Molecular and Genomic Analysis of Bacterial Pathogenicity - Term Paper Example

Molecular and Genomic Analysis of Bacterial Pathogenicity Introduction "Analysis," in the Oxford dictionary, is defined as "resolution into simple elements." In genetic analysis we must be clear about what we resolve and into what simpler elements. Mainly as a consequence of the development of microbial genetics, genetic analysis has increased enormously its resolving power in recent years, so much so that it now goes beyond that of physical or chemical techniques applied to biological organization (Pontecorvo, 1958). The essential process on which genetic analysis is based is recombination. Consider the analogy with microscopy, which is based instead on diffraction. The resolving power attained in microscopy depends on the quality of the microscope and on other technical details, but we know that it has a theoretical limit set by the wavelength of the light used. So far, in genetic analysis the resolving power has been limited only by the refinement of techniques. In the type of recombination on which classical genetic analysis is based, these structures are the chromosomes and their linearly arranged elements. The latter are recognized as genes as a consequence of their specific activities in metabolism and development (Pontecorvo, 1958). Complete genome sequences are now available for multiple strains of several bacterial pathogens and comparative analysis of these sequences is providing important insights into the evolution of bacterial virulence. Recently, DNA microarray analysis of many strains of several pathogenic species has contributed to our understanding of bacterial diversity, evolution and pathogenesis (Fitzgerald & Musser, 2001). Comparative genomics has shown that pathogens such as Escherichia coli, Helicobacter pylori and Staphylococcus aurues contain extensive variation in gene content whereas Mycobacterium tuberculosis nucleotide divergence is very limited. Overall, these approaches are proving to be a powerful means of exploring bacterial diversity, and are providing an important framework for the analysis of the evolution of pathogenesis and the development of novel antimicrobial agents (Fitzgerald & Musser, 2001). It is of little relevance whether the agents of risk are organic or inorganic; their effects both relate to processes of ‘contamination’ and ‘spreading’. They can both be understood as ‘actors’. In the discourses that have brought viruses to our attention, pathogen motivation is of crucial importance. Viruses make us ill because they are replicating themselves; like waste, they are virulent objects of modernity. However, unlike waste, they ‘take over’ bits and pieces of our bodies because they are motivated by self-replication. That is, they borrow bits of genetic material (DNA or RNA) and ribosome from their hosts (Cann, 1997; Levine, 1992). Popular culture can indeed be seen as playing a crucial role in the social and symbolic organization of risk management; expositions of newly emergent pathogen virulence have fully embraced the technological culture of the risk society. However, our exploration would not be able to escape the ironic turning-inward if it would merely circulate on the plane of textual analysis. Therefore, we turn to more sociological explanations of infections and epidemics to argue that pathogen virulence is part of a wider network of actors (humans, animals, technologies, and spirits). Moreover, it allows us to understand the social in terms of a complex spatialization of body politics and biopolitics, in which pathogen virulence constitutes a particularly effective medium of both ‘sense-making’ and the management of body boundaries (Joel Cracraft, Michael M. Miyamoto, 1991). Bacterial Pathogenesis Molecular Mechanisms De Bary (1879) broad definition of symbiosis includes parasitism and disease, areas in which significant discoveries are being made. This has been most evident in bacterial pathogenesis. During the past decade, scientists have introduced innovative approaches and concepts from disciplines such as bacteriology, cell biology, and immunology in an attempt to understand the molecular basis of infectious diseases. Stanley Falkow calls such attempts the "Zen" of bacterial pathogenesis, believing, in the Zen Buddhist tradition, that true enlightenment is achieved through meditation and insight (Falkow, 1988, 1990)."Virulence" and "pathogenicity" refer to the ability of bacteria to cause disease. The traditional criteria for establishing that a bacterium is responsible for a disease have been Koch's postulates, which were developed in 1882. Although serving well for many years, these postulates have limitations: (1) not all bacteria can be cultured, (2) not all members of a species are equally virulent, and (3) adequate animal hosts are not always available. Host susceptibility is an important virulence factor for bacteria. Falkow has proposed the "molecular Koch's postulates" to examine the potential role of genes and gene products in pathogenesis. The new postulates are: 1. The gene or its product should be found in strains of bacteria that cause the disease and not in bacteria that are avirulent. 2. A mutation of a virulent gene should reduce its virulence, and, conversely, introduction of a virulent gene into an avirulent bacterial strain should make it virulent. 3. The gene should be expressed by the bacterium during the infection process. 4. Antibodies to a gene product should be protective, or the gene product should elicit an immune response. Molecular biology techniques now make it possible to detect and identify bacteria and their genes even though they cannot be cultured (Finlay and Falkow, 1997). The term parasite-specified endocytosis describes microbial entry into phagocytic cells such as macrophages (Moulder, 1985). Alternatively, the bacterial entry may be through a host-specified endocytosis. Phagocytic cells have many lysosomes with hydrolytic enzymes that can digest microbial pathogens. Once a pathogen is engulfed, it is contained in a vacuole called a phagosome. The phagosome fuses with a lysosome to make a phagolysosome, and digestion of the engulfed microbes then occurs by one of the following mechanisms: 1. Oxygen-dependent phagocytosis, which requires increased oxygen consumption by phagocytic cells and conversion of oxygen into toxic intermediate forms such as the superoxide anion, hydrogen peroxide (H 2 O 2 ), singlet oxygen, and hydroxyl radicals. 2. Oxygen-independent mechanisms, which involve the activities of lysosomal enzymes such as phospho-lipases, proteases, RNase, and DNase. 3. Nitrogen-dependent mechanisms, which use reactive forms of nitrogen intermediates such as nitric oxide (NO), nitrite and nitrate. Nitric oxide is the most effective killing agent of the three and is produced from arginine when the phagocytes are stimulated by interferons or tumor necrosis factor (Lancaster, 1992). Lactic acid production, a result of altered metabolism in phagocytes, lowers pH and enhances the activities of many lysosomal enzymes. Escherichia Coli: A Versatile Human Pathogen Escherichia coli can cause many different types of diseases in humans. Most strains of E. coli are avirulent because they lack virulence genes. E. coli is classified into the following five "virotypes" on the basis of how the bacterium attaches to host cells, how host cells are affected following attachment, and the kind of toxins produced: 1. Enterotoxigenic E. coli (ETEC). Strains of ETEC resemble Vibrio cholerae in that they adhere to the mucosa of the small intestine and produce diarrhea in the host by secreting toxins. They are unable to invade the host tissue. Infants and children are most susceptible to this disease, the adult version of which is called traveler's diarrhea. A choleralike toxin, called heat-labile toxin, and a diarrheal toxin, heat-stable toxin, are involved. Genes for these toxins are carried on plasmids. 2. Enteroaggregative E. coli (EAggEC). The bacteria adhere to the mucosal cells in clumps and produce a heatstable--like toxin and a hemolysinlike toxin. The EAggEC strains cause diarrhea in children. 3. Enteropathogenic E. coli (EPEC). The EPEC strains produce dramatic changes in the host mucosal cells. The epithelial cells to which the bacteria bind lack the normal microvilli and instead have a cup-shaped pedestal under each bacterium. EPEC strains are the major cause of an often fatal diarrhea in children and infants. The EPEC strain invades cultured host cells in three stages. First, bacteria bind to the host cell surface via bundleforming pili (BFP). Second, a signal transduction pathway is triggered that activates host cell tryosine kinases and increases Ca2+ levels. Third, the host actin cytoskeleton is rearranged, and a pedestal where the bacterium becomes firmly attached is formed (Donnenberg et al., 1997). 4. Enterohemorrhagic E. coli (EBEC). The EHEC strain such as 0157:H7 causes dysentery and hemolytic uremic syndrome, which can result in death from acute kidney failure. EBEC strains produce a toxin similar to shiga toxin, the gene for which is located on a temperate phage. 5. Enteroinvasive E. coli (EIEC). The EIEC strains cause a disease identical to those caused by Shigella sp. but do not produce shiga toxin. The genes for virulence are located on a large plasmid and are regulated in a manner similar to those of Shigella species. Helicobacter Pylori: Molecular Mimicry between Pathogen and Host Until 1991, medical microbiologists had been teaching that the stomach environment was too harsh to support microbes. This changed following the discovery of Helicobacter pylori, which opened an exciting new chapter in our understanding of bacterial life (Rabeneck and Ranshoff, 1991). Helicobacter pylorus not only grows and thrives in the stomach, but it may also be responsible for 90% of gastric and duodenal ulcers, though most infections are asymptomatic. Helicobacter pylori produce large amounts of urease, which converts urea into ammonia and carbon dioxide. Bacteria survive the stomach environment by being surrounded with ammonia molecules, which neutralize stomach acids. Many bacteria invade the mucin layer, but only H. pylori have the adhesins that bind to mucosal cells. These adhesins include Lewis blood group O antigens, phosophatidyl-ethanolamine, sialic acid, and laminin. H. pylori also produce cytotoxins that cause the symptoms of peptic ulcers. The cytotoxins produce vacuoles within the mucosal cells lining the stomach and small intestine. When the injected mucosal cells die, gastric acids and digestive enzymes cause the formation of ulcers. H. pylori isolated from patients with peptic ulcer disease and with gastric cancer contained a 38-kb fragment of DNA that was not present in asymptomatic carriers. A cytotoxin-associated gene, the CagA gene, occurs in this stretch of DNA called the pathogenicity island. Another virulent gene, the vacuolating toxin gene, the VacA, is located 300 kb away from CagA. The genes are coexpressed in the most severe forms of gastroduodenal diseases. Strains lacking the CagA gene also lack the vacuolating activity of VacA. Mutations of CagA show that virulence in H. pylori has evolved through the inheritance of one or more DNA insertions (Covacci et al., 1997). Molecular mimicry is observed between pathogen and the host when certain protein sequences are compared. The LPS of H. pylori is unusual in that it expresses Lewis x and y blood group antigens. Such antigens are also expressed on the host mucosal cell. Sequence similarity exits between the vacuolating toxin of H. pylori and the host gastric H+K+-ATPase. Gastric H+K+-ATPase is the principal target of the autoimmune response in pernicious anemia. Whether H. pylori play a role in triggering antibodies against host gastric proteins remains to be determined (Appelmelk et al., 1997). The DNA sequence of Helicobacter pylori strain 26695 has also provided new insights into its pathogenesis ( Zhongming and Taylor, 1999). The H. pylori genome contains the two best-known virulence determinants, the vacuolating cytotoxin allele and the 38-kb cagA pathogenicity island. Though H. pylori is a Gram-negative pathogen, many of its proteins correspond to proteins in eukaryotes, archaea, and Gram-positive bacteria. This suggests that horizontal gene transfer from disparate phylogenetic groups into H. pylori lineage occurred during evolution (Berg et al., 1997). Techniques Employed to Analyze Genes The history of the Human Genome Project is a history of mapping projects. In its course, geneticists and molecular biologists surveyed the human chromosomes with cytogenetic, genetic, and physical markers. The maps featuring these landmarks were subsequently often collated or mapped onto one another, and eventually biologists began to “sequence” human DNA, a process customarily explained as mapping the hereditary material at the highest possible resolution. But even this final phase of the Human Genome Project occurred not once, but twice. In February 2001, independent research groups described preliminary drafts of the human DNA sequence in separate publications: a consortium of laboratories commonly known as the Human Genome Project published its draft in Nature (International Human Genome Sequencing Consortium 2001). Visionary molecular biologists have always conceived of the human genome as a single natural object. Dubbing their fantastic plan of determining the sequence of its chemical building blocks as the “holy grail of genetics, ” they predicted that the human genome sequence would eventually become “the central organizing principle for human genetics in the next century” (Waterston and Sulston 1998:53). Arguably, the publication of two human DNA sequences threatens to spoil these aspirations. Making a virtue out of necessity, one team of scientists concluded in February 2001: “We are in the enviable position of having two distinct drafts of the human genome sequence. Although gaps, errors, redundancy and incomplete annotation mean that individually each falls short of the ideal, many of these problems can be assessed by comparison” (Aach et al. 2001:856). Relating the discoveries of the Human Genome Project and Celera Genomics as imperfect yet comparable representations of the same natural object is a common move, but by no means the only way of making sense of these discoveries. Alternatively, one might conceive of the two versions of the human genome as separate objects. This is not to say that the drafts are irreconcilable in principle, but for the time being there are good reasons to speak of them as distinct objects in scientific practice. First, independent research groups working with different methods produced them: The Human Genome Project pieced together its version of the human genetic code in a “map-based sequencing” operation. Celera Genomics, in contrast, employed a “whole-genome shotgun.” Second, the two drafts differ in ways that go beyond superficial sequence similarities and discrepancies, such as their topology. Third, it remains to be seen whether the two drafts of the human genome will be reconciled at all (Gaudillière, & Rheinberger, 2004). Sequence Analysis Comparative biology has had a long and noble history (Rieppel, 1988). Its central goal has been to understand the bewildering diversity of form observed across the world's organisms. Concepts such as taxa (in particular, species) and homology have been the core intellectual instruments for facilitating this understanding. Thus, comparative biologists have labored to sort the world's organisms into species (however construed), largely based on their characteristics of form, and to describe their similarities and differences. For several hundred years now, this effort to compare has led to the realization that similarities and differences among species are best ordered in terms of a hierarchy of relationships, which we now take to represent the primary pattern of life's history as it has diversified over the last 4 billion years (Joel Cracraft, Michael M. Miyamoto, 1991). Systematic is the science of comparative biology and the primary goal of systematists is to describe taxic diversity and to reconstruct the hierarchy, or phylogenetic relationships, of those taxa (Nelson and Platnick, 1981). The overall importance of this goal is that corroborated hypotheses of relationship are the prerequisites for all inferences regarding what needs to be explained (i.e., various patterns) within the context of an historical perspective. This might involve repeated patterns of geographic distribution from one group to another (Nelson and Platnick, 1981), the distribution across lineages of certain behavioral, ecological, or morphological attributes, or the sharing among taxa, or perhaps among spatially segregated populations, of variation at the level of DNA. Just as importantly, it is only by having an hypothesis for the hierarchical pattern of shared attributes--with its implications for understanding character polarity--that one can begin to formulate explanations for derived novelties that have arisen and become fixed in populations (Joel Cracraft, Michael M. Miyamoto, 1991). Procedures for constructing phylogenetic hypotheses have been most fully developed by the discipline of phylogenetic systematics, or cladistics, which presently dominates the field of systematics (Hull, 1989). The emergence of cladistics can be related to its direct connection to genealogy in that only shared derived characters are used as evidence to support hypotheses about phylogenetic relationships. With respect to those hypotheses, similarities due to the retention of primitive features are ignored because they are uninformative regarding relationships. The cladistic approach is thus conceptually coherent in that only synapomorphies are considered to provide evidence for monophyly, and only monophyletic groups have objective reality as historical entities. The increasing acceptance of cladistics can also be attributed to its placement of systematic methodology within a broader scientific context. Thus, one of the more important philosophical advances has been to require that phylogenetic hypotheses conform to observations as closely as possible (Farris, 1983). Stated differently, those hypotheses which make the fewest ad hoc assumptions about shared patterns of character-state distributions are to be preferred (Wiley, 1981), yet the hypotheses themselves will always remain vulnerable to testing by additional observations (Joel Cracraft, Michael M. Miyamoto, 1991). Comparative Genomics Comparative genomics is a rabidly advancing discipline that is currently being energized by the availability of genome sequences for multiple strains of pathogenic bacterial species and by the advent of DNA microarray technology (Fitzgerald & Musser, 2001). “The large size and complexity of the human genome have limited the identification and functional characterization of components of the innate immune system that play a critical role in front-line defense against invading microorganisms. However, advances in genome analysis have reduced the obstacles to discovery of novel host resistance genes. Study of the genomic organization and content of widely divergent vertebrate species has shown a remarkable degree of evolutionary conservation and enables meaningful cross-species comparison and analysis of newly discovered genes. Application of comparative genomics to host resistance will rapidly expand the understanding of human immune defense by facilitating the translation of knowledge acquired through the study of model organisms. (Malo, 1999). “Two major elements underlie a thorough understanding of the pathogenesis of virtually any infectious disease: identification and characterization of the virulence factors and in vivo survival mechanisms of the invading microorganism and understanding of the components of the host response that lead to elimination of the invading pathogen and resolution of disease. The traditional approach to human infectious diseases has been to focus research on the study of important pathogens. The outcome of investigation of relevant bacteria, viruses, fungi, and parasites has led to the production of protective vaccines, antimicrobial agents, and effective strategies for control and elimination of disease outbreaks. A principal advantage of microbiologic research is the relative ease with which the organisms may be obtained, manipulated, and analyzed in the laboratory. Because microbial genomes are smaller, complete cloning and DNA sequencing of several microorganisms have been achieved and have paved the way for comprehensive study of gene expression and genome organization. In contrast are relatively limited advances in our understanding of the molecular basis of host defense. The study of host immune defense in humans is inherently complex; obstacles to greater understanding include limited opportunities for controlled observation and experimental manipulation, a large genome, and until recently, a lack of molecular techniques capable of facilitating genome-wide analysis” (Malo, Danielle, 1999). Advantages and Limitations of Techniques used to Analyze Genes The growing importance of DNA sequences for phylogenetic inference and for analysis of evolutionary processes at the molecular level requires that investigators strive for complete gene sequences whose accuracy has been verified by sequencing both complements. Such efforts will enhance the overall value of these comparative data to biology as a whole. With regard to systematic, a more detailed understanding of molecular evolution will facilitate the development of improved methods of phylogenetic reconstruction (Cracraft, & Miyamoto, 1991). Continued technological developments (with respect to collecting sequence data), coupled with the vast phylogenetic information contained in nuclear and extra-nuclear genomes, and virtually guarantees that nucleotide sequences will become the primary source of systematic data in the near future. Congruence provides the ultimate test of reliability in the absence of revealed truth, and as such, adoption of this methodological criterion can be regarded as crucial to the continued development of the field. Despite the growing importance of sequence data, it cannot be stressed strongly enough that there remains a pressing need to enlarge our nonmolecular database of systematic characters. If we are to gain meaningful insights into the "what’s," "how’s," and "whys" of the history of life, phylogenetic studies will have to rely on all available comparative information, both molecular and nonmolecular (Cracraft, Miyamoto, 1991). Comprehensive understanding of infectious disease pathogenesis necessitates identification and classification of host genes that control the reaction to virulent microorganisms. Through evolutionary assortment, a sequence of innate immune defense mechanisms has developed to defend the host in opposition to the constant danger of microbial injury as well as direct the progress of specific adaptive immune responses. Genetic analysis of naturally taking place variation in the host response among model organisms has productively recognized novel genes such as Nrampl, Lyst, and Btk, thus offering new approaching into the molecular nature of host resistance. Rapid progresses are now being made in the creation as well as incorporation of dense genetic maps of model organisms and humans. Comparative genomics will play a progressively more significant role in aiding the transfer of new information from experimental models to a more inclusive understanding of human host resistance (Malo, 1999). Conclusion Much progress has been made toward understanding the molecular basis of infectious diseases. Koch's postulates, the standard test for determining if a bacterium causes disease, have limitations. A new set of postulates, the molecular Koch's postulates, has been proposed and focuses on the role of genes and gene products in disease. Adherence to a host cell is an important step in bacterial pathogenesis. This is accomplished by means of pili and surface proteins such as adhesions and invasions. Attachment of bacteria to eukaryotic integrins involves three strategies: lectin binding, masking, and mimicry. Bacteria use siderophores to obtain the iron they need from a host. Bacterial pathogens produce exotoxins that disrupt host functions and cause cell death. Digestion of microbes in a phagosome occurs by several methods including nitrogen- and oxygen-dependent phagocytosis. Intracellular pathogens include Yersinia pestis (plague), which uses different ways to enter into host cells. About 60 genes are in-volved in virulence of Salmonella, and many of them occur in clusters called pathogenicity islands. When Shigella invades macrophages, it causes apoptosis. Up to 5-10% of the human population may be asymptomatic carriers of Listeria monocytogenes. Mycobacterium tuberculosis causes one of the most common of human diseases. Legionella pneumophila occupies a unique niche inside a macrophage, which engulfs the pathogen by means of a novel mechanism called coiling phagocytosis. The gene which codes for diphtheria toxin is carried by temperate phages, and only strains of Corynebacteria diptheria that carry the phage can cause the disease. The main virulence factors in Vibrio cholerae are pili and cholera toxins. Outbreaks of cholera in Peru have been linked to planktonic blooms (copepods) caused by El Niño. New strains of V. cholerae arise from genetic recombination and horizontal gene transfer. Escherichia coli is now classified into five virotypes. Pseudomonas aeruginosa is an opportunistic pathogen in humans. Helicobacter pylori responsible for most human gastric and duodenal ulcers shows molecular mimicry between pathogen and the host protein sequences, suggesting horizontal gene transfer. Reference: Applemelk, B. J., R. N. Negrini, A. P. Moran, and E. J. Kuipers. ( 1997) "Molecular mimicry between Helicobacter pylori and the host." Trends in Microbiology 5: 70-71. Berg, D. A., P. S. Hoffman, B. J. Appelmelk, and J. G. Kusters. ( 1997) "The Helicobacter pylori genome sequence: genetic factors for long life in the gastric mucosa." Trends in Microbiology 4: 468-474. Falkow, S. ( 1988) "Molecular Koch's postulates applied to microbial pathogenicity." Review of Infectious Diseases 10: S274S276. Falkow, S. ( 1990) "The Zen of bacterial pathogenicity." 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