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Prokaryotic Chromosomes and Disease

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Science  08 Aug 2003:
Vol. 301, Issue 5634, pp. 790-793
DOI: 10.1126/science.1086802


Recent insights into bacterial genome organization and function have improved our understanding of the nature of pathogenic bacteria and their ability to cause disease. It is becoming increasingly clear that the bacterial chromosome constantly undergoes structural changes due to gene acquisition and loss, recombination, and mutational events that have an impact on the pathogenic potential of the bacterium. Even though the bacterial genome includes additional genetic elements, the chromosome represents the most important entity in this context. Here, we will show that various processes of genomic instability have an influence on the many manifestations of infectious disease.

Roughly 5000 bacterial species have been described, representing a mere 0.5 to 1% of the total number of prokaryotes. Only an extremely small portion of these microbes, about 200 species, are known to cause disease in humans. Yet, for some of the most feared diseases, the infection dose required may be exceedingly small: it takes on average only 10 microbes of Yersinia pestis to cause bubonic plague and only 100 microbes of certain Shigella species to initiate severe dysentery. Considering the impact that pathogenic microorganisms had on human history and considering that infectious disease is still the principal threat to human health today, it is important to ask how pathogenic bacteria cause disease.

Virtually every niche of the human body that can be colonized by bacteria is prone to infection. Fortunately, most bacteria residing in or on the human body are harmless commensals, for example, those that occur in the intestine. Current theory holds that the majority of disease-causing bacteria from the intestine may have been derived from commensals that have acquired genes from foreign sources turning them into pathogens. Another important mechanism by which harmless bacteria may turn into pathogens is change of host or host niche, upon which their virulence potential is frequently revealed to its full extent. Certain bacterial diseases caused by Y. pestis, Salmonella enterica, Borrelia burgdorferi, or multiresistant enterococci are dramatic examples underscoring the relevance of the host side of infection.

With the advent of DNA sequencing, it has become possible to correlate infectious disease with prokaryotic genome structure. The sequence data of more than 50 fully annotated genomes of pathogenic and nonpathogenic bacteria have allowed the identification of unifying patterns as well as differences among genomes of pathogenic and closely related, nonpathogenic bacteria. It has also revealed mechanisms that promote genome plasticity, such as horizontal gene transfer, genome reduction, genome rearrangements, and the generation of point mutations. Moreover, the discovery of super-integrons has altered our understanding of infectious disease. Here, the relationships between genome evolution and disease that have emerged recently are discussed.

Evolution of Pathogens

Horizontal gene transfer (HGT) is the process by which genetic information is passed from one bacterial genome to another (1, 2) (Fig. 1). HGT is especially important in the evolution of pathogenic lifestyles as infection-related factors can be transmitted in a single-step integration event. The three most important characteristics are as follows:

Fig. 1.

Evolution of different variants of pathogenic and symbiotic γ-proteobacterial variants by acquisition and loss of genetic information from a common bacterial ancestor (e.g., S. enterica, Shigella spp., uropathogenic E. coli and the endosymbionts of aphids, B. aphidicola, and ants, Blochmannia spp. Abbreviations are as follows: cadA, lysine decarboxylase-encoding gene; ompT, outer membrane protein T-encoding gene; PAI, pathogenicity island; EIEC, enteroinvasive E. coli; UPEC, uropathogenic E. coli.

Antibiotic resistance. Resistance determinants of Gram-negative bacteria are often associated with mobile or transferable genetic elements such as plasmids, integrons, super-integrons, and complex transposons. Furthermore, pathogenic variants of Gram-positive cocci (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis) causing severe cases of sepsis and catheter-associated infections in hospitals carry genomic islands encoding methicillin resistance and/or large transposons responsible for, e.g., vancomycin resistance (35). Integrons are natural cloning and expression systems that incorporate open reading frames and convert them into functional genes. This allows the accumulation of large arrays of gene cassettes that can eventually be transferred as a whole between different replicons (6). They are also the primary system for antibiotic resistance and virulence gene capture in Gram-negative enterobacteria. Super-integrons represent another type of integron that occurs in many genera of the γ-Proteobacteria and are far superior in their ability to “stockpile” gene cassettes of different functions, including virulence traits.

Pathogenicity. Virulence genes are frequently located on mobile or formerly mobile genetic elements including pathogenicity islands (PAIs) that are present in Gram-negative and Gram-positive bacteria (79). PAIs represent large chromosomal regions of horizontally acquired DNA that are believed to have evolved from former lysogenic bacteriophages and plasmids. The subsequent bacterial acquisition of virulence-associated factors encoded on different mobile genetic elements indicates a functional interdependency between such factors. Accordingly, the virulence factor SseI encoded by the Gifsy 2 phage in S. enterica sv. Typhimurium is secreted by a type III secretion system that is itself encoded on the pathogenicity island SPI-2. Moreover, it is becoming increasingly clear that independent transfer events can have synergistic effects. For example, this phage also encodes another virulence factor, GtgE, (10) and a superoxide dismutase, SodC, which acts as a fitness factor. The combination of phage- and PAI-encoded factors, both offensive and defensive, supports infections due to S. enterica sv. Typhimurium.

The integration of newly acquired genetic elements into general regulatory circuits as well as the coordination of their expression is a prerequisite for optimal function. The genes mgtC and sopD2, involved in the invasive phenotype of S. enterica, are regulated by the two-component regulatory systems phoP/Q- and ssrA/B, respectively (11, 12). In the first case, the horizontally acquired gene comes under the control of preexisting regulators. In the latter case, the regulator itself was introduced on a pathogenicity island and has come to control the regulation of transcription of phage-encoded genes. The mechanisms by which newly acquired elements are harnessed by preexisting networks and by which compatibility of different genetic systems is ensured are as yet unkown.

Fitness traits. Many horizontally acquired determinants are involved in metabolic adaptation and increasing survival of the bacterium. These traits are found in commensal and pathogenic bacteria alike. For example, the so-called “high pathogenicity island” initially described in the highly virulent Yersiniae has subsequently been found in nonpathogenic enterobacteria (13, 14). Comparative analysis of the complete genome sequences of Escherichia coli and S. enterica variants has revealed that none of the phenotype traits that distinguish the two species are attributable to individual point mutations. Instead, speciesspecific traits derive from functions encoded either by horizontally acquired genes (e.g., lactose, citrate, and propanediol utilization, indole production) or from the loss of ancestral genes (e.g., alkaline phosphatase).

There is evidence that HGT between bacteria can occur during infection, for example with Campylobacter jejunii, which causes diarrhea (15), or even during passage of Y. pestis through an insect vector (16). Although these and other observations suggest that environmental stress can stimulate HGT, the signals that trigger this event in vivo are, for the most part, unknown. Transfer RNA (tRNA) genes frequently serve as integration sites for mobile elements, but the mechanism for this selective integration is unknown. Because tRNA gene sequences are highly conserved, they may increase the host range of a mobile element. Additionally, tRNA genes are generally transcriptionally active ensuring immediate expression of acquired genes. After integration of mobile genetic elements into tRNA genes by site-specific recombination, the tRNA genes remain functional. They also exhibit symmetric nucleotide sequences in the stem loops facilitating the binding of integrases. The association with particular so-called “minor” tRNAs may also have modulatory effects on the translational efficiency of target genes (17).

Genome Reduction in Pathogenic and Symbiotic Bacteria

Because bacterial genomes are not growing ever larger in size, the acquisition of foreign genetic elements must be counterbalanced by the loss of native genes. Deletional bias is a major force shaping bacterial genomes. In some cases, the loss of gene function may provide a selective advantage, as exemplified by the beneficial loss of metabolic genes (termed “black holes”) (18). Many unexpressed pseudogenes of the pathogen Y. pestis are functional in other Yersinia species (19), implying that gene loss contributes to the adaptation of Y. pestis to its insect vector, which is a prerequisite for transmission of this pathogen from rodents via pest fleas to humans.

Analysis of genome sequence information of various obligately intracellular bacteria (pathogenic or symbiotic), such as Chlamydia spp., Rickettsia spp., Buchnera aphidicola, and Blochmannia spp., shows that these bacterial genomes have lost large amounts of DNA. This phenomenon also emphasizes the similar mechanisms between pathogens and symbionts (20, 21). Genes that confer metabolic traits necessary for niche adaptation are maintained, whereas those that do not provide a selective benefit are lost. Eventually, the optimization of these processes shapes the genome architecture of a microorganism (Fig. 1). In bacteria that have been associated with hosts for evolutionarily long periods of time, the genome structure frequently reflects the lifestyle of the bacterium (22). Accordingly, intracellular symbionts contain genes encoding beneficial functions that may supplement nutrition of their hosts, whereas intracellular parasites eventually cause host damage.

It is tempting to speculate that the loss of genetic information is programmed in some way to ensure long-term persistence in the host. This hypothesis is supported by the observation that the virulence potential of uropathogenic E. coli isolated from acute infections differs markedly from those recovered from chronic infections. This phenotypic modulation under in vivo conditions leads to an irreversible loss of genes, gene blocks, or even entire PAIs during infection (7). Apparently, less virulent variants are better adapted for a long-term colonization than their highly pathogenic counterparts. The signals and enzymes involved in the directed loss of genetic information or “phase variation,” i.e., the switch between an “on” and “off” status of gene expression, during the course of the infection remain to be resolved.

DNA Rearrangements

Bacterial genomes constantly undergo rearrangements. DNA repeats and gene paralogs can mediate intragenomic recombination events that can simultaneously alter the expression of disease-associated genes. Genome rearrangements often play a role in surface structure variation to circumvent confrontation with the host immune system (Fig. 2). Phase variation has been described for type 1 fimbriae (Fim) expression in pathogenic E. coli. Type I fimbriae production is increased during urinary tract infection promoting colonization by uropathogenic E. coli strains. Phase variation results from a stimulation of FimB recombinase expression in vivo (23); however, the stimuli for the preferential in vivo “on” status of the fim switch are not known. Transposition and precise excision of accessory genetic elements [e.g., insertion sequences (IS)] can also cause phase variation, e.g., for biofilm formation of the nosocomial pathogen S. epidermidis (24).

Fig. 2.

Mechanisms contributing to chromosomal variability of pathogens. Chromosomal variations can result from (A) phenotypic modulation, e.g., the deletion of a pathogenicity island encoding α-hemolysin (hly) and P-fimbriae (pap) in uropathogenic E. coli (UPEC); (B) phase variation, e.g., the inversion of DNA elements such as the fim promoter switch directing type 1 fimbriae (Fim) expression in UPEC; (C) “slipped strand mispairing,” e.g., phase variation by point mutation within the siaD gene required for capsule expression in N. meningitidis; (D) phase variation of biofilm formation by insertion or excision of an insertion sequence element (IS256) into the extracellular polysaccharide-encoding ica gene cluster of S. epidermidis; and (E) antigenic variation by DNA rearrangements in a variable surface-exposed lipoprotein gene cassette vlsE of B. burgdorferi. Abbreviations are as follows: C, cytosin; P, promoter; PAI, pathogenicity island.

The genome of B. burgdorferi, the causative agent of lyme disease, undergoes dynamic rearrangements within the chromosome and among the 12 linear and 9 circular plasmids. A substantial fraction of the genome is made up of paralogous genes. About 5% of the chromosomal genes and an estimated 15% of plasmid genes as well as many pseudogenes encode for lipoproteins. Lipoproteins are important surface structures and targets for the host immune response. Borrelia apparently uses recombination to vary its surface structures with both homologous and nonhomologous mechanisms being involved in switching or recombining of these paralogs (25).

The most striking feature of pathogenic Neisseria species (N. gonorrhoeae and N. meningitidis, which cause gonorrhea and meningitis, respectively) is the amount of repetitive DNA in the chromosome. Repeat-mediated rearrangements facilitate cell surface genes moving around on the chromosome, allowing “silent genes” to be positioned next to “on” switches where they become active. Other repeat sequences may facilitate rearrangements of DNA within cell surface genes. Internal shuffling of these genes changes the encoded proteins, and each generation of bacteria presents a different appearance to the immune system. Phase variation by slipped-strand mispairing (SSM) causes changes in the number of repeats thus changing the coding frame of the gene. Examples include capsule-, hemoglobin receptor (HmbR)-, or opaque-protein expression in Neisseria (26), which seem to be variably induced in different stages of disease. Additionally, antibiotic resistance can be caused by DNA rearrangements, such as remodeling of penicillin-binding protein encoding genes that result in penicillin resistance of Streptococcus pneumoniae, a severe pathogen of the respiratory tract (27).

Adaptation and Variation of Mutation Rates

The majority of bacteria seem to pass repeatedly through periods of increased mutation rates during their evolutionary history. However, a link between high mutation rates and virulence potential cannot be generalized at this point (28). In E. coli and S. enterica sv. Typhimurium, mutS mutators that are deficient in DNA mismatch repair accelerate the mutation rates and relax the barriers that normally restrict homologous recombination. Interestingly, the mutS gene belongs to a recombinational hot spot within the E. coli and Salmonella chromosome (mutS-rpoS region), suggesting that mutS itself may also be subject to horizontal transfer. Rescue of defective mutS alleles with wild-type sequences by HGT may be a mechanism for stabilizing adaptive changes promoted by mutS mutators and has been reported to occur in nature (29, 30).

More than one-third of cystic fibrosis (CF) patients harboring Pseudomonas aeruginosa are infected by mutator strains (31), whereas no mutator strains were found among P. aeruginosa isolated from lungs of non-CF patients in this study. It is noteworthy that a correlation has been observed between high mutation rates and multiple antibiotic resistance. CF patients are also infected with S. aureus mutator strains (32). Differences in mutation rates affect SSM and, therefore, phase-variable expression of hemoglobin receptor genes in N. meningitidis (33). Thus, mutator bacteria may gain an advantage in certain pathologies.

Point mutations resulting in single-nucleotide polymorphisms (SNPs) can lead to genetic alterations that provide a selective advantage during the course of a single infection, epidemic spread, or the long-term evolution of virulence. Allelic variations of fimbrial adhesins in E. coli and S. enterica sv. Typhimurium can determine host specificity and tissue tropism and can serve as a molecular bridge from commensal to pathogenic lifestyles. For example, naturally occurring point substitutions in FimH alleles, coding for the type 1 fimbrial adhesin of uropathogenic E. coli, result in higher affinity for monomannose (and type IV collagen) receptors than most intestinal commensal isolates. This correlates with an increased tropism for uroepithelium and bladder colonization. In S. enterica sv. Typhimurium, SNPs in the type 1 fimbrial adhesin gene produce important differences in HEp-2 cell binding, biofilm formation, and host-colonization (34, 35). These findings underscore the great impact of mutations as generators of diversity.

Future Challenges

New insights regarding the mechanisms of infectious disease have been gained in the wake of large-scale genome sequencing. Most importantly, comparative and functional genomics have helped unravel the magnitude of horizontal gene transfer and its impact on prokaryotic genome evolution. The continued understanding of these processes provides us with a vision of how genome dynamics may contribute to infectious disease. It has also become apparent that evolutionary events are accelerated during infection. In figurative terms, disease can be regarded as an “evolutionary pressure cooker” rather than Darwin's “warm little pond.” The research accomplishments of the past few years have provided, for the first time, insights into the evolutionary origins of infectious disease. Future questions that must be addressed are: What is the in vivo relevance of horizontal gene transfer during the course of an infection? Is HGT a programmed event, how is it regulated, and what might the signals be? By what mechanisms does the genome maintain stability and at the same time flexibility in the face of environmental challenge and how does it protect function? What is the function of the large number of unknown genes that are located on horizontally acquired elements? In summary, we are able, for the first time, to illuminate the dynamical processes of genome evolution and to correlate these findings with infectious disease.

References and Notes

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