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Category: Applied and Industrial Microbiology; Food Microbiology
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Genomic sequencing initiatives from around the world have resulted in the complete sequencing of a large number of food- and waterborne pathogens, including most of the bacterial agents associated with significant disease in human populations. In this contributed work, the authors review these genome sequencing initiatives, explaining how they continue to build our understanding of the ecology, adaptation, and evolution of individual pathogens. In addition, the authors explain how our growing understanding of pathogen genomics has the potential to help ensure the safety of global food and water supplies.
Each chapter focuses on a specific food- or waterborne pathogen that represents a major public health threat. Moreover, each chapter has been written by one or more authors who have made major contributions to the genomic discovery of the particular pathogen. These authors explore all the findings, potential applications, and new questions that have arisen from genomic discovery efforts. They also examine how global trends such as climate change, growing human populations, and increasing levels of pollution are stressing the ecosystems that food- and waterborne pathogens encounter, forcing them to adapt.
Genomes of Foodborne and Waterborne Pathogens not only presents the latest findings from genome sequencing studies: it also translates this information into both scientific and public health opportunities. It is therefore recommended not only for microbiology and genomics researchers but also for public health officials, regulators, food scientists, and other professionals tasked with protecting our food and water supplies from dangerous pathogens.
Hardcover, 352 pages, full-color insert, illustrations, index.
In this chapter, the knowledge of the genome of E. coli O157:H7 may be used in the control of serious foodborne pathogen, E. coli strains are associated with gastrointestinal and extraintestinal illness. The E. coli O157:H7 genome provides us with information about the evolution and emergence of pathogen and the diversity that exists within populations of E. coli O157:H7. Most of E. coli prophage are defective and cannot form infectious phage. This chapter provides a brief review of subtyping methods used to characterize E. coli O157:H7 strains. Differences in biochemical utilization can be used to distinguish large categories of E. coli strain. Following the generation of cDNA from the bacteria’s mRNA using the enzyme reverse transcriptase, the level of gene expression is inferred from the intensity of the label signal from gene-specific microarray spots following hybridization. This procedure is the foundation of the new science of transcriptomics. Typically, quantitative PCR assays are also carried out on selected genes to measure mRNA levels to verify the significant changes in expression of genes observed in microarray-based transcriptomics studies. It is also evident that our understanding of E. coli O157:H7 gene regulatory systems will be enhanced through genomic sciences. E. coli O157:H7 and other bacterial pathogens have evolved through the acquisition of gene clusters borne on plasmids, bacteriophages, and genomic islands. Finally, with the arrival of the genomics revolution researchers are able to examine the "pan-genome" of the species and specific groups within species such as enterohemorrhagic E. coli(EHEC) and E. coli O157:H7.
Although all Shigella species share similar pathogenic properties, each species exhibits peculiar epidemiological characteristics. S. dysenteriae, S. flexneri, and S. boydii are most common in developing countries, whereas S. sonnei is more prevalent in developed countries. A remarkable difference between Shigella and E. coli genomes is the presence of pseudogenes in the former. The second remarkable feature of the Shigella genome is the presence of enormous copies of IS elements, which are likely the cause of genomic rearrangements, including deletions, inversions, and translocations that may effectively disrupt the colinearity among different Shigella genomes. After the acquisition of the ancestral forms of the virulence plasmid by Shigella/enteroinvasive E. coli (EIEC), genome reduction by inactivation of the pathway-specific antivirulence loci (AVL) is vital for adaptation in the cytosolic niche. Similar to obligate pathogens, reduced selection pressure might have played an important role in Shigella genome reduction, which may have further accelerated terminal evolution and resulted in the increased host specificity. Numerous genes responsible for cell motility cell envelope, carbohydrate transport, and metabolism that are present in E. coli were frequently lost in Shigella. A combination of various strategies using the basic information provided by genomic research will be helpful in efficient control and prevention of Shigella infections.
A small number of Salmonella enterica serovars are host specific, only causing infection in one host species or a few closely related species. The diseases caused by broad-host-range and host-restricted Salmonella serovars also differ. Rearrangements can occur via recombination between short repeat sequences, but most large chromosomal rearrangements in bacteria occur by recombination between repeated sequences with several kbp of homology. IS200 is the most common IS element in Salmonella, it transposes infrequently. Thus, in contrast to many bacteria that have a high background of transposition-mediated rearrangements, most genome rearrangements in Salmonella are due to recombination. In contrast to the broad-host-range serovars, isolates of host-specific S. entericaserovars have large-scale chromosomal rearrangements resulting from recombination between the rrn operons. Inversions and translocations change the order of the chromosomal regions between the rrn operons from the conserved arrangement type found in the broad-host-range serovars to one of over fifty arrangement types identified so far in host-specific serovars. Comparative genomics has revealed that many host-specific pathogens show greater genome plasticity than closely related bacteria that live in a wider variety of environmental conditions. Although the genome rearrangements in other bacteria are often mediated by active transposons, it seems likely that the observed differences in genome plasticity are simply determined by the selective constraints of their distinct ecological niches. Genome rearrangements also have practical implications for foodborne pathogens, as they can complicate the identification and tracking of outbreak strains.
Campylobacteraceae is composed of three main genera, Campylobacter, Sulfurospirillum, and Arcobacter, and is represented currently by 43 described or proposed species. Unlike Campylobacter spp., that are associated with warm-blooded avian and mammalian hosts, members of the Sulfurospirillum and Arcobacter genera are non-host-associated, free-living, environmental organisms. Arcobacter spp. can grow aerobically, unlike campylobacters, which are restricted to microaerobic and/or anaerobic environments. The organisms whose genomes have been or are being sequenced are (i) host-associated or free-living; (ii) nonpathogenic, human commensals, or human pathogens (oral or gastrointestinal); or (iii) food or environmental isolates. Analysis of these genomes alongside comparative genomics will provide clues pertaining to the genetic nature of pathogenicity, host association, environmental adaptation, and evolution. The chapter addresses the topics related to the Campylobacter and Arcobacter genomes. The coding sequences (CDSs) with assigned function are fairly consistent across the sequenced Campylobacteraceae genomes, with an assigned function/total CDSs average of 45%. It is possible that many Campylobacter species are composed of multiple host-associated phylogenetic clusters. Comparative genomics of the Campylobacter and Arcobacter genomes has not identified any pathogenicity islands containing toxin-encoding genes or other virulence determinants. Campylobacter and Arcobacter species provide fertile ground for comparative genomics. Therefore, comparative genomics of Campylobacter and Arcobacter should provide insights into pathogenicity, host adaptation and specificity, evolution, and environmental adaptation and survival. The degree of variation within Campylobacter and Arcobacter is an important consideration.
Vibrio vulnificus, a gram-negative marine bacterium, has been recognized as an important pathogen of both humans and eels for decades. V. vulnificus infection is characterized by the rapid spread of this organism from intestine or skin into deeper tissue and even the bloodstream to result in septicemia and/or necrotic skin lesions. One of gene regions, region XII, was found to be associated with one of the two lineages of V. vulnificus divided based on the multilocus sequence typing (MLST) data of six housekeeping genes. The superintegron is unique to each Vibrio species, containing different kinds of gene cassettes. The functions of gene cassettes and open reading frames (ORFs) in superintegrons remain largely unknown. The publication of nucleotide sequences of two BT1 V. vulnificus genomes and the BT2 virulence plasmid has opened up opportunities for mining new virulence genes for mice and eels as well as developing new diagnostic methods. Moreover, the complete genome information can be applied to epidemiology study and food safety monitoring. Because of the high mortality rate of systemic infection with V. vulnificus, an effective vaccine against this organism is desired, particularly for individuals at high risk. Contaminated bivalve molluscan shellfish, including oysters, clams, and mussels, are major sources of per os infection by V. vulnificus. The vast information generated from the genomes of major representative Vibrio species has enabled comparative analysis and provided an opportunity to investigate the biology of this group of marine bacteria.
This chapter talks about Vibrio parahaemolyticus that can cause fatal septicemia in immune-compromised hosts. The Kanagawa phenomenon (KP) has long been recognized as an effective marker for discriminating pathogenic from nonpathogenic V. parahaemolyticus strains and is used extensively as a clinical indicator for the virulence of this bacterium. Thermostable direct hemolysin (TDH) is considered an important virulence factor for V. parahaemolyticus. Genes classified in transcriptional regulation have specific roles in response to environmental changes. The most notable finding of the genome sequencing of V. parahaemolyticus was the presence in the genome of two sets of genes for the type III secretion system (T3SS). T3SS2 may also be useful for novel preventive and therapeutic methods, for example, as a target for vaccines or drugs against V. parahaemolyticus infections. The novel preventive and therapeutic methods discussed here are highly selective for this pathogen and may therefore contribute to therapies that are distinct from conventional treatments based on the administration of antibiotics. Studies on V. parahaemolyticus have mainly focused on the role of virulence factors TDH and thermostable direct related hemolysin (TRH), but genome sequencing of this bacterium has shed light on novel aspects of this organism. A notable finding in this context is the discovery of functional T3SS genes in the genome of V. parahaemolyticus, which are not found in the genomes of cholera-toxin-producing V. cholerae and V. parahaemolyticus.
The main symptom of cholera, profuse rice water stool, is primarily caused by the action of cholera toxin (CT), a very potent AB-type enterotoxin that consists of five binding B subunits and one active A subunit. Many bacterial pathogens attain or increase their virulence potential through HGT of genetic material carried on mobile and integrative genetic elements (MIGEs) such as plasmids, bacteriophages, pathogenicity islands (PAIs), integrons, and integrative conjugative elements (ICEs). The impact of horizontal gene transfer (HGT) on bacterial evolution--particularly its role in the emergence of many human gastrointestinal pathogens--is unquestionable. This chapter gives a brief overview of the features that differentiate integrative genetic elements such as integrases, transposases, phage structural genes, or plasmid conjugal transfer genes, from one another. In evolutionary terms, while phylogenetic analysis of housekeeping gene mdh groups V. cholerae and V. mimicus strains separately, both species group together based on the nanH gene, which indicates recent HGT between the species. The authors identified 24 regions, gaps in the genome atlas, of greater than 10 kb that were unique to RIMD2210633. 22 members of the family Vibrionaceae in the genome database were examined, and only 5 species encode neuraminidase: V. cholerae pathogenic isolates, V. mimicus, V. parahaemolyticus strain 16, V. shilonii AK1 and Vibrio sp. strain MED222. The last two are the only strains to encode sialic acid scavenging, synthesis, and catabolism genes.
The study of the Y. pestis genome indirectly reveals enteric features of Y. pseudotuberculosis (and Y. enterocolitica). This chapter describes the salient features of postgenomic studies on Y. enterocolitica and Y. pseudotuberculosis and relates this to our understanding of the diversity and evolution of virulence for these species. The enteropathogenic yersiniae are ubiquitous in the environment, and are a common cause of animal infections, and they have been isolated from cattle, sheep, pigs, domesticated animals, and avian species. Various other adhesion- and invasion-associated factors such as Ail and the Myf fimbriae have also been reported in enteropathogenic yersiniae. B12 is an essential cofactor for several reactions, including 1,2-propanediol degradation. Although the true significance of tetrathionate respiration operon in Y. enterocolitica is unknown, in Salmonella, 1,2-propanediol is an important source of energy, and mutants unable to make B12 are significantly attenuated in their ability to grow in macrophages. With respect to the enteropathogenic yersiniae, the natural deselection highlights many known genes required for the enteropathogenicity by Y. pseudotuberculosis (and Y. enterocolitica), but also highlighted many previously unsuspected candidates. Perhaps the most striking aspect of the evolution of Yersinia is the extremely rapid emergence of Y. pestis from Y. pseudotuberculosis, and genome analysis shows how this has happened.
Although there is an obvious advantage to a pathogen that can disrupt the host immune system, there is a gap in knowledge as to the exact etiology of the disease. First, Staphylococcus aureus that harbors enterotoxin genes was determined to reside in the healthy gut of more than 50% of the nonenteritis-associated patients examined in a recent study, suggesting that the pathogenicity is dictated in part by "to be determined" host factors that lead to pathogen spread and heavy enterotoxin production. Second, there is a lack of evidence for the exact origin of symptoms. There are over 20 known staphylococcal proteins that share high similarity to the staphylococcal enterotoxins (SEs). The SEs have classically been organized into families based on their percentage identity or similarity. Methicillin-resistant S. aureus (MRSA) was first identified in food-producing animals in the 1970s, being identified as a cause of mastitis in dairy cattle. A logical extension of concerns regarding MRSA in food animals was investigation of MRSA contamination of food products. There are three main areas of concern relating to the potential routes of transmission: MRSA as a cause of classical enterotoxin-associated food poisoning, contaminated food as a source of nasal colonization, and contaminated food as a source of extraintestinal infection. Nasal passages are the most common site of MRSA colonization in humans, and the nose is a frequent hand-contact site. Good food handling and hygiene practices could minimize the risks for food handlers.
Analyses of over 400 strains suggested that Listeria monocytogenes populations adapted to different niches exist. This study identified one L. monocytogenes 1/2a strain that was dominant among the strains collected from the food-processing plants. It is clear that the patterns and markers identified by these different genomic techniques in L. monocytogenes strains are an invaluable basis for developing powerful tools for rapidly tracing listeriosis outbreaks and for conducting effective surveillance of food-processing environments. The ongoing sequencing projects aimed at determining the complete genome sequence of one representative of each species of the genus Listeria by the Institut Pasteur and the German PathoGenomiK network. The determination of the complete genome sequence of an additional 18 Listeria strains of by the Broad Institute can be the driving force for understanding the function of the many factors encoded by the genome, whether involved in virulence or not, and to understanding strain-specific differences in niche adaptation and virulence. The sequencing of hundreds of genomes and communities, like those resident in food production environments, will allow us to move from population genetics to population genomics.
It is expected that the increasing genetic information on Bacillus cereus in genome databases will pave the way for investigating its evolution, ecology, and virulence, and, finally, will contribute to the development of new strategies to control and prevent foodborne diseases caused by B. cereus. This chapter talks about toxins and population structure of the B.cereus group. Many bacterial genome sequences, including B. cereus strains, are available in draft versions only and are missing a few percent of the sequence. The chapter deals with only complete genome sequences, and illustrates that species definition in the B. cereus group remains an open question and it may well be that the discussion will gain momentum with daily growing genome information, especially of "borderline strains". It focuses on pan genome, core genome, accessory genome, and mobilome of the B. cereus group. B. cereus geomics suggest that the mobilome of this species group is important not only to model its evolution, but also to differentiate and detect the different pathotypes. It is clearly visible that the high number of B. anthracis and B. cereus strains already known is improving research tools to study pathogenicity, ecology, and host and environmental adaptation.
Bacillus cereus is the putative ancestor of all Bacillus anthracis strains, which obtained two virulence plasmids (pXO1 and pXO2) and at least one chromosomal mutation that inactivated the plcR gene. Identification of the molecular signals that trigger germination and the spore surface receptors involved is critical to understanding the pathogenesis of B. anthracis. Sporulation and pathogenesis are opposite processes, while germination and virulence gene expression are synergistic, since transition from dormant spores to vegetative cells is essential for the virulence of B. anthracis. One of the enzymes within the basal layer of B. anthracisspores is an alanine racemase capable of converting the spore germinant L-alanine to the germination inhibitor D-alanine. Comparative genomic studies have contributed significantly to our understanding of the virulence properties, host specificity, ecology, and adaptations of B. anthracis and other species comprising the B. cereus group. While all Banthracis strains examined so far contain four prophages of the lambda family inserted at defined loci, most of the other sequenced B. cereus group genomes do not contain homologous prophages inserted at these sites. With the advent of next-generation sequencing technologies, which promise to deliver even more sequence data over shorter periods of time, and with metagenomics and community genomics approaches on the rise, the future of pathogen genomics is bright and growing fast.
Genetic analysis focused on BoNT genes and flanking genes, which elucidated the botulinum locus encompassing the genes encoding BoNT, associated nontoxic proteins, and the regulator Bot/R, and provided evidence of its variation among the different toxinotypes. Whole genomes sequencing is now available for eight Clostridium botulinum strains and is in progress for several other strains. These sequence data will allow us to get deeper insights into the lifestyle of C. botulinum—in particular, its survival in the environment and in food products, as well as its regulation of toxin production and also permit us to better understand its relationship with related nontoxigenic clostridia and the modes of transfer of toxin genes. The neurotoxin and ANTP genes are clustered in close vicinity and constitute the botulinum locus. Indeed, two main types of botulinum locus can be distinguished, the HA locus containing ntnh and ha genes and the OrfX locus containing orf X, p47, and ntnh genes in addition to the bont gene. The upstream regions of the botulinum locus are identical in the genome of the three C. botulinum A1 strains and contain a flagellin gene. Bacteriophages mediate the neurotoxin gene transfer in group III of C. botulinum. In vitro transcription, transcripts were observed when BotR/A-Core was incubated only with DNA templates containing ntnh-bont/A and ha35 promoters. Thus, BotR/A is an alternative sigma factor required for specifc expression of the botulinum locus operons.
Clostridium perfringens is one of the most frequent causative agents of human food-poisoning worldwide. Strains of C. perfringens are divided into five distinct toxin types on the basis of their differential production of four major C. perfringens extracellular toxins. The published genomes of C. perfringens ATCC 13124, SM101, and strain 13 contain single circular chromosomes of 3,256,683, 2,897,393, and 3,031,430 bp, respectively. The genome sequence of C. perfringens strain 13 was examined to identify catabolic pathways that may utilize such sugar substrates as energy sources. Importantly, the fermentative pathways were predicted to lead to the production of CO2 and H2 gasses that may be involved in the establishment of an anaerobic environment within an infected host suitable for C. perfringens growth. One of the hallmark characteristics of clostridial species is their capacity to form endospores, which are essentially highly modified dormant cells resistant to extremes of heat and radiation. In C. perfringens strain 13, 49 genes involved in capsule production were found to be organized into a single large gene cluster. Beyond broadening one's appreciation for toxin genes, the genome sequences highlighted additional factors that may facilitate the pathogenic potential of this organism, for example, genes involved in capsule production and in sporulation. Future studies will explore the precise functional roles of these genes in the virulence of C. perfringens and may yield avenues to combating C. perfringens infections.
Mycobacterium avium subspecies paratuberculosis, the causative agent of Johne’s disease, is distributed worldwide in farmed ruminant animals such as cattle, sheep and goats, and in wildlife such as rabbits, deer, antelopes, and bison. The major impact of this disease is on the world's milk industry. When the focus is on a particular mutant phenotype such as attenuation in cultured macrophages or in mice, then some defined virulence determinants have recently been identified. The key genomic attributes of the bovine isolate of M. aviumsubsp. paratuberculosis, designated strain K-10, are discussed in this chapter. The chapter provides some applications of genome sequences. None of the studies have been applied genomic tools to directly address the role of M. avium subsp. One strain typing study examined caprine isolates of M. avium subsp. paratuberculosis using three molecular techniques: PFGE, IS900 RFLP analysis and IS1311 PCR-restriction enzyme analysis. This study found that PFGE analysis was more discriminatory than the other two methods, enabling a resolution of 13 different PFGE profiles among the 44 isolates evaluated. Researchers have also investigated this method but found more consistent expression of M. avium subsp. paratuberculosisproteins using the traditional recombinant protein production approach.
The noroviruses (NVs) cause approximately one illness for every 30 persons per year on a worldwide basis and are now recognized as the number one cause of foodborne illness in the United States, causing about 9 million foodborne infections per year. Human calciviruses fall into two major genera, Norovirus and Sapovirus. Sapoviruses also cause gastroenteritis, principally in infants, and are less commonly associated with foodborne transmission than noroviruses (NVs). NVs are nonenveloped, icosahedral viruses ranging from 27 to 35 nm in diameter. The icosahedral virion has T=3 symmetry and is composed of 90 dimers of the single capsid protein VP1. The first 225 amino acids form the shell domain, which is critical for icosahedron formation. One or two copies of VP2 are believed to be within the capsid. The high isoelectric point of VP2 suggests the possibility of an interaction between the positively charged virus protein and the viral RNA during virus assembly. STAT-1 mice are deficient for interferon production and interferon-mediated responses. On the surface, this information suggests that the innate immune response may be critical for successful defense against NV. For a protective immune response in normal mice inoculated with MNV-1, it has recently been shown that both adaptive CD4 and CD8 T-cell responses are critical for a protective immune response. The high degree of variability of different NV strains suggests that there is significant evolutionary immune selection on NVs. Currently, outbreaks from virus strains from the GII.4 cluster account for approximately 80% of outbreaks worldwide.
In the early 1940s, "infectious" and "serum" hepatitis was identified based on mode of transmission. Enteric hepatitis includes two types: hepatitis A virus (HAV) and hepatitis E virus (HEV), both of which can be foodborne and waterborne. The genetic characteristics of the enteric hepatitis viruses are discussed in this chapter. Enteric hepatitis infections may also develop asymptomatically; chronic disease has so far never been reported. The absence of a lipid envelope helps ensure the stability of the enteric hepatitis agents in the presence of biliary salts, which is not the case for serum hepatitis viruses. Alum and chlorine treatment prevented bacterial infections in 1955 and 1956, but 30,000 cases of hepatitis occurred among the population. Irrigation of vegetables as well as washing and processing of any food that is consumed raw with HAV- or HEV-contaminated water could lead to enteric hepatitis outbreaks. Enteric hepatitis viruses may become water contaminants through the discharge of untreated sewage and treated wastewater. Molecular epidemiology points to a geographical distribution of hepatitis A and E viruses. Globalization, climate change, and the inherent genetic variability of these viruses may promote the emergence of new variants. All this calls for the need to trace and characterize circulating enteric hepatitis viruses.
Food microbiologists often overlook the importance of fungi as a threat to food safety and security even though fungi produce a wide array of toxic compounds. Aspergillus flavus is known to produce over 14 described mycotoxins. It is common for strains of A. flavus to produce both aflatoxin and cyclopiazonic acid (CPA) and for commodities to contain both mycotoxins. Other than A. flavus and A. minisclerotigenes, no other fungal genera are known to produce aflatrem; however, structurally related tremorgenic mycotoxins, such as penitrem from Penicillium spp., are prevalent in other fungi and likely share common enzymes. Fungi have the capacity to produce many diverse secondary metabolites, and over 300 fungal secondary metabolites are described as mycotoxins. It is now possible to predict whether potential toxins produced by secondary metabolism clusters may be present in food. With respect to the regulation of aflatrem biosynthesis, researchers have shown that the gene called veA, previously shown to control aflatoxin and sclerotial production in A. parasiticus was found to not only be necessary for the production of aflatoxins B1 and B2 and sclerotia, but also regulated the synthesis of the mycotoxins cyclopiazonic acid and aflatrem. New tools for genomewide gene profiling and functional analysis will surely reveal additional information on aflatoxin production and the regulation of the process. This knowledge will empower researchers to find effective strategies for controlling aflatoxin contamination of food and feed. Gradually, newly developed next-generation sequencing technologies will become common research tools for functional genomics studies.
Cryptosporidium species cause one of the common opportunistic infections in immunocompromised persons. Due to lack of effective treatment, Cryptosporidium infection can cause prolonged and often life-threatening diarrhea in AIDS patients. The use of molecular biologic tools has made significant contributions to one's understanding of the biology and epidemiology of Cryptosporidium species. This chapter provides a snapshot of the Cryptosporidium genomes and discusses what has been learned about cryptosporidiosis from the genomic data. It discusses the key features of the Cryptosporidium genomes. Cryptosporidium hominis used to be one of the genotype of C. parvum but was renamed as a separate species a few years ago based largely on the host specificity, pathogenicity, and sequence differences from type II C. parvum at various genomic loci. The C. hominis genome shares an extremely high degree of similarity with that of C. parvum. The major metabolic pathways encoded by the compact Cryptosporidium genome are highly streamlined by the absence of virtually all de novo synthetic capacities, such as those for amino acids, nucleotides, and fatty acids. The chapter deals with genomic data associated with host-pathogen interactions and pathogenesis, genomics and the development of molecular diagnostic and epidemiologic tools, and genome sequencing and public health. The lack of progress in research on the treatment of and vaccines against cryptosporidiosis is a particular public health concern. Better collaboration among molecular biologists, parasitologists, clinicians and other public health researchers, and the support of public health institutions, funding agencies, and policy decision makers will increase this progress.
This chapter provides an update on Giardia genome research and highlights studies that utilized genomics and functional genomics to understand the phylogeny, biochemistry, molecular biology, and epidemiology of Giardia lamblia. Earlier observations of a highly compact G. lamblia genome containing a disproportionate number of genes coding for variant surface proteins (VSPs), structural proteins, and ribosomal RNA have been confirmed by genomewide sequence analysis. Many cellular processes of G. lamblia (e.g., DNA replication, transcription, and RNA processing), as well as metabolic pathways, are simple compared to higher eukaryotes. To ensure its survival inside and outside its host, G. lamblia must exert precise control over the transcription of genes involved in encystation, excystation, and trophozoite replication. RNA polymerases and transcription initiation factors are similar in some respects to those in eukaryotes but also contain peptides unique to Giardia. G. lamblia is of special interest to those attempting to understand eukaryotic evolution. Transfection of CHO cells with a second cyst wall protein (cwp2) promoter region fused to the gene for firefly luciferase was used to study regulation of cwp2 transcription by sterol regulatory element binding proteins (SREBPs). This study showed that, in the absence of cholesterol, cwp2 transcription was increased and that SREBP binds to the cwp2 promoter to activate transcription of this cyst wall protein. A third cyst wall protein (cwp3) was identified by searching the G. lamblia database with amino acid sequence of a first cyst wall protein (cwp1) and cwp2 leucinerich repeat regions.
Cyclospora cayetanensis is one of 18 currently recognized species of Cyclospora. The internal transcribed spacer (ITS) regions have long been used in the study of plant genomics and evolution. Van der Heijden and colleagues attempted to use the ITS-1 region to subtype Histomonas meleagridis but like many found the A/T rich fluorograms too complex to discern meaningful patterns. Of the four future directions John Barta proposed for the classification of protozoan parasites, two are especially applicable to C. cayetanensis. First, there is an overemphasis on species of medical and veterinary importance. Thus, it is essential that other Cyclospora species are collected, examined, and sequenced so as to further resolve this genus's phylogenetic place. Second, sole reliance on the 18S rRNA is not prudent and other genes should also be examined. It has also been suggested that looking at other genomes within the organism would be wise. The similarity of C. cayetanensis to other coccidia, coupled with its maddening differences, drives us to devise and try new methods of sequence analysis, even to examine our assumptions regarding taxonomic organization. The study of C. cayetanensis vividly illustrates and thus reminds us of the interdependence between ecology, epidemiology, and genomics. Knowledge of the ecology of C. cayetanensis would also reveal the conditions leading to oocyst infectivity, which would enable epidemiologists to formulate public health recommendations to interrupt disease transmission.
This chapter provides an assessment of the extent to which the Toxoplasma gondii genome has fulfilled its promise. It describes the available genomic resources, and provides an overview of those aspects of toxoplasmosis research that have grown most as a consequence of genomic resources. To provide an estimate and description of the "attributable fraction" of progress owed to genomic biology, most of this chapter reviews the hundred (or so) papers that acknowledge several key developments in Toxoplasma gondii genomics. The chapter discusses some additional perspectives intended to expand upon and supplement the documented record of developments made possible by genomic biology, including a discussion of literature that may only indirectly establish that causal link. Finally, the chapter considers the impact that genomics has made on our understanding of T. gondii toxoplasmosis, and broader issues in eukaryotic Microbiology. Recombinant vaccine candidates for N. caninum have been identified by searching T. gondii genomic and expressed sequence tags (ESTs) databases. A genomewide survey of SNP polymorphism provided a means to more fully substantiate and explain earlier observations, that most isolates of T. gondii studied to date can be classified as members of one of three multilocus types, and that only a few ancestors could have contributed all of the diversity represented among them. In the case of T. gondii, the genomic data have encouraged the engagement of a creative and inquisitive cadre of research scientists.
Elaborate regulation of membrane trafficking is essential for the targeting of molecules necessary for pathogenesis (e.g., lectin and CPs) to precise intracellular compartments such as the surface membrane and lysosomes. This chapter discusses the diversification of cysteine proteases (CPs) and Rab in the context of their diverse localization and function. Encystation and excystation are fundamental processes required for the transmission and development of amebiasis. The current assembly predicts that the genome contains 8,160 genes, almost 1.5-fold more than the number of genes in Plasmodium falciparum (5,268) or Saccharomyces cerevisiae (5,538), and close to that in Dictyostelium discoideum (12,500). The majority of ribosomal protein genes are well conserved, and only the gene for the large subunit protein L41 has not been identified. The chapter focuses only on several important pathways closely related to the parasite's molecular epidemiology, biology, and virulence. It also talks about the insights obtained from comparing their genome information with that of E. histolytica to understand the conservation and unique evolution of the Entamoeba species. Insights into the developmental regulation of CPs and Rab small GTPases during encystation are also discussed as examples of transcriptomic approaches to understand the biology and pathogenesis of this group of enteric protozoa. To gain insights into the mechanisms of encystation-specific membrane trafficking, the authors also investigated the transcriptomic changes of Rab genes during the encystation of E. invadens. The kinetics of the steady-state levels of mRNA for individual Rab genes were categorized into cyst-specific, trophozoite-specific, or constitutive genes.
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