responsive factors.
121 citations
Cites background from "Characteristics of the biologically..."
...coli (138); Helicobacter pylori (55); Haemophilus influenzae (86); Yersinia enterocolitica (60, 84); Salmonella typhimurium (44, 68); Streptococcus pneumoniae (66); Klebsiella pneumoniae (28); Streptococcus pyogenes (55, 69) 21 BA1290 Camelysin Bacillus cereus (52) 27 BAS5205 Collagen adhesin Staphylococcus aureus (106, 150, 151); Pneumoviridea virulence (88) 34 BA2944 Polysaccharide deacetylase Streptococcus pneumoniae (14, 143) 40 BA0331 Polysaccharide deacetylase 36 BA0165 Prolyl olygopeptidase Streptococcus pneumoniae (113); Salmonella enterica (102); Porphyromonas gingivalis (10); Trypanosoma cruzi (53) 38 BA1952 NlpC/P60 family protein Listeria monocytogenes (92); Staphylococcus aureus, Clostridium acetobutylicum, Mycobacterium tuberculosis (3); Corynebacterium diphtheriae (149) 41 BA2793 Chitin-binding protein Staphylococcus areus (54); Listeria monocytogenes (34) 46 BA0599 NprB Listeria monocytogenes (26); Bacillus cereus (110) 49 BA3737 Alanine amidase Clostridium, Enterococcus faecalis, Pseudomonas aureginosa, Neisseria meningitidis, Streptococcus agalactiae, Staphylococcus, Yersinia pestis (122); Salmonella enterica (40); Listeria monocytogenes (21); Bacillus anthracis, immunodominant membranal proteins (8, 25) 50 BA0898 Alanine amidase...
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TL;DR: This review presents the state of the art in the design of such enzyme inhibitors with potential therapeutic applications, as well as recent advances in the use of some of these proteases in therapy.
Abstract: Proteases of the serine-, cysteine- and metallo- type are widely spread in many pathogenic bacteria, where they play critical functions related to colonisation and evasion of host immune defences, acquisition of nutrients for growth and proliferation, facilitation of dissemination, or tissue damage during infection. Since all the antibiotics currently used clinically share a common mechanism of action, i.e., inhibition of bacterial cell wall biosynthesis, resistance to these pharmacological agents represents a serious medical problem, which might be resolved by using a new generation of antibiotics with a different mechanism of action. Bacterial protease inhibitors constitute an interesting possibility, due to the fact that many specific and ubiquitous proteases have recently been characterised in some detail in both Gram-positive and Gram-negative pathogens. Unfortunately, at this moment few potent, specific inhibitors for such bacterial proteases have been reported, except for signal peptidase, clostrip...
119 citations
Cites background from "Characteristics of the biologically..."
...Following cleavage of the amino-terminal 24 amino acid signal sequence, the 55 kDa inactive zymogen is secreted to the external medium, whereas mature active protease possessing a molecular mass of 36 kDa is formed after further processing of the N-terminal 180 amino acids [114,115]....
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...The specificity of cleavage by the Mpl protease has not been defined and no specific inhibitors have been designed, although a model of its active site has recently been reported on the basis of its homology with thermolysin [115]....
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References
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TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.
225,085 citations
"Characteristics of the biologically..." refers methods in this paper
...Protein concentration was determined by the method of Bradford (1976)....
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TL;DR: The menu-driven operation of WHAT IF, combined with the use of default values wherever user input is required, makes it very easy to use for a novice user while keeping full flexibility for more sophisticated studies.
3,495 citations
"Characteristics of the biologically..." refers methods in this paper
...A three-dimensional model of the metalloprotease was constructed on the basis of the well defined thermolysin structure (Holmes and Matthews 1982) using the program WHAT IF (Vriend 1990)....
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TL;DR: Improved methods for detecting and enumerating the organism in foodstuffs are now available, including those based on the use of monoclonal antibodies, DNA probes, or the polymerase chain reaction, which can help in the prevention and control of human infection.
2,410 citations
"Characteristics of the biologically..." refers background in this paper
...monocytogenes has a maximum growth temperature as low as 45 °C (Farber and Peterkin 1991)....
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...The tracing of recent epidemics of listeriosis to food contaminated with L. monocytogenes suggests that the natural route of infection is the gastrointestinal tract (Farber and Peterkin 1991; Falkow et al. 1992)....
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...L. monocytogenes has a maximum growth temperature as low as 45 °C (Farber and Peterkin 1991)....
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...monocytogenes suggests that the natural route of infection is the gastrointestinal tract (Farber and Peterkin 1991; Falkow et al. 1992)....
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Book•
15 Jan 2001
TL;DR: P.H. North: Prevention of unwanted proteolysis V.V.C. Price: Proteinase as probes of conformation J.M. Pratt: Proteases as membrane probes P.E. Butler: Solubilization of membrane proteins P.B. Neurath: The diversity of proteolytic enzymes G. DeMartino, R.S. Sarath, & F. Wagner : Proteax assay methods.
Abstract: H. Neurath: The diversity of proteolytic enzymes G. DeMartino: Purification of proteolytic enzymes G. Sarath, R.S. de la Motte, & F. Wagner: Proteax assay methods B.M. Dunn: Determination of protease mechanism G. Salvesen: Inhibition of proteolytic enzymes M.H. North: Prevention of unwanted proteolysis V. Kasche: Proteases in peptide synthesis A.V. Flannery, R.J. Beynon, & J.S. Bond: Proteolysis of proteins for sequence analysis N.C. Price: Proteinase as probes of conformation J.M. Pratt: Proteases as membrane probes P.E. Butler: Solubilization of membrane proteins P.B. Gordon: Exogenous control of catabolism N.P. Birch: Proteases in protein maturation
816 citations
TL;DR: The results imply that the motile mechanism involves continuous polymerization and release of actin filaments at the bacterial surface and that the rate of filament generation is related to the rates of movement.
Abstract: The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen capable of rapid movement through the host cell cytoplasm. The biophysical basis of the motility of L. monocytogenes is an interesting question in its own right, the answer to which may shed light on the general processes of actin-based motility in cells. Moving intracellular bacteria display phase-dense 'comet tails' made of actin filaments, the formation of which is required for bacterial motility. We have investigated the dynamics of the actin filaments in the comet tails using the technique of photoactivation of fluorescence, which allows monitoring of the movement and turnover of labelled actin filaments after activation by illumination with ultraviolet light. We find that the actin filaments remain stationary in the cytoplasm as the bacterium moves forward, and that length of the comet tails is linearly proportional to the rate of movement. Our results imply that the motile mechanism involves continuous polymerization and release of actin filaments at the bacterial surface and that the rate of filament generation is related to the rate of movement. We suggest that actin polymerization provides the driving force for bacterial propulsion.
530 citations
"Characteristics of the biologically..." refers background in this paper
...…in the host cytoplasm (Marquis et al. 1993), and intracellular motility and cell-to-cell spread without an extracellular phase (Dabiri et al. 1990; Theriot et al. 1992; Correspondence to: Tjakko Abee, Tjakko Abee, Laboratory of Food Microbiology, Wageningen University, Bomenweg 2, 6703 HD,…...
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