Genome Plasticity and Infectious Diseases

Genome plasticity leads to the generation and selection of fitter mutants via the key processes of Darwinian evolution. Urinary tract infections (UTIs) are considered to be the most common bacterial infection in industrialized countries. Genotypic and phenotypic comparisons of closely related E. coli isolates which demonstrated the impact of genome plasticity, i.e., gene acquisition and gene loss, on bacterial evolution as different members of sequence type (ST) 73 represent highly uropathogenic as well as commensal or ABU E. coli isolates. Data on genome-wide changes and adaptation during long-term growth of E. coli in vitro have started to accumulate only recently. Bacterial adaptation to prolonged in vivo growth in different host backgrounds has been analyzed by comparing the genome structure and virulence- and fitness-related phenotypes of E. coli 83972 and three selected reisolates from deliberate human therapeutic urinary bladder colonization. In individual hosts, E. coli 83972 used different strategies to optimize proliferation. The observed increased genome plasticity of in vivo-grown reisolates of E. coli 83972 relative to control isolates from the in vitro experimental evolution study may in part result from stress-induced increased genome plasticity.

The chapter begins with an overview of epidemiology and clinical pathogenesis of Enterohemorrhagic Escherichia coli (EHEC). EHEC causes diarrhea, bloody diarrhea, and hemolytic-uremic syndrome (HUS). The major virulence factor of EHEC responsible for the microvascular endothelial injury is Shiga toxin (Stx). In additon to Stx, several other potential virulence determinants have been identified in EHEC. Next, the chapter focuses on genomes of EHEC. The lability of horizontally acquired genomic elements, such as pathogenicity islands, and especially of bacteriophages, increases the possibility of genomic alterations during human infections. Most EHEC strains possess one or more large plasmids. To investigate genetic changes of EHEC in patients with HUS, consecutive stool samples from such patients was analyzed by detecting stx and eae genes and by using a cytotoxicity assay to detect free Stx. Further, the chapter talks about interserotype differences in conversion of EHEC to EHEC-LST, and consequences of in vivo genetic changes in EHEC. To identify both EHEC and EHEC-LST in stool samples, detection of stx must be complemented by detection of stx-independent target(s) which are common to both pathotypes. Genetic changes in EHEC during infection might influence clinical outcome and have impacts on diagnosis, epidemiology, and evolution. For clinicians, awareness of a potential conversion of the pathotype of the infecting EHEC during infection is essential in making decisions about correct management of the patients and appropriate therapies.

Genomic fluidity associated with Helicobacter pylori has important consequences for clinical management of the gastroduodenal diseases caused by colonization with this significant pathogen. This chapter discusses the attributes of the genomes of different Helicobacter strains and the roles of strain-specific genes from the genomic plasticity region. Researchers analyze two core genome data sets, one at the genus level and the other at the species level. Helicobacter genomes were compared at the biochemical level, based on the presence of enzymes in their metabolic pathway. The Helicobacter genome was found to be subdivided into two clades, highlighting the fact that they have two distinct modes of biochemical transformation. It would be very interesting if such varied metabolic repertoires indeed represent genomic fluidity across these two Helicobacter clades. In H. pylori, three types of genomic islands coding for the type IV secretion system were identified: (i) the cytotoxin-associated gene pathogenicity island (cagPAI), (ii) the competence island (comB gene cluster), and (iii) the plasticity zone. Geneticists think that the comB transformation apparatus has evolved conservatively in H. pylori and is typically present in all the strains. This conservation explains why genomic fluidity in H. pylori is so common, especially when the deletions and rearrangements due to natural transformation and transposition are described as frequently occurring phenomena. H. pylori-induced chronic gastritis is a definitive risk factor for the development of gastric cancer.

Coagulase-negative staphylococci (CoNS) are primarily commensals residing on the skin and mucosa of humans and animals. This chapter summarizes the most recent findings in the genomics of CoNS and discusses the mechanisms and factors contributing to the extraordinary flexibility of these pathogens. It first discusses fitness and virulence-associated factors of CoNS. All CoNS were found to be devoid of superantigen and toxin genes, but a number of genes and factors were identified which are associated with the commensal lifestyle and also the virulence of CoNS. An interesting finding of CoNS sequencing projects was that many of the species-and virulence-specific genes are located in a certain region of the staphylococcal genome around the chromosomal origin of replication. CoNS harbor a great diversity of mobile genetic elements which comprise, in addition to plasmids, mainly bacteriophages, genomic islands, transposons, and insertion sequence elements (IS). Horizontal gene transfer by mobile genetic elements has a major impact on enhancing the biological fitness of CoNS. Biofilm formation is a major pathomechanism of CoNS, notably in Staphylococcus epidermidis. Different mechanisms to modulate biofilm formation are indeed detectable during CoNS infections, and the process is therefore supposed to be critically involved in the establishment of device-associated S. epidermidis infections. In most cases, hypervariability of biofilm formation was detected which was also accompanied by genome rearrangements, reflecting a significant flexibility of the staphylococcal genome during the infection process.

This chapter starts with a description of the general characteristics of the genome sequences of Legionella pneumophila and Legionella longbeachae. It then highlights the characteristic features and common traits of the two main human-pathogenic Legionella species. Emphasis is given to putative virulence and Legionella life cycle-related functions. In the second part, the focus is on the comparison of these genome sequences, in order to learn about the plasticity of the Legionella genomes and the possible mechanisms involved. In the third part, the possible evolution of the identified virulence factors are analyzed and discussed. Finally, future perspectives in Legionella genomics are presented. L. longbeachae NSW 150 encodes four T4SS, a rather exceptional number and L. longbeachae D-4968 encodes two T4SS. The authors found that IcmE/DotG of L. longbeachae (1,525 amino acids) is 477 amino acids larger than that of L. pneumophila (1,048 amino acids). Most of the T4SS are located on regions of genome plasticity; some even show plasticity on the gene/protein level, as shown above for DotG. The authors undertook a phylogenetic analysis for the L. longbeachae protein Llo2643, which contains PPR repeats, a protein family typically present in plants. In the last few years, genome analyses, as well as comparative and functional genomics, have demonstrated that genome plasticity plays a major role in differences in host cell exploitation and niche adaptation of Legionella.

Genome plasticity in Salmonella was first detected in Salmonella enterica serovar Typhi. It has been observed in a number of Salmonella serovars since then. Mechanisms that can lead to alterations in the genome include changes at the single-nucleotide level, gene loss, and genome rearrangements. Genome rearrangements including inversions and translocations can lead to genome plasticity, contributing to the divergence of Salmonella strains. Salmonella pathogenicity islands (SPIs) are large regions of DNA, which are most likely acquired as a result of horizontal gene transfer (HGT) and are often associated with virulence. The virulence plasmids of Salmonella strains contribute to the adaptation of the organism, and in some cases allow the transfer of genes. It appears that expansion of host range is linked to lateral gene transfer (LGT) of genes involved in host-pathogen interactions. A common theme for the variable regions between serovars was the diversity in sugar metabolism, highlighting the redundancy of these systems. The majority of coding regions unique to serovar Enteritidis encode prophage-related functions. Although the genome sequence of S. enterica serovar Pullorum is found to be very similar to those of other Salmonella serovars, the genetic arrangement was significantly different, with three major inversions and one translocation found between strains. Understanding the phenomenon of genome plasticity in this species is important to characterize the relationship between genetic variation and host adaptation, as well as the ability to cause a relatively minor gastrointestinal disease or a potentially life-threatening systemic fever.

This chapter on mechanisms of genome plasticity in Neisseria meningitidis initially gives a short overview over the genetic variability at the population level and some peculiarities of meningococcal genome organization as revealed by genome sequencing projects. Later, the focus is on genetic mechanisms and genomic features that are paramount for the generation of genomic flexibility, and a brief account of the genetic basis of virulence in Neisseria meningitidis as far as it is known today. Exogenous and endogenous stress induces DNA damage in the meningococcal genome that must be repaired, and DNA repair mechanisms are therefore likely to have a key role in meningococcal genome dynamics. So far, Escherichia coli has served as the prime model organism for DNA repair systems in other microorganisms such as N. meningitidis. The majority of strong mutators found in a number of bacterial species have a defective MMR pathway due to the inactivation of mutS or mutL genes. In addition to global mutation and phase variation, intragenomic as well as intergenomic recombination is of pivotal importance for the generation of genome flexibility in N. meningitidis, and one of the most striking characteristics of the meningococcal genomes is the abundance and diversity of repetitive DNA serving as potential target sites for homologous recombination or replication slippage.

This chapter reviews the genome plasticity that has led to distinct subpopulations of enterococci, as well as the evidence for loss of genome defenses as precipitating events in their emergence. It presents evidence as it currently exists describing the role of genome plasticity in the virulence and persistence of hospital-adapted, multidrug-resistant enterococcal lineages. It also highlights the mechanistic and comparatively unappreciated role that loss of endogenous genome defenses, such as restriction modification and clustered, regularly interspaced, short palindromic repeats (CRISPR) systems, may have played in the emergence of these lineages. To the authors knowledge, the extent to which insertion sequence (IS) element inactivation of chromosomal genes contributes to the success of enterococci as pathogens has not been explored. However, there is evidence that intragenomic recombination at IS elements has contributed substantially to genome plasticity of the enterococci. The chapter focuses on genome defense mechanisms in the enterococci, including restriction-modification and CRISPR. Much work remains to be done before understanding whether and how genome defense mechanisms influence enterococcal ecology and evolution.

This chapter describes the scale and driving force behind the profound genomic plasticity of human immunodeficiency virus (HIV). It discusses the intense interplay between the virus and the immune system of the host. It also speculates on the impact of this variability on the prospects for the development of effective vaccines and novel therapies. The evolution of HIV is, as with all organisms, driven predominantly by Darwinian natural selection that comprises two fully independent but essential components. The first is the production of random mutations that form a pool of organisms differing slightly in pheno-type. Second, is selective pressure that drives the rapid and spectacular evolution of HIV. Neutralizing antibodies produced are relatively type specific and targeted to one or two regions of the envelope glycoprotein that are relatively free to mutate without the virus taking a significant fitness hit. The options open to the virus for escaping cytotoxic T lymphocyte (CTL) recognition are numerous. First, it may alter one of the amino acids in the epitope necessary for recognition by the corresponding CTL T-cell receptor. In addition to the extreme selection pressure exerted on HIV by the immune system, there is an additional "artificial" pressure driving HIV evolution, particularly in industrialized countries.

This chapter on genome plasticity of influenza viruses discusses the current knowledge of viral factors. Influenza viruses have caused devastating pandemics and epidemics in the past, and they continue to be a major health problem causing a huge economic burden worldwide. Thus, it is important to understand the characteristics of influenza viruses and to elucidate the extensive interplay between virus and host. Besides the eight structural proteins, influenza A virus encodes the three nonstructural proteins NS1, NEP, and PB1-F2. Influenza viruses pose a major problem for human health and thereby cause a substantial economic burden. The influenza pandemics which occurred in the past century share the fact that new subtypes of influenza A viruses were introduced into the human population. There are two classes of US Food and Drug Administration-approved drugs against influenza: inhibitors of the ion channel M2 and NA inhibitors. The first group comprises the adamantanes, rimantadine and amantadine, which act by inhibiting the viral ion channel M2 and thereby block the step of uncoating during virus entry. It becomes clear that circulating influenza viruses need to be closely monitored for resistance to the available drugs. Within the same host species genetic polymorphisms may occur and influence the ability of the virus to use the host proteins. For influenza viruses to survive, they need to be transmitted from host to host. The development of reverse genetics techniques has greatly advanced understanding of the virus and its replication cycle.

With approximately 3% of the world's population (~130 million people) infected with hepatitis C virus (HCV), the World Health Organization has declared HCV a global health problem. The genotypes are divided into multiple epidemiologically distinct subtypes differing by 20 to 25% from one another. In public databases, the consensus sequence of the quasispecies is normally presented as the most predominant residue at any given position. Virtually all patients undergo reinfection of the donor organ. This reinfection is associated with a dramatic shift in the complexity of the quasispecies population, suggesting that only a limited number of viral variants establish reinfection. Importantly, the sequences from the infection source are located close to the center of the phylogenetic tree whereas the viruses from different patients continuously evolve away from the center. It is noteworthy that samples for the subsequent infection of the next animal were taken during the acute phase before antibodies became detectable. In a study it was found that a virus harboring an escape mutation in an HLA-B8 restricted epitope in NS3 was transmitted to a host who was HLA-B8 negative and therefore was not able to mount the same T-cell response.

The epidemic and sporadic forms of gastroenteritis are common causes of morbidity in developed countries and of morbidity and mortality in developing countries. Although the current review focuses on viral causes of acute gastroenteritis, it can also be caused by bacteria or parasites. Recent studies employing molecular and antigenic methods for detection of enteric viruses showed that the majority of acute viral gastroenteritis cases worldwide are caused by noroviruses (NVs). The molecular cloning and genomic characterization of other viral strains has greatly facilitated our understanding of the genetic structure and classification of NVs. The RNA genome is organized into three open reading frames (ORFs). Several studies were undertaken in recent years to improve understanding of the mechanisms and biological advantages of genotype GII.4 epidemic strains. A study showed that GII.4 NVs evolve stepwise by highly significant preferential accumulation and fixation of nucleotide and amino acid mutations in the protruding part of the capsid protein. Another study showed that the NV capsid protein accumulates mutations more rapidly in healthy immunocompetent individuals than in immunocompromised individuals. In this study, 66 P2 sequences from viruses isolated during outbreaks occurring between 1997 and 2006 in the United Kingdom showed diversity of up to 20%. Further high-resolution structural studies are necessary to determine the role of the P2 subdomain in host interactions and to understand its possible role in NV strain diversity.

Rotaviruses (RVs) are a major cause of acute gastroenteritis (AGE) in infants and young children worldwide, and in the young of a large variety of animals (mammals and birds). This chapter describes the basic facts of genome diversity of RVs and discusses several mechanisms driving their evolution. An attempt is made to judge how the diversity of cocirculating wild-type (wt) RVs may be influenced by the ongoing vaccination programs. RVs are subdivided into different groups based on differences in antigenic properties of VP6, of which groups A through E are well distinguished. For the evolution of human RVs, the following genetic mechanisms have been found to be operative: accumulation of point mutations, genome reassortment, genome rearrangements (recombination), zoonotic transmission, and combinations of the above factors. Genome rearrangements have been found in combination with point mutations, and reassortment is frequently combined with zoonotic transmission. The conditions of genome diversity of RVs are very reminiscent of those of influenza viruses. Whole-genome sequencing, resulting in the assignment of genotypes to all 11 individual RV RNA segments, has allowed further insights into RV evolution. Since 2006, two RV vaccines have been licensed in various countries, and millions of doses have been distributed in universal mass vaccination (UMV) programs.

The human papillomavirus type 16 (HPV-16) genome is typical for the vast majority of all papillomaviruses. In contrast to RNA viruses, papillomaviruses replicate based on the same mechanisms and enzymes that support replication of the chromosomes of the host cell. Humans are presently known to carry at least 125 different human papillomavirus types, i.e., virus genomes differing by more than 10% from one another. In phylogenetic trees, these 125 human papillomaviruses form minor and major clusters that, under present taxonomic rules, are identified as species and genera. On the higher taxonomic level of the genus, the human papillomaviruses belong to five remotely related genera: Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus, and Nupapillomavirus. The most extreme examples of papillomavirus-host linkage are the three and two papillomaviruses which have so far been identified in birds and reptiles, respectively, and which form phylogenetic out-groups to all mammalian papillomaviruses and to one another. The molecular strategies encoded in the replication initiator E1, the transcription/replication regulator E2, the major capsid protein L1, and the minor capsid protein L2, with its important role in the infection process, are apparently very successful and indispensable to the papillomavirus life cycle. The majority of papillomaviruses have these genes, but one or two of the genes are lacking in many animals and even three human papillomavirus types. The stability of the HPV-16 and HPV-18 genomes makes it unlikely that new viral variants will emerge in response to vaccination.

Herpesvirus virions contain a double-stranded DNA (dsDNA) genome of 108 kbp (bovine herpesvirus 4) to 248.5 kbp (anguillid herpesvirus 1) in length. To date, eight different human herpesviruses (HHV) are known. Among human-pathogenic herpesviruses, human cytomegalovirus (HCMV) has the longest genome at ~230 kbp. Importantly, selected host proteins, such as actin, annexin, CD55, and CD59 become "deliberately" incorporated into herpesvirus particles as well. Based on virion morphology criteria, herpesviruses have been found in a variety of vertebrate classes, such as mammals, reptiles, fish, and birds, and even in invertebrates such as oysters. Herpesviruses are the only known viruses capable of deploying two separate transcriptional programs upon infection of a target cell: productive (and usually lytic) infection and latent infection. As observed in other DNA viruses like the Poxviridae, herpesviruses seem to steal genes from their host species and use them for their own purposes, a strategy called molecular piracy. Among herpesviruses, HHV-8 seems to be "the unchallenged master of molecular piracy". A section in the chapter explains leading genetic paradigms combined with selected findings from certain herpesviruses, to put the most important principles of herpesvirus genome plasticity and also their limitations into a broader perspective. The chapter presents examples that highlight the ability of herpesviruses to rapidly mutate under selecting conditions, indicating a remarkable potential of genetic plasticity and adaptability within a handful of in vivo passages.

Malaria parasites infect an impressively broad range of vertebrate hosts, with over 100 species identified in mammals, reptiles, and birds. The majority of malaria parasites are transmitted by mosquitoes; human malaria parasites are transmitted by anopheline mosquitoes exclusively. The study of genome diversity and plasticity, particularly at chromosome ends, is a robust area of research within the malaria community. A class of polymorphisms contributing to genome diversity and plasticity is deletions and insertions (indels) of short repetitive DNA sequences, known as microsatellites (MS) or minisatellites depending on the size of the repeated unit. Gene copy number variation (CNV) is yet another factor contributing to genome diversity in malaria parasites. In Plasmodium falciparum CNV has been detected for many genes by using high-density microarray and/or real-time PCR. Malaria parasites have relatively high recombination rates compared with other organisms studied, such as humans and mice. The degree and extent of genome diversity is tightly linked to parasite origins and evolutionary history. Genome-wide polymorphic single nucleotide polymorphisms (SNPs) can be applied to mapping genes or mutations that are associated with parasite phenotypes of interest. The P. vivax genome is at least as diverse as that of P. falciparum, according to estimates of the nucleotide substitution rate. An understanding of the genome diversity of malaria parasites provides valuable insights into parasite evolution, population structure, dynamics of transmission, mechanisms of drug resistance, immune evasion, and parasite development.

As a parasite that causes a variety of chronic human and livestock diseases in Africa and elsewhere, the trypanosome needs to overcome a number of grand challenges mounted, directly or indirectly, by its wide range of hosts. In general, the adaptations are all to do with the generation, diversification, and regulation of hypervariant, multigene families, the most important of which encodes thousands of variant surface glycoprotein (VSG) isoforms. To understand how the various interlinked processes in antigenic variation contribute to and are served by genome adaptations, it is necessary first to describe what we know, phenotypically and genotypically, about this variation system. Uniquely to the African trypanosomes that use antigenic variation, there is also a set of minichromosomes, Trypanosoma brucei number ~100. There is a set of potential transcription units, known as bloodstream expression sites (BES), adjacent to the telomeres of some of the megabase chromosomes and numbering 5 to 15 per genome, depending on the strain. The main function of BES is to provide transcription loci for VSG. Mechanisms for singular and differential expression of VSG center on the BES, which emphasizes the pivotal role of the expression site in antigenic variation. Importantly, short indels of a few bases also occur, creating frame-shifting: pseudogene formation. The other types of VSG locus also display signs of change through recombination, although fewer data are available.

This chapter describes the different mechanisms of genome plasticity in Candida albicans and their impact on phenotypic plasticity, with an emphasis on recent advances in antifungal drug resistance. The average divergence between C. albicans genetic groups is approximately 2 million years. As a consequence, the recombination and genetic exchanges are most likely due to ancient mating events in C. albicans and not due to recent mating events. The requirement of sex to repair DNA damage may be moot in a diploid because sequences on homologous chromosomes can be used as templates to repair DNA breaks by an effective homologous recombination mechanism. The chapter gives a brief overview of some of the hypotheses that may particularly apply to C. albicans. A source of genome plasticity associated with recombination at MRS loci is chromosome translocation. The possibility exists that recombinations at the MRS can alter its structure and affect filamentation. In conclusion, the development of resistance to fluconazole can involve mutations at TAC1 and ERG11, as well as several genome plasticity events leading to loss of eterozygosity (LOH) and aneuploidy that affect these two genes as well as additional genes on chromosome 5.

The chapter on genome plasticity of Aspergillus species focuses on the genome of various Aspergillus species. Aspergilli have an important impact on humankind, both beneficial and detrimental. On the one hand, some Aspergillus species are used industrially for the production or refinement of beverages, enzymes, food additives, or pharmaceuticals. The main genome features of fully sequenced Aspergillus genomes are summarized. The likelihood of finding genes belonging to these functional categories in the chromosomal center is six times higher than that of finding them within the subtelomeric regions. The function of most secondary metabolites in the producing organism is not known yet. As biologically active compounds they might protect the fungus against other soil inhabitants and may also contribute to weakening of the host immune system. Genes involved in the production of secondary metabolites are often organized in a cluster. Many of the clusters for biosynthesis of secondary metabolites contain regulatory genes. Secondary metabolite gene clusters are located predominantly in plasticity zones; in A. fumigatus only the DHN melanin biosynthesis cluster and the Pes-1-associated cluster are not part of a plasticity zone. In eukaryotes, intragenic tandem repeats (ITRs) are not equally distributed in protein-encoding genes but tend to be biased to the end of the protein.

The fungal pathogen Aspergillus fumigatus and the human cytomegalovirus (HCMV) represent a great challenge for immuno suppressed patients. This chapter summarizes our current knowledge about the increasing number of genetic markers influencing the pathobiology of and susceptibility to A. fumigatus and HCMV. Further complications that arise after exposure to A. fumigatus include allergic bronchopulmonary aspergillosis (ABPA) with hypersensitivity reactions that often occur in immunocompetent hosts with asthma or cystic fibrosis. The promoter of IL-10 contains several polymorphisms from which SNPs at positions -1082, -819, and -592 have been intensively investigated. HCMV is one of the most demanding complications after allo-SCT. The highest risk for patients after transplantation exists when stem cells from an HCMV-negative donor are transferred into an HCMV-positive recipient. Researchers found no significant association between 71 of 80 markers and the occurrence of HCMV reactivation or disease in patients after allo-SCT. Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is an important negative regulator of T-cell activation. The hope for early identification of patients at risk for A. fumigatus and/or HCMV infection is legitimate and might have an impact on individualization of antifungal and antiviral prophylaxis and treatment in the near future.

Microbial determinants of acute-disease severity and tissue damage have been extensively studied, but less is known about genetic variation influencing host susceptibility. This chapter discusses two candidate genes with strong effects on the innate immune response and the antibacterial defense in the urinary tract and with major but opposite effects on urinary tract infection (UTI) susceptibility. The chapter explains that defects in TLR4 expression are protective and associated with asymptomatic bacteriuria (ABU) while defects in CXCR1 expression promote acute pyelonephritis (APN) and renal scarring. C3H/HeJ mice, then known as lipopolysaccharide (LPS)-nonresponder mice, had an increased susceptibility to UTI, as shown by delayed bacterial clearance. It also had an impaired innate immune response, suggesting that defects in innate immunity are of great importance for the antibacterial defense of the urinary tract. Studies of the murine model showed that the antibacterial defense of the urinary tract mucosa relies on innate immunity and that TLR4 plays a central role in the early host defense against infection. The results suggest that genetic variation of the TLR4 promoter is an essential, largely overlooked mechanism to influence TLR4 expression and UTI susceptibility. It was found that the protein expression was reduced and additionally the level of CXCR1 transcript and protein expression was lower in this new subset of pediatric patients. There is a great clinical need to identify genetic variants that improve resistance or increase susceptibility to infectious pathogens.