Microbial genome sequencing and pathogenesis

The year 1997 saw the publication of the complete nucleotide sequence of Helicobacter pylori and Escherichia coli. It is conceivable that the complete nucleotide sequence for all the major human bacterial pathogens will be available by the end of the century. Database alignments have been used to ascribe the putative functions of open reading frames in the sequenced isolates and to define the differences between bacterial species at the nucleotide level. The most striking finding from all genome projects has been the high proportion of open reading frames that have no known function. Experimental data demonstrating the utility of the genome sequencing projects are only just beginning to emerge.     

Microbial pathogenesis and type III interferons

The innate immune system possesses a multitude of pathways to sense and respond to microbial pathogens. One such family are the interferons (IFNs), a family of cytokines that are involved in several cellular functions. Type I IFNs are appreciated to be important in several viral and bacterial diseases, while the recently identified type III IFNs (IFNL1, IFNL2, IFNL3, IFNL4) have been studied primarily in the context of viral infection. Viral and bacterial infections however are not mutually exclusive, and often the presence of a viral pathogen increases the pathogenesis of bacterial infection. The role of type III IFN in bacterial and viral-bacterial co-infections has just begun to be explored. In this mini review we discuss type III IFN signaling and its role in microbial pathogenesis with an emphasis on the work that has been conducted with bacterial pathogens.     

Microbial pathogenesis and tyrosine dephosphorylation: surprising'bedfellows'

The importance of parasite-directed phosphatases in such diseases as smallpox and the bubonic plague emphasizes the need to understand the molecular events associated with the normal function of protein tyrosine phosphatase in eukaryotic cells.     

Microbial pathogenesis: mechanisms of infectious disease

The FASEB Summer Research Conference on Microbial Pathogenesis: Mechanisms of Infectious Disease was held in Colorado, USA in July 2007. The central theme was the interplay between pathogenic microbes and their mammalian hosts. Here, we review the presented research that highlights this theme, including studies of both short-term and long-term interactions between microbes and their hosts.     

Serine proteases and metalloproteases associated with pathogenesis but not host specificity in the Entomophthoralean fungus Zoophthora radicans

The protease activity of a Zoophthora radicans strain that was highly infective toward Pieris brassicae (cabbage butterfly) larvae was compared with that of isogenic strains that were adapted to Plutella xylostella (diamondback moth) larvae through serial passage. All strains produced three distinct serine proteases ranging in size from 25 to 37 kDa; however, the original strain from P. brassicae also produced large amounts of an approximately 46 kDa metalloprotease. Subsequently, a cDNA encoding a 43 kDa (mature enzyme) zinc-dependent metalloprotease, ZrMEP1, was isolated from the original fungal strain and most likely corresponds to the 46 kDa protease observed with in-gel assays. ZrMEP1 possessed characteristics of both the fungalysin and thermolysin metalloprotease families found in some pulmonary and dermal pathogens. This is the first report of this type of metalloprotease from an entomo pathogenic fungus. A cDNA encoding a trypsin-like serine protease, ZrSP1, was also identified and was most similar to a serine protease from the plant pathogen Verticillium dahliae. In artificial media, ZrMEP1 and ZrSP1 were found to be differentially responsive to gelatin and catabolite repression in the fungal strains adapted to P. brassicae and P. xylostella, but their expression patterns within infected larvae were the same. It appears that while these proteases likely play a role in the infection process, they may not be major host specificity determinants.     

Microbial pathogenesis: lipid rafts as pathogen portals

The route of initial entry influences how host cells respond to intracellular pathogens. Recent studies have demonstrated that a wide variety of pathogens target lipid microdomains in host cell membranes, known as lipid rafts, to enter host cells as an infectious strategy.     

Microbial pathogenesis: new niches for salmonella.

Salmonella occupies a vacuolar compartment inside cells of its host. Recent studies have shown that the fate of this vacuole is different in various cell types, and that the outcome of colonization is determined by both the infecting bacterium and defense mechanisms of the host cell.     

Haemophilus paragallinarum secretes metalloproteases

Haemophilus paragallinarum secretes metalloproteases into different culture media lacking serum. Secreted proteins, concentrated by precipitation with 70% ammonium sulphate ((NH(4))(2)SO(4)) or methanol, displayed proteolytic activity at >100 kDa molecular mass in 10% polyacrylamide gels co-polymerized with porcine gelatin (0.1%). They were active in a broad pH range (4-9); pH 7.5 being the optimum. Protease activity was inhibited by 20 mmol EDTA/L and reactivated by calcium. The proteolytic activity was heat-stable at 40, 50, and 60 degrees C, but its activity diminished at 70 degrees C or higher. Secreted proteins partially degraded chicken immunoglobulin G (IgG) and cross-reacted with a polyclonal serum against a high molecular mass protease secreted by Actinobacillus pleuropneumoniae. Extracellular proteases could play a role in infectious coryza caused by H. paragallinarum.     

Physiology and pathophysiology of matrix metalloproteases

Matrix metalloproteases (MMPs) comprise a family of enzymes that cleave protein substrates based on a conserved mechanism involving activation of an active site-bound water molecule by a Zn²⁺ ion. Although the catalytic domain of MMPs is structurally highly similar, there are many differences with respect to substrate specificity, cellular and tissue localization, membrane binding and regulation that make this a very versatile family of enzymes with a multitude of physiological functions, many of which are still not fully understood. Essentially, all members of the MMP family have been linked to disease development, notably to cancer metastasis, chronic inflammation and the ensuing tissue damage as well as to neurological disorders. This has stimulated a flurry of studies into MMP inhibitors as therapeutic agents, as well as into measuring MMP levels as diagnostic or prognostic markers. As with most protein families, deciphering the function(s) of MMPs is difficult, as they can modify many proteins. Which of these reactions are physiologically or pathophysiologically relevant is often not clear, although studies on knockout animals, human genetic and epigenetic, as well as biochemical studies using natural or synthetic inhibitors have provided insight to a great extent. In this review, we will give an overview of 23 members of the human MMP family and describe functions, linkages to disease and structural and mechanistic features. MMPs can be grouped into soluble (including matrilysins) and membrane-anchored species. We adhere to the ‘MMP nomenclature’ and provide the reader with reference to the many, often diverse, names for this enzyme family in the introduction.     

Extracellular metalloproteases from bacteria

Bacterial extracellular metalloproteases (BEMPs) are a large group of metal-containing proteases secreted by heterotrophic bacteria. In this review, the diversity, structural characteristics, mechanisms of maturation, physiological roles, and applications of BEMPs are described. BEMPs are distributed among nine families of metalloproteases because of differences in primary sequences and structural characteristics. Until now, all of the BEMPs identified have been endoproteases harboring one catalytic Zn²⁺ in the active centers. BEMPs are usually synthesized as inactive zymogens with a propeptide that is covalently linked to and inhibits the catalytic domain. The removal of the propeptides of BEMPs is dependent on other proteases or an autocleavage process. The main physiological function of BEMPs is to degrade environmental proteins and peptides for bacterial heterotrophic nutrition. As extracellular proteases, BEMPs vary greatly in enzymology properties to adapt to their respective environments. BEMPs have been widely used in the food and pharmaceutical industries. In order to broaden the application of BEMPs, it is essential to explore novel BEMPs and apply gene/protein engineering to improve the production and properties of promising BEMPs.     

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.     

Bacterial extracellular zinc-containing metalloproteases.

Extracellular zinc-containing metalloproteases are widely distributed in the bacterial world. The most extensively studied are those which are associated with pathogenic bacteria or bacteria which have industrial significance. They are found practically wherever they are sought in both gram-negative and gram-positive microorganisms, be they aerobic or anaerobic. This ubiquity in itself implies that these enzymes serve important functions for the organisms which produce them. Because of the importance of zinc to enzymatic activity, it is not surprising that there is a pervasive amino acid sequence homology in the primary structure of this family of enzymes regardless of their source. The evidence suggests that both convergent and divergent evolutionary forces are at work. Within the large family of bacterial zinc-containing metalloendopeptidases, smaller family units are observed, such as thermolysin-like, elastase-like, and Serratia protease-like metalloproteases from various bacterial species. While this review was in the process of construction, a new function for zinc-containing metalloproteases was discovered: the neurotoxins of Clostridium tetani and Clostridium botulinum type B have been shown to be zinc metalloproteases with specificity for synaptobrevin, an integral membrane protein of small synaptic vesicles which is involved in neurotransmission. Additional understanding of the mode of action of proteases which contribute to pathogenicity could lead to the development of inhibitors, such as chelators, surrogate substrates, or antibodies, which could prevent or interrupt the disease process. Further studies of this broad family of metalloproteases will provide important additional insights into the pathogenesis and structure-function relationships of enzymes and will lead to the development of products, including "designer proteins," which might be industrially and/or therapeutically useful.     

Roles of metalloproteases in metastatic niche

Metalloproteinases (MMPs) are a cluster of at least 23 enzymes belonging to the more wide family of endopeptidases called Metzincins, whose structure is characterized by the presence of a zinc ion at the catalytic site. Although the general view of MMPs as physiologic scissors involved in extracellular matrix (ECM) degradation and tissue remodeling is still valid, additional functions have recently emerged, including the ability to cleave non ECM molecules such as growth factors, cytokines and chemokines from their membrane-anchored proforms. These functions are utilized by tumor cells and are fundamental in the determination of tumor progression and invasion. The effect of MMPs activity in cancer progression has been traditionally associated with the acquisition by tumor cells of an invasive phenotype, an indispensable requisite for the metastatic spreading of cancer cells. In addition to the traditional view, a new role for MMPs in creating a favourable microenvironment has been proposed, so that MMPs are not only involved in cell invasion, but also in signaling pathways that control cell growth, inflammation, or angiogenesis. Finally, recent evidence suggest a role of MMPs in the so called "pre-metastatic niche" that is the hypothesis of an early distant modification of the premetastatic site by primary cancer cells. This new hypothesis is changing our traditional view about MMPs and provides important insights into the effective time window for the therapeutic use of MMP inhibitors. In this review we provide the main available data about the ability of MMPs in creating a suitable microenvironment for tumor growth in metastatic sites and we indicate the implication of these data on the potential use of MMP inhibitors in the metastatic therapy.     

Pathogenic role of matrix metalloproteases and their inhibitors in asthma and chronic obstructive pulmonary disease and therapeutic relevance of matrix metalloproteases inhibitors.

Matrix metalloproteinases (MMPs) are an at least 23 member family of calcium and zinc dependent enzymes implicated in many physiological and pathological processes. Asthma, chronic obstructive pulmonary disease (COPD) and emphysema are diseases associated with an inflammation of the airways and lung parenchyma. In this review, we focus on the role played by MMPs in the pathogenesis of inflammation, airway remodelling and alveolar destruction, depicting the observational studies in humans and the experimental studies in animal models. During the course of asthma, MMP-2,-8,-9 and TIMP-1 are expressed at baseline and the allergen exposure or exacerbations of the disease lead to an increase of MMP-9 secretion being at this time much higher than that of TIMP-1, allowing temporarily a matrix damage, possibly followed by abnormal repair. Animal models suggest a predominant role for MMP-9 and MMP-12 in the pathogenesis of pulmonary inflammation and link an absence of MMP-2 to an increased parenchymal inflammation. In COPD and emphysema, human studies indicate an over-secretion of MMP-2,-8,-9 and animal models pointout MMP-1 and MMP-12 as being key players in the pathogenesis of emphysema. Taken together, these data identify specific MMP inhibition as appropriate target for therapeutic intervention in asthma or COPD/emphysema They also strongly argue against the widespread use of large spectrum non specific inhibitors that could be detrimental.     

The snake venom metalloproteases berythractivase and jararhagin activate endothelial cells

PIII snake venom metalloproteases (SVMPs) are metalloproteases structurally related to ADAMs (a disintegrin and metalloprotease human family of proteins). Berythractivase and jararhagin are PIII SVMPs with 69% homology that have different hemostatic properties. In order to clarify these differences and further characterize the biological effects of these proteins, we have analyzed the effect of both proteases on human umbilical-vein endothelial cell functions. We found that both proteins enhanced nitric oxide generation, prostacyclin production and interleukin-8 release. Berythractivase but not jararhagin increased the expression of decay accelerating factor. Jararhagin decreased cell viability in a concentration-dependent manner and induced cellular apoptosis, while berythractivase did not modulate cell survival. Our results show for the first time that, besides the known anti-aggregating or procoagulant effects of PIII SVMPs, these proteins trigger endothelial cell effector responses. Although structurally related, berythractivase and jararhagin induce a dissimilar generation and release of endothelial molecules that may account for their different hemorrhagic activity.