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The obligate intracellular pathogens Chlamydiaceae are the agents of several human diseases. They cannot be genetically manipulated and they survive and replicate in a unique intracellular organelle called the inclusion. In the past five years, publications of the genome sequences of several strains have opened new areas of research. Some of these new advances are presented here.
MOTIVATION: There is a growing literature on wavelet theory and wavelet methods showing improvements on more classical techniques, especially in the contexts of smoothing and extraction of fundamental components of signals. G+C patterns occur at different lengths (scales) and, for this reason, G+C plots are usually difficult to interpret. Current methods for genome analysis choose a window size and compute a chi(2) statistics of the average value for each window with respect to the whole genome. RESULTS: Firstly, wavelets are used to smooth G+C profiles to locate characteristic patterns in genome sequences. The method we use is based on performing a chi(2) statistics on the wavelet coefficients of a profile; thus we do not need to choose a fixed window size, in that the smoothing occurs at a set of different scales. Secondly, a wavelet scalogram is used as a measure for sequence profile comparison; this tool is very general and can be applied to other sequence profiles commonly used in genome analysis. We show applications to the analysis of Deinococcus radiodurans chromosome I, of two strains of Helicobacter pylori (26695, J99) and two of Neisseria meningitidis (serogroup B strain MC58 and serogroup A strain Z2491). We report a list of loci that have different G+C content with respect to the nearby regions; the analysis of N. meningitidis serogroup B shows two new large regions with low G+C content that are putative pathogenicity islands. AVAILABILITY: Software and numerical results (profiles, scalograms, high and low frequency components) for all the genome sequences analyzed are available upon request from the authors.
The ability of bioinformatics to characterize genomic sequences from pathogenic bacteria for prediction of genes that may encode vaccine candidates, e.g. surface localized proteins, has been evaluated. By applying appropriate tools for genomic mining to the published sequence of Haemophilus influenzae Rd genome, it was possible to identify a putative vaccine candidate, the outer membrane lipoprotein, P6. Proteomics complements genomics by offering abilities to rapidly identify the products of predicted genes, e.g. proteins in outer membrane preparations. The ability to identify the P6 protein uniquely from entries in a sequence database from the expected peptide-mass fingerprint of P6 demonstrates the power of proteomics. The application of proteomics for identification of vaccine candidates for another pathogenic bacterium, Helicobacter pylori using two different approaches is described. The first involves rapid identification of a series of monoclonal antibody reactive proteins from N-terminal sequence tags. The other approach involves identification of proteins in outer membrane preparations by 2-D electrophoresis followed by trypsin digestion and peptide mass map analysis. Our combined studies demonstrate that utilization of genome sequences by application of bioinformatics through genomics and proteomics can expedite the vaccine discovery process by rapidly providing a set of potential candidates for further testing.
DNA from five isolates of Entamoeba histolytica were examined for their pathogenicity by polymerase chain reaction. Three isolates SH-3,SH-6,SH-8 were isolated from patients with acute amoebic dysentery, whereas SH-5 and SH-7 were isolated from asymptomatic cyst passers. Gel electrophoresis of PCR products showed that primers P11, P12 for pathogenic strains could amplify genomic DNA extracted from SH-8, and primers P13, P14 for nonpathogenic strains could amplify genomic DNA extracted from SH-3,SH-5,SH-6 and SH-7. Furthermore, zymodeme analysis and the reactivity of McAb 4G6, which recognizes the 30 kDa antigen of pathogenic E. histolytica indicated that only SH-8 was pathogenic, while the others were nonpathogenic. The results of the genotypic analysis by PCR were in accord with the phenotypic properties. It is suggested that there are differences in genomic DNA between pathogenic and nonpathogenic strains. PCR is a highly sensitive and specific method for genomic DNA analysis of E. histolytica.
Since the publication of the complete genome of a pathogenic bacterium in 1995, more than 50 bacterial pathogens have been sequenced and at least 120 additional projects are currently underway. Faced with the expanding volume of information now available from genome databases, vaccinologists are turning to epitope mapping tools to screen vaccine candidates. Bioinformatics tools such as EpiMatrix and Conservatrix, which search for unique or multi-HLA-restricted (promiscuous) T cell epitopes and can find epitopes that are conserved across variant strains of the same pathogen, have accelerated the process of epitope mapping. Additional tools for screening epitopes for similarity to 'self' (BlastiMer) and for assembling putative epitopes into strings if they overlap (EpiAssembler) have been developed at EpiVax. Tools that map proteasome cleavage sires are available on the Internet. When used together, these bioinformatics tools offer a significant advantage over traditional methods of vaccine design since high throughput screening and design is performed in silico, followed by confirmatory studies in vitro. These new tools are being used to develop novel vaccines and therapeutics for the prevention and treatment of infectious diseases such as HIV, hepatitis C, tuberculosis, and some cancers. More recent applications of the tools involve deriving novel vaccine candidates directly from whole genomes, an approach that has been named 'genome to vaccine'.
Many fungal genomes have now been sequenced and thousands of genes are being discovered. Gene disruption or inactivation technology offers an important tool not only for elucidating the function of the many unknown genes but also for the identification of genes essential for fungal growth and pathogenesis. A variety of gene-silencing methods that inhibit genes at the post-transcriptional level are now being used in both non-pathogenic and human pathogenic fungi. We focus on the recent advances in RNA-mediated gene silencing technologies and their potential for functional genomics studies in fungi.
It has been proposed that a multivalent malaria vaccine is necessary to mimic the naturally acquired resistance to this disease observed in humans. A major experimental challenge is to identify the optimal components to be used in such a multivalent vaccine. Expression library immunization (ELI) is a method for screening genomes of a pathogen to identify novel combinations of vaccine sequences. Here we describe immune responses associated with, and the protective efficacy of, genomic Plasmodium chabaudi adami DS expression libraries constructed in VR1020 (secretory), monocyte chemotactic protein-3 (chemoattractant), and cytotoxic T lymphocyte antigen 4 (lymph node-targeting) DNA vaccine vectors. With splenocytes from vaccinated mice, specific T-cell responses, as well as gamma interferon and interleukin-4 production, were observed after stimulation with P. chabaudi adami-infected erythrocytes, demonstrating the specificity of genomic library vaccination for two of the three libraries constructed. Sera obtained from mice vaccinated with genomic libraries promoted the opsonization of P. chabaudi adami-infected erythrocytes by murine macrophages in vitro, further demonstrating the induction of malaria-specific immune responses following ELI. Over three vaccine trials using biolistic delivery of the three libraries, protection after lethal challenge with P. chabaudi adami DS ranged from 33 to 50%. These results show that protective epitopes or antigens are expressed within the libraries and that ELI induces responses specific to P. chabaudi adami malaria. This study further demonstrates that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.
The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.
Salmonella typhi, the etiologic agent of typhoid fever, is adapted to the human host and unable to infect nonprimate species. The genetic basis for host specificity in S. typhi is unknown. The avirulence of S. typhi in animal hosts may result from a lack of genes present in the broad-host-range pathogen Salmonella typhimurium. Genomic subtractive hybridization was successfully employed to isolate S. typhimurium genomic sequences which are absent from the S. typhi genome. These genomic subtracted sequences mapped to 17 regions distributed throughout the S. typhimurium chromosome. A positive cDNA selection method was then used to identify subtracted sequences which were transcribed by S. typhimurium following macrophage phagocytosis. A novel putative transcriptional regulator of the LysR family was identified as transcribed by intramacrophage S. typhimurium. This putative transcriptional regulator was absent from the genomes of the human-adapted serovars S. typhi and Salmonella paratyphi A. Mutations within this gene did not alter the level of S. typhimurium survival within macrophages or virulence within mice. A subtracted genomic fragment derived from the ferrichrome operon also hybridized to the intramacrophage cDNA. Nucleotide sequence analysis of S. typhimurium and S. typhi chromosomal sequences flanking the ferrichrome operon identified a novel S. typhimurium fimbrial operon with a high level of similarity to sequences encoding Proteus mirabilis mannose-resistant fimbriae. The novel fimbrial operon was absent from the S. typhi genome. The absence of specific genes may have allowed S. typhi to evolve as a highly invasive, systemic human pathogen.
To identify putative new virulence factors of avian pathogenic Escherichia coli (APEC) strains, a genomic subtraction was performed between the APEC strain MT512 and the non-pathogenic E. coli strain of avian origin EC79. Seventeen DNA fragments were cloned that were specific for the APEC strain. Among them, nine were identified that were more frequent among pathogenic than non-pathogenic isolates in a collection of 67 avian E. coli. Chromosome or plasmid location, and the nucleotide sequence of these nine fragments were characterized. Four fragments were plasmid-located. The nucleotide sequence of two of them exhibited identity with the sequence of the RepF1B replicon of E. coli plasmids, and the amino-acid deduced sequences from the two other fragments exhibited similarity to the products of genes sitA of Salmonella Typhimurium and iroD of E. coli, which are involved in iron metabolism. Of the five chromosome-located fragments, three were predicted to encode parts of proteins that were significantly homologous to previously described proteins: TktA (transketolase) of Haemophilus influenzae, a FruA (fructokinase) homologue of Listeria innocua and Gp2 (large terminal subunit) of phage 21. The putative products of the two other chromosome-located fragments were homologous to proteins with unknown functions: Z0255 of E. coli strain EDL933 (EHEC) and RatA of Salmonella Typhimurium strain LT2. Both these chromosomal fragments, whose presence is correlated with serogroups O1 and O2 and to the virulence of APEC strains belonging to these serogroups, are good candidates for being part of novel virulence determinants of APEC. Moreover, several fragments were shown to be located close to tRNA selC, asnT or thrW, which suggests they could be part of pathogenicity islands. Six fragments that were shown to be part of whole ORFs present in the APEC strain MT 512 were also present in extra-intestinal pathogenic E. coli (ExPEC) strains of human and animal origin. Thus, the putative novel virulence factors identified in this study could be shared by ExPEC strains of different origins.
Relatively small genomes and high replication rates allow viruses and bacteria to accumulate mutations. This continuously presents the host immune system with new challenges. On the other side of the trenches, an increasingly well-adjusted host immune response, shaped by coevolutionary history, makes a pathogen's life a rather complicated endeavor. It is, therefore, no surprise that pathogens either escape detection or modulate the host immune response, often by redirecting normal cellular pathways to their advantage. For the purpose of this chapter, we focus mainly on the manipulation of the class I and class II major histocompatibility complex (MHC) antigen presentation pathways and the ubiquitin (Ub)-proteasome system by both viral and bacterial pathogens. First, we describe the general features of antigen presentation pathways and the Ub-proteasome system and then address how they are manipulated by pathogens. We discuss the many human cytomegalovirus (HCMV)-encoded immunomodulatory genes that interfere with antigen presentation (immunoevasins) and focus on the HCMV immunoevasins US2 and US11, which induce the degradation of class I MHC heavy chains by the proteasome by catalyzing their export from the endoplasmic reticulum (ER)-membrane into the cytosol, a process termed ER dislocation. US2- and US11-mediated subversion of ER dislocation ensures proteasomal degradation of class I MHC molecules and presumably allows HCMV to avoid recognition by cytotoxic T cells, whilst providing insight into general aspects of ER-associated degradation (ERAD) which is used by eukaryotic cells to purge their ER of defective proteins. We discuss the similarities and differences between the distinct pathways co-opted by US2 and US11 for dislocation and degradation of human class I MHC molecules and also a putatively distinct pathway utilized by the murine herpes virus (MHV)-68 mK3 immunoevasin for ER dislocation of murine class I MHC. We speculate on the implications of the three pathogen-exploited dislocation pathways to cellular ER quality control. Moreover, we discuss the ubiquitin (Ub)-proteasome system and its position at the core of antigen presentation as proteolysis and intracellular trafficking rely heavily on Ub-dependent processes. We add a few examples of manipulation of the Ub-proteasome system by pathogens in the context of the immune system and such diverse aspects of the host-pathogen relationship as virus budding, bacterial chromosome integration, and programmed cell death, to name a few. Finally, we speculate on newly found pathogen-encoded deubiquitinating enzymes (DUBs) and their putative roles in modulation of host-pathogen interactions.
Goose astrovirus (GAstV) is a significant pathogen affecting goslings by inducing visceral gout, yet no commercial vaccine is currently available. This study involved the serial passaging of the GAstV-GXNN strain in LMH cells to investigate alterations in viral replication, genomic stability, and pathogenicity, as well as to assess the potential of a vaccine candidate. The findings indicated that the viral titer increased progressively with each passage, reaching 107.35 TCID50/mL by the 120th passage (GAstV-GXNNP120). Whole-genome sequencing revealed the presence of 6, 19, 26, and 28 nucleotide mutations at the 30th, 60th, 90th, and 120th passages, respectively. Pathogenicity assays demonstrated a reduction in virulence with successive passages, culminating in the complete attenuation of GAstV-GXNNP120, which did not induce clinical signs or lesions in one-day-old goslings. Following five successive passages in goslings, the attenuated strain exhibited stable genetic characteristics without any reversion to virulence. Goslings aged one day, inoculated with GAstV-GXNNP120 at dosages ranging from 102.0 to 105.0 TCID50, developed neutralizing antibodies by the third day post-vaccination. Antibody levels increased in a dose-dependent manner, peaking at day 21 and remaining elevated through day 42. Challenge experiments utilizing the virulent GAstV-GXNN strain revealed that groups vaccinated with doses of 103.0 TCID50 and above achieved complete protection. These groups exhibited no clinical symptoms or pathological damage post-challenge, and both tissue viral loads and virus shedding levels were significantly reduced compared to the control group. Consequently, the minimum effective vaccination dose was established at 103.0 TCID50. These results provide a crucial foundation for the development of a live attenuated GAstV vaccine.
An analysis of genome polymorphism of the Y. pestis strains by using the method of polymerase chain reaction (PCR) for the tandem repeats of bacteriophage M13 DNA revealed a species similarity of both typical and atypical (according to diagnostic signs) plague-microbe strains. Strain Y. pestis A-1726 with the atypical differential-and-diagnostic properties, without the amplicon specified for Y. pestis and sized 1000 b.p., was identified among 27 analyzed Y. pestis strains. The amplicon profiles of the basic Y. pestis subtype were found to be different from such profiles of other Y. pestis subtypes.
The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.
Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium-properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain-Barré syndrome. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.
The study of microbial intracellular pathogenesis has benefited from the application of immunofluorescence microscopy to characterize interactions of the pathogen with host cells. Unfortunately, immunofluorescence microscopy is impractical for screening the large number of bacterial mutants necessary to represent the entire genome of the pathogen. Screening has been limited due to the lack of materials suitable for high-throughput processing (e.g. 96-well plates) that also possess the optical features needed for high resolution fluorescence microscopy. Recently marketed 96-well Special Optics (SO) plates provide both the 96-well template ideal for high-throughput analysis and optical features suitable for fluorescence microscopy. Until this work, mutants needed for the study of a fluorescence-based virulence phenotype could not be obtained by direct screening approaches. In this study, SO plates were used to examine 11520 individual Salmonella typhimurium MudJ mutants for the loss of the ability to disrupt host cell endocytic compartments. The direct application of the fluorescence phenotype for screening allowed us to obtain a set of mutants to characterize the formation of lysosomal membrane glycoprotein (lgp) containing tubules upon Salmonella infection of HeLa epithelial cells. This approach will facilitate the characterization of a wide range of microbial phenotypes detectable by fluorescence microscopy.
Identification of novel drug targets is required for the development of new classes of drugs to overcome drug resistance and replace less efficacious treatments. In theory, knowledge of the entire genome of a pathogen identifies every potential drug target in any given microbe. In practice, the sheer complexity and the inadequate or inaccurate annotation of genomic information makes target identification and selection somewhat more difficult. Analysis of metabolic pathways provides a useful conceptual framework for the identification of potential drug targets and also for improving our understanding of microbial responses to nutritional, chemical and other environmental stresses. A number of metabolic databases are available as tools for such analyses. The strengths and weaknesses of this approach are discussed.
Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic P. aeruginosa strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from P. aeruginosa strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human P. aeruginosa isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for "P. aeruginosa genomic island 1"). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of P. aeruginosa isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow P. aeruginosa to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the P. aeruginosa chromosome, which provides a selective advantage for strains that have acquired this genomic island.