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Laboratory of Genetics and Genome Center,1 Animal Health and Biological Sciences and Genome Center,4 Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706,6 Infectious Disease Division, Massachusetts General Hospital, Boston, Massachusetts 02114,2 Department of Enteric Infections, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910,3 Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712-10955
Received 18 October 2002/ Returned for modification 3 January 2003/ Accepted 18 February 2003
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Shigella is pathogenic only for humans. It causes disease by invading the epithelium of the colon, resulting in an intense acute inflammatory response (51). Shigella strains are unusual among enteric bacteria in their ability to gain access to the epithelial cell cytosol, where they replicate and spread directly into adjacent cells. Shigella strains contain a large virulence plasmid that is known to encode genes required and sufficient for invasion of epithelial cells (61). However, chromosomal genes present in "pathogenicity islands" also participate in the pathogenic process directly or contribute to survival in the environments encountered during infection (2, 21, 22, 49, 58, 70). The genetic bases for several aspects of the pathogenic process and intracellular lifestyle of Shigella, including the mechanisms of species specificity, tissue tropism, and restriction of the immune response, are still poorly understood (Table 1) and probably involve chromosomally encoded proteins. In common with other enteric bacteria, Shigella survives the proteases and acids of the intestinal tract by uncertain means. Highly tissue-specific disease results from a very low infectious dose (10 to 100 bacteria) and in the absence of flagellum-based motility. We selected the virulent strain 2457T of Shigella flexneri serotype 2a (33) for sequencing because it has been widely used for genetic research and for clinical challenge studies. Although Shigella spp. have been regarded as distinct from Escherichia coli, as early as 1972, DNA hybridization studies estimated that Shigella and E. coli are taxonomically indistinguishable at the species level (5). Recent work of the Reeves group (34, 56, 57) based on multilocus enzyme electrophoresis and sequencing of a small number of genes places Shigella clearly within the genus Escherichia and arising several times independently. Comparison of the complete S. flexneri genome sequence with that of E. coli K-12 establishes the precise genetic relationship of S. flexneri to E. coli. Given the markedly different lifestyles of intracellular Shigella and extracellular E. coli, the comparison should also reveal important genetic differences expected to underlie pathogenesis, other than the presence or absence of the virulence plasmid.
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MATERIALS AND METHODS |
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Genomic DNA preparation, libraries, and sequencing. Bacteria were grown in Luria-Bertani (LB) medium at 37°C, and genomic DNA was prepared by R. A. Welch at the University of Wisconsin. The genomic DNA was released from bacteria embedded in agarose to prevent shearing during preparation (44). Whole-genome libraries in M13Janus (7) and pBluescript KS- (Stratagene) were prepared by using nebulization to randomly shear genomic DNA extracted from agarose by digestion with Gelase (Epicentre) (44). Random clones were sequenced by Applied Biosystems Prism dye-terminator chemistry, and data were collected with ABI377 and 3700 automated sequencers. Sequence reads (66,219 with an average length of 502 nucleotides [nt]) were assembled by Seqman Genome Edition (DNASTAR). Additional PCRs and sequencing reactions were performed to close gaps, improve coverage, and resolve sequence ambiguities. The final coverage was 7.2X. A whole-genome optical map (38) for restriction enzyme XhoI was prepared to aid the ordering of contigs during assembly and so that the end points and lengths of inversions could be confirmed.
Sequence analysis. Potential open reading frames (ORFs) were defined by GeneMark.hmm (42) or Genequest (DNASTAR). All predicted proteins larger than 30 amino acids were searched against the nonredundant and local databases. tRNAs were identified with tRNAscan-SE (40). Alternative translation start sites were chosen to conform to the annotated MG1655 sequence. Frameshifts and point mutations were carefully verified for authenticity, and disrupted genes with homologs in K-12 were annotated as "pseudogenes." Predicted backbone proteins were considered to be orthologs when matches to the corresponding K-12 protein exceeded 90% amino acid identity, alignments included at least 90% of both proteins, and no equivalent match was found elsewhere in the 2457T genome. The protein-level matches were also individually inspected to include genes with lower similarities within colinear regions of the genomes. The genome sequence was compared with that of MG1655 by the modified maximal exact match (MEM) alignment utility that was used for the comparison of EDL933 and K-12 (54). The genomic comparison with strain 301 was performed by a new multigenome comparison tool, Mauve.
Nucleotide sequence accession number. The complete, annotated sequence was deposited in GenBank under accession no. AE014073.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ISs.
The S. flexneri chromosome was known to be rich in insertion sequences (45, 53). The IS elements we identified (Table 2) make up 6.7% (309.4 kb) of the chromosome, in contrast to the typical ranges of 0 to 
4%. The archaeon Sulfolobus solfataricus is a significant exception, because 10% of its 2.99-Mb genome is composed of ISs, which is unusual even among archea. In the sequenced E. coli genomes, the IS content is <1.5%, and in Y. pestis, the IS content is 3%. The virulence plasmid of S. flexneri also has an extremely high IS content (53% of the plasmid-encoded genes) (69). Of the 284 IS elements in 2457T, 108 are IS1X1 copies. The intact IS1 elements in this genome are typically families with 98 to 100% nucleotide sequence identity. Forty-six IS1 elements still have detectable flanking direct repeats, indicating recent acquisition (20 are full length, 9 bp; 24 are 8 bp; and 2 are 7 bp), and relatively little amelioration has occurred within these IS1 sequences. Comparative genome analysis with E. coli K-12 showed that 156 IS elements are involved in deletions or inversions associated with backbone rearrangements or with presumed horizontal transfer. The arrangements of several nested clusters of IS indicate that at each cluster, one integrated IS has acted as a target for subsequent insertions, resulting in multiple disrupted elements, with only the most recently acquired IS remaining intact.
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150 single-nucleotide differences distinguishing them. In the course of assembling the genome sequence of S. flexneri 2457T, we also unexpectedly identified a fourth plasmid of 165 kb. This was an S. enterica serovar Typhi R27-like plasmid, which we named "pSf-R27." The R27 plasmid (62) was thought to be limited to Salmonella, in which it is implicated in the accumulation and spread of antibiotic resistance, but more recently, the similarity noted between R27 and pMT1, the large virulence plasmid of Y. pestis, suggested that there may have been a common ancestral plasmid. Sequence comparison showed that in pSf-R27, Tn10 (carrying tetracycline resistance genes), IS30, and a citrate uptake locus are absent, while the rest of the plasmid is 99.7% identical to R27. PCR was used to screen 142 S. flexneri isolates, including 57 of serotype 2a, for R27 sequences. The sequenced strain, 2457T, was the only strain to give a positive result. 2457T isolates from two other research groups that had obtained the strain from the same source were screened; the plasmid was found in one but not the other. Since 2457T was originally isolated before antibiotic usage had become widespread, it is possible that pSf-R27 may represent a primordial state of the R plasmid subsequently lost from the negative isolate, although we cannot formally exclude the possibility that pSf-R27 was accidentally introduced shortly after the strain was first isolated. Pseudogenes. While islands represent insertions into the S. flexneri genome, there are also a large number of gene disruptions and deletions. Disruptions resulted in 372 pseudogenes (8.1% of the genome), caused by several mechanisms, including single-nucleotide indels, point mutations, and IS elements. (IS alone accounts for 27 disruptions and 85 truncations.) Larger IS-mediated deletions and insertions are also seen. In total, 879 genes of K-12 are either absent or are pseudogenes in S. flexneri. Many types of function are missing (Table 4). The missing function is sometimes supplied by a plasmid- or island-encoded gene. The chromosomal fepE is a pseudogene; FepE is a homolog of Cld in K-12, encoding an O-antigen chain-elongation factor. An intact homolog is found on one of the small multicopy S. flexneri plasmids, and this FepE function is required for virulence (23, 65). Similarly, the mhp operon of K-12 is involved in catabolism of small aromatic molecules. Although it is missing from S. flexneri, an alternative system with similar activity is encoded by the hpa locus present on an island. This locus is also found in E. coli C and W and Y. pestis, but not K-12. K-12 genes missing from the S. flexneri backbone are clustered in K-12, suggesting either a single deletion event for each group in S. flexneri or their absence from a common ancestor, with later acquisition by K-12 via horizontal transfer. As an example, the island at tRNA leuX is completely different in K-12, EDL933, CFT073, and 2457T. Clearly, the four strains acquired these islands by distinct events, even if some could have been replacements rather than insertions. Phenotypic tests that have been widely used to distinguish E. coli from S. flexneri are largely explained by pseudogenes, which account for loss of flagellar motility; utilization of mucate, acetate, various sugars, and glycerol; and the requirement for NAD.
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The genome of 2457T shows rearrangements relative to strain 301 (Fig. 4) as well as, and distinct from, those relative to K-12. Around the origin of replication, strain 301 is colinear with K-12, whereas 2457T is not. Around the terminus, a large inversion in 2457T relative to strain 301 was followed by reinversion of most of the DNA within the rearrangement (Fig. 4), leaving two small patches of inverted sequence marking the end points of the initial event. These recombinations were apparently mediated by IS elements.
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The annotated strain 301 genome sequence shows 254 pseudogenes, compared with 372 pseudogenes in 2457T. Some of these differences are due to individual annotation criteria and styles, but 159 are pseudogenes in both strains, of which 42 have unknown functions. Each strain has its own unique set of pseudogenes. Those with known or predicted functions are listed in Table 5: 100 pseudogenes in 2457T and 20 in strain 301. The significance, if any, of the backbone and pseudogene complements of the two strains remains unclear.
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The IpaH proteins encoded on the virulence plasmid of S. flexneri (8, 68) consist of a conserved C-terminal domain and a variable N terminus containing a leucine-rich repeat (LRR). They are secreted by the plasmid type III secretion system, and at least one (IpaH7.8) has been shown to aid S. flexneri in escaping from the macrophage vacuole and is considered to be a virulence factor (16). There are five copies of ipaH on the plasmid, and we found seven more in the 2457T genome, of which four are intact, containing both the LRR domain and the conserved region. The genome sequence of strain 301 (25) also revealed four complete and three incomplete genomic copies. Figure 5 illustrates the differences between the ipaH genes in the two genomes. In both genomes, the incomplete copies are disrupted by insertion sequences or frameshifting mutations. One of the incomplete 2457T copies is highly divergent from all of the other ipaH genes.
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DISCUSSION |
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While a specific host cell receptor may not be the only valid explanation for host specificity, it is consistent with experimental data and in vivo observations. We emphasize that there are clear differences among the consequences of infection of cultured mammalian cells and inoculation of mice or humans. When grown as a nonpolarized and nonconfluent monolayer, cells from a wide variety of hosts and anatomic origins are readily invaded by S. flexneri. When grown as a polarized and confluent monolayer, S. flexneri invades cells only at the basolateral membrane (50). However, in the context of an intact animal host, only cells of the human or monkey colonic mucosa or mouse respiratory epithelium have been shown to be infected by S. flexneri. S. flexneri strains have not been shown to cause intestinal disease in nonprimates, and in mice, S. flexneri strains appear not to invade the colonic mucosa (M. B. Goldberg, unpublished data). Thus, while alternative explanations of S. flexneri species and tissue specificity exist, a specific receptor on polarized primate colonic cells might be involved in the specific invasion of this tissue. In particular, such a receptor might be important to S. flexneri gaining access to the basolateral sides of these cells.
Expression of receptor candidate proteins in nonpathogenic E. coli and screening for adherence to appropriate human tissue (24) might then allow the unique human cellular receptor to be identified (36). From there, the construction of a transgenic mouse model for S. flexneri infection is possible, as reported for Listeria monocytogenes (37), another human-specific intestinal pathogen that causes disease in humans but not mice. An improved animal model will greatly facilitate evaluation of candidate genes with possible roles in virulence.
Experimental evidence suggests that IpaH proteins may play a role in modulating the host response to infection. IpaH7.8 on the invasion plasmid was shown to help S. flexneri escape from macrophage vacuoles (16). Mutations in two ipaH genes on the invasion plasmid induce an exaggerated keratoconjunctivitis response with greater-than-normal inflammation in guinea pig eyes, and IpaH9.8 encoded on the plasmid was shown to translocate to the host nuclei in tissue culture cells (67), but the precise functions of these proteins remain unknown. Unlike the ipaH genes on the invasion plasmid, the genome-encoded ipaH genes are mostly associated with prophage-like islands, reminiscent of the Salmonella lambda-like Gifsy prophages, which encode effector proteins of the YopM/IpaH family (48). Lysogenic conversion with these phages is responsible for much of the diversity of the effector protein repertoires observed among Salmonella spp. (48). The finding that ipaH genes on the plasmid and chromosome may show strain-specific differences in sequences is a novel observation and might suggest that, like in Salmonella, the ipaH gene family might contribute to diversity of effector molecules. This remains to be tested.
IpaH proteins belong to the superfamily of LRR-containing proteins, which includes members from bacteria, plants, and vertebrates (6, 27). The conservation level of these proteins indicates that the LRR probably has structural or functional significance. IpaH-like proteins are found in the animal pathogens Salmonella, Yersinia, and Listeria, as well as the plant pathogens Rhizobium, Bradyrhizobium, and Ralstonia, again often associated with prophage (9, 18, 20, 35). In many host organisms, including plants, receptors involved in recognizing invading pathogens are also LRR proteins: for example, mammalian Toll-like receptors and the NB/LRR family in plants (1, 26). Experimental evidence accumulating from various studies of host-pathogen interactions is beginning to suggest that the bacterial effector proteins might interfere with or modulate the host receptor activity, presumably enabling the pathogen to evade the host's defensive response.
Acquisition of new traits by horizontal transfer has enabled microorganisms to survive in new niches. A complementary loss-of-function mechanism has been proposed (52, 64) by which virulence is enhanced through mutation of ancestral genes encoding factors that interfere with the expression or function of traits necessary for success in the new environment. Acquisition of the virulence plasmid enabled S. flexneri to enter the highly specialized intracellular environment in human intestinal epithelial cells. In this new niche, genes that were required in the intestinal lumen may be deleterious or are no longer beneficial and may accumulate mutations without a selective force to maintain them. Lysine decarboxylase (CadA) produces cadaverine, which inhibits the escape of S. flexneri from the vacuole into the cell cytosol (15, 46). Since S. flexneri replication and spread are dependent upon its access to the cytosol, biosynthesis of cadaverine attenuates virulence. In 2457T, cadA and cadC, which encodes a transcriptional activator of the cad operon, are deleted (entirely absent from the genome). Lack of surface structures such as flagella, fimbriae, and curli in S. flexneri provides the advantage of fewer antigens that can be easily recognized by the host immune system. In 2457T, of 14 dysfunctional genes of flagellar biosynthesis, 11 (fliF, fliJ, fliP, flgC, flgE, flgF, flgK, flgL, flhA, flhB, and cheR) contain frameshifts and 1 (fliA) contains a point mutation, while IS1 elements truncate flhD and flhE.
Although invasion and intercellular spread are well studied (51), many of the signaling and gene expression controls that orchestrate these processes are unknown (Table 1) and might provide new points of therapeutic intervention. Although S. flexneri is an intracellular pathogen, adaptive immunity to S. flexneri may be restricted to B-lymphocyte-dependent humoral responses. Human adaptive immunity is serotype specific, and exposure induces production of specific immunoglobulins (17, 59). In mouse models, adaptive immunity is completely independent of T-lymphocyte function (72). However, the mechanism by which S. flexneri modulates T-lymphocyte responses is unknown. With the sequence known, gene chips could now be used to interrogate expression profiles during infection, identifying all of the genes responding to the various changing conditions of particular interest, including oxidation, temperature shift, and iron depletion, which are specifically induced in the intracellular environment.
The high incidence of shigellosis and the proliferation of drug resistance have spurred serious efforts in vaccine development. Some success has been reported with live attenuated bacteria with mutations in the plasmid gene virG (necessary for intercellular spread), both alone and in combination with chromosomal deletions of aroA (aromatic amino acid synthesis), iuc (aerobactin), set (enterotoxin), or guaBA (purine biosynthesis pathway) (29-31). New candidate genes, when characterized, will provide alternative routes to further attenuation while maintaining antigenicity.
Because of its ability to enter into the cytosol of mammalian cells, S. flexneri strains have been developed as a delivery vehicle of antigens to major histocompatibility complex class I for immunization or of DNA into target cells for gene therapy (3, 12, 14, 63). Again, optimization of these approaches will require sufficient attenuation of the S. flexneri vehicle, specific binding to target cells, and controlled modulation of the immune response.
Knowledge of all the proteins encoded in the 2457T genome provides the entire repertoire of surface proteins that are potential vaccine targets, and candidates found to be adequately antigenic could therefore be used singly or in combination, engineered for expression from recombinant constructs, or even used directly in DNA vaccines. The sequence will also facilitate identification of many of the corresponding vaccine candidate genes in other S. flexneri serotypes, both type specific or in common. Comparison with the genome of nonpathogenic E. coli will reveal factors that, like cadaverine, block or limit survival of S. flexneri in host tissue. Thus, functions no longer active (pseudogenes) in S. flexneri but expressed in nonpathogenic E. coli may lead to the development of novel S. flexneri-specific therapies by virtue of a suppressive effect on bacterial growth or tissue invasion. These genome-driven research activities will serve as starting points for a new phase of vaccine and molecular pathogenicity investigation.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grants AI-44387 to F.R.B and AI-43562 to M.B.G.
J.W., M.B.G., and V.B. contributed equally to this work.
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FOOTNOTES |
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Paper no. 3603 from the Laboratory of Genetics.
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