Mobile genetic elements and pathogenicity islands encoding bacterial toxins

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Mobile genetic elements and pathogenicity islands encoding bacterial toxins Ulrich Dobrindt1, Sarah Tjaden1, Sadrick Shah1, and Jörg Hacker2 Institute of Hygiene, University of Münster, Münster, Germany; 2National German Academy of Sciences Leopoldina, Halle/Saale, Germany

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Introduction: The genome structure of prokaryotes Pathogenicity correlates with the expression of disease-related factors that are present in pathogenic bacteria but usually absent from nonpathogenic bacteria. Analyses of complete prokaryotic genome sequences have shown that the bacterial genome size and organization is considerably variable. Different numbers of circular or linear chromosomes, extrachromosomal linear or circular replicons, as well as different combinations thereof, exist in bacteria [1]. The bacterial chromosome is considered to be composed of a conserved “core” gene pool harboring genetic information required for essential cellular functions and of a “flexible“ gene pool. The latter encodes additional traits that contribute to the adaptation of microbes under certain circumstances, such as with resistance to antibiotics, production of toxic compounds, and other virulence factors. The chromosomal organization of the core regions is similar in closely related species and is not transferable per se. Genes located within this chromosomal backbone exhibit relatively homogenous G + C content and a specific codon usage [2]. In contrast, the flexible gene pool consists of variable chromosomal regions which may be beneficial under certain circumstances, such as with bacterial toxins and other virulence factors. This gene pool includes mobile and accessory genetic elements such as bacteriophages, plasmids, genomic islands (GEIs), insertion sequence (IS) elements, transposons, and integrons. GEIs represent a group of distinct genetic entities. Depending on the functions and their role for a specific lifestyle of a bacterium, GEIs may be called pathogenicity, symbiosis, fitness, metabolic, or resistance islands [3–5]. Furthermore, the presence of identical genes in pathogenic and nonpathogenic variants of one species (e.g., in extraintestinal pathogenic and commensal Escherichia coli) implies that some of these encoded factors contribute more to general adaptability, fitness, and competitiveness than to particular virulence traits [6–8]. Other accessory components have been described, like the transposable IS elements and transposons [9,10]. They are restricted to moving themselves, and sometimes additional sequences, by recombination events from one site of their genome to other sites of the same genome [11]. In addition to the The Comprehensive Sourcebook of Bacterial Protein Toxins. DOI: http://dx.doi.org/10.1016/B978-0-12-800188-2.00002-1 © 2015 Elsevier Ltd. All rights reserved.

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

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chromosomes, many prokaryotes can contain genetic information on plasmids, which are extrachromosomal DNA elements with the ability of autonomous replication. The genetic information encoded by plasmids can contain valuable genes, which may be beneficial under certain conditions, such as toxin genes, but also resistance determinants and genes coding for specific metabolic properties. Many different plasmid types can be transferred between bacteria. Some have the ability to become integrated into the chromosome, thereby losing control of their own replication [12,13]. Other genetic elements frequently found in association with the genome are integrons, retrophages, and lysogenic bacteriophages. Transferable genetic elements, such as bacteriophages and plasmids, can function as vehicles laterally transporting genetic information, thus playing an important role in bacterial evolution. Bacteriophages, as viruses that infect prokaryotes, can genetically modify their host in becoming part of the genome. They may carry genes that bring about new functions or modify existing ones. Interestingly, toxin-specific genes are often located on bacteriophage genomes [14–17,304]. With respect to the abovementioned structure of bacterial chromosomes and the different classes of mobile elements frequently found in prokaryotic genomes, it can be concluded that the genome of prokaryotes constantly undergoes structural variations due to the potential of mobile elements to integrate into different sites of the chromosome and to promote chromosomal rearrangements via recombinational mechanisms. Genome plasticity (i.e., the frequently occurring acquisition or loss of genetic information) is very important for adaptive evolution of disease-causing bacteria, as simultaneous acquisition of many genes by horizontal gene transfer (HGT) allows the inheritance of complex, disease-related characteristics, including protein toxins, in a single step [18–21]. Next-generation sequencing allows the accumulation of prokaryotic gene and genome sequences in a so far unprecedented way and has shed light on many aspects of microbiology. Comparative genomics reveals that a striking correlation exists between the distribution of protein toxins and movable genetic elements in bacteria. A great number of bacterial protein toxin determinants are associated with plasmids, bacteriophages, or pathogenicity islands (PAIs). Together with gene loss and other genomic alterations, gene acquisition plays an important role for adaptive evolution of prokaryotes. Considerable insight has been gained into the contribution of mobile and accessory genetic elements to these processes. The wealth of prokaryotic genome sequences available offers us deeper insights into the variability of bacterial protein toxin-encoding genes and demonstrates that there is a structural and functional interdependence between many bacterial protein toxin genes and mobile and accessory genetic elements that promotes adaptive evolution of pathogenic bacteria.

Protein toxins encoded by mobile genetic elements Protein toxins encoded by plasmids Many bacterial pathogens harbor plasmids carrying protein toxin determinants. They frequently contribute to specific combinations of virulence factors present in these

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Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

strains. This argues for a coevolution of specific factors in different pathotypes. Some of these plasmids have been shown to integrate into the chromosome [22,23]. Important plasmid-encoded protein toxins are given in Table 2.1.

Gram-negative bacteria Intestinal E. coli bacteria may cause different types of diarrheal diseases. The differences in the clinical pictures reflect the different pathotypes of intestinal E. coli, such as enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), and others (see Table 2.1). A main feature of the different intestinal E. coli pathotypes is the presence of pathotype-specific plasmids, which often encode protein toxins. ETEC strains cause diarrhea through the action of two different plasmid-encoded types of enterotoxins, the heat-labile enterotoxin (LT) and the heat-stable enterotoxin (ST). These strains usually contain the determinant for an LT only, an ST only, or both toxin types [28–30]. Heatlabile toxins (LTs) are closely related to the cholera toxin of Vibrio cholerae [31,32]. Two unrelated classes of plasmid-encoded heat-stable toxins without sequence homology exist (STa and STb). The STb-encoding gene (estB) is found on heterogenous plasmids, which may also contain other properties [33–35]. Whole-genome sequence comparison of ETEC isolates identified globally distributed lineages, which carry distinct virulence factor profiles including enterotoxin genes. The authors propose that for at least some of these lineages, the specific chromosome and plasmid combinations could promote fitness and transmissibility. This study demonstrates that plasmid acquisition is a major driving force for the evolution different ETEC clades [36]. The low-molecular-weight ST called EAST1 of enteroaggregative E. coli (EAEC) strains [37,38] exhibits 50% protein identity to STa and is encoded on so-called EAF-plasmids, which range from 50–70 MDa in size [39]. Due to a sequence homology with the enterotoxic domain of heat-stable enterotoxin a (STa) as well as an EAST1-mediated increase of cGMP levels in rabbit ileal tissue, a similar cyclic guanosine monophosphate (cGMP)–mediated mechanism was assumed for EAST1 [38]. Nevertheless, treatment with EAST1-positive strains neither induced increased cGMP values in T84 cells nor caused diarrhea in piglets [40,41]. EAST1 is encoded by the 117 bp-astA gene. After its initial discovery in prototypic EAEC strain 17-2, an EAST1 gene variant was identified in EAEC isolate 042 [42]. The upstream and downstream regions of astA differ between EAEC strains 042 and 17-2. In EAEC isolate 042, astA is located on the 110-kb plasmid pAA and lies adjacent to pet, which encodes for a secreted autotransporter protease. On pAA, a DNA stretch including astA and pet separates the aggregative adherence fimbriae II (aafII)–encoding gene cluster into two halves, indicating that the acquisition of the astA and pet genes involved recombination of different mobile genetic entities. The E. coli 042 astA nucleotide sequence differs by one nucleotide at the 21st codon position (ACA→GCA) from that of the EAEC 17-2 astA allele. This single nucleotide polymorphism (SNP) results in an amino acid exchange (namely, threonine to alanine; [42]). In a volunteer study, three of five adults infected with EAEC strain 042 developed diarrhea, whereas ingestion of EAEC strain 17-2 caused only one case of mild diarrhea in 19 volunteers [43]. To date, astA has been detected in one or multiple

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

Table 2.1 

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Protein toxins encoded by bacterial plasmids

Organism

Pathotype1 Plasmidencoded property

Gene symbol

Other plasmidencoded virulence factors

E. coli

ETEC

elt, etx

ST, CFAs, drug resistance, colicins LT, CFAs, drug resistance, colicins Type IV adhesin

ETEC EAEC, ETEC EHEC EPEC ExPEC EHEC

ExPEC, ETEC EIEC

Yersinia spp. Shigella spp.

E. faecalis S. aureus

Heat-labile enterotoxin (LT) Heat-stable enterotoxin (ST)2 EAST1

EAST1 Alpha-hemolysin (Hly) Enterohemolysin (Ehx)

ast

hly ehx

Subtilase (SubAB)

subAB

CNF2

cnf2

EIEC enterotoxin (=Shigella enterotoxin 2) Yop proteins

sen

Shigella enterotoxin 2 (shET2-2, OspD2) Shigella enterotoxin SenA (OspD3) Cytolysin (Cyl) Enterotoxin types B, D, J, R, S, T Exfoliative toxin B

C. perfringens

est

ADP-ribosyltransferase EDIN-C (epidermal cell differentiation inhibitor) Beta-toxins (CPB), epsilon-toxin (ETX), iota-toxins (ITX), necrotic enteritis toxin B (NetB), TpeL

yop

senA

cyl seb, sed, selj, ser, ses, set etb edin-C

cpb, cpb2, etx, ia, ib, netB, tpeL

bundle-forming pili – Catalase KatP, adhesin ToxB, protease EspP Adhesin Saa, adhesin Sab, autotransporter EpeA F17-, AFA/Dr adhesins, CDT Genes required for invasion, Ipa proteins, T3SS Genes required for invasion, Yop proteins, T3SS Genes required for invasion, Ipa proteins, T3SS – Penicillin and cadmium resistance Cadmium resistance, bacteriocin see above

–

(Continued)

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Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

Table 2.1  (Continued) Protein toxins Organism

C. tetani C. botulinum

B. anthracis

encoded by bacterial plasmids

Pathotype1 Plasmidencoded property Tetanus neurotoxin (TeNT) Botulinum neurotoxin (BoNT)

Anthrax toxin (lethal lactor, edema factor, protective antigen)

Gene symbol

Other plasmidencoded virulence factors

tentX

Collagenase (ColT)

bont/A3, /A4, /B1, /B2, /B5, /G lef, cya, pag

Bacteriocin (Boticin G), hemagglutinin protein (HA) Bacteriocin

1

EAEC, enteroaggregative E. coli; EHEC, enterohemorrhagic E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli. 2 Genes encoding for STa (estA) and STb (estB) have been found on Tn1681 [24] and Tn4521 [25–27], respectively.

copy numbers in E. coli strains of human and animal origin. Its role in the cause of disease, as well as the mechanism by which EAST1, (alone or in combination with other virulence factors) contributes to pathogenensis, must be further investigated. Despite its initial discovery in EAEC, the astA sequence is not restricted to the EAEC pathotype. This gene is widely distributed among other human diarrheagenic E. coli strains [39, 45–47, 306], as well as in porcine and bovine pathogenic strains [48,303]. The EAST1 gene sequence was also detected in uropathogenic E. coli (UPEC). Consequently, it was hypothesized that certain extraintestinal pathogenic E. coli may have received intestinal virulence genes by plasmid uptake [49,50,308]. Subsequently, additional astA variants with varying nucleotide sequence were identified in different diarrheagenic E. coli isolates [51,52], in addition to the reported EAST1 variants of EAEC strains 042 and 17-2. Although EAEC strains 17-2 and 042 possess only a single astA copy on a virulence plasmid, specific ETEC strains may carry multiple copies on plasmids, as well as on the chromosome [46,53]. Furthermore, individual strains can possess different EAST1 alleles: the gastroenteritis outbreak strain E. coli O166:H15 carries one astA variant on the chromosome, while another astA allele is located on a plasmid [52]. Due to the high prevalence of the EAST1-encoding gene in diarrheagenic E. coli, it has been hypothesized that astA is located on a transposon [46]. One EAST1 allele described in enterohemorrhagic E. coli (EHEC) was also identified within IS285 of Yersinia pestis. This demonstrates evolutionary crossspecies gene transfer and the involvement of mobile genetic elements [54]. In ETEC strain 27D, astA is located on a plasmid and was described to be embedded into a putative transposase-encoding gene of IS1414 [55]. IS1414 comprising astA, has also been identified in multiple copies in Salmonella enterica serovar Abortusovis, representing an intriguing example of intergeneric virulence gene transfer via an IS element into Salmonella [56].

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

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The Shigella enterotoxins 1 (ShET1) and 2 (ShET2) alter the electrolyte and water transport in the small intestine [57–59]. Whereas the Shet1-encoding set gene has been detected on the chromosome of clinical Shigella flexneri serotype 2 isolates and also in some EAEC, enteroinvasive E. coli may carry the ShET2-encoding gene sen on the 140-MDa pInv-plasmid [44,60]. ETEC strains and extraintestinal E. coli from animals often carry the structural gene for CNF2 (cnf2) on the F-like Vir-plasmid [61,62]. The Vir plasmid also carries several fimbrial adhesin-encoding determinants, as well as the cdtIII gene, which codes for the cytolethal distending toxin (CDT) and the remnants of the α-hemolysin operon [63–65]. Interestingly, cnf1, which is a homologous gene of cnf2, is located on PAIs of UPEC strains [66,67]. Extraintestinal pathogenic E. coli, a major cause of urinary tract infections (UTIs), sepsis, and newborn meningitis (NBM) in humans, often carry PAIs that contain the α-hemolysin gene cluster (hly) [68]. Some E. coli strains contain hly determinants on plasmids, which are heterogenous in size (50–160 kb), conjugational behavior, and incompatibility group [69]. The plasmid- and chromosomally encoded hly determinants show a high overall homology [69,70]. Nevertheless, the sequences of the upstream regions differ significantly [71,72]. The plasmid-associated α-hemolysin gene cluster can also be found in some ETEC, Enterohemorrhagic E. coli (EHEC), and EPEC. The nucleotide sequence comparison of 11 plasmid-derived α-hemolysinencoding operons indicated that the plasmid-located hly determinants include the hlyR regulatory gene. The latter is absent from the chromosomal hly gene clusters. Furthermore, the plasmid-derived hly gene clusters are often associated with the IS elements IS1 or IS2 (Burgos and Beutin, 2010). The enterohemolysin determinant (ehxABD) of EHEC strains is encoded on plasmids (pO157) that range in size from 93–104 kb [73,74]. Although this toxin has a high overall similarity to α-hemolysin, the amino- and carboxy-termini of both proteins are different [75,76]. EHEC strains of different serogroups, including O157:H7, O26:H11/H-, O145:NM, O111:NM/Hand O103, possess large virulence plasmids, which carry the genes coding for EHEC virulence factors, such as ToxB involved in adherence, the serine protease EspP, the catalase peroxidase KatP, and the enterohemolysin (Ehx) [77,78]. The locus of enterocyte effacement (LEE)–negative EHEC O113 strain EH41 carries pO113, a 116-kb plasmid coding for Ehx, EspP, and the STEC agglutinating adhesin Saa. In addition, pO113 includes the subtilase cytotoxin (subAB), Sab autotransporter adhesin (sab), and the EpeA autotransporter (epeA) [79]. Virulence plasmids of pathogenic Yersinia, Shigella, and Salmonella species all have in common that they lack genes encoding conventional protein toxins. The Yersinia and Shigella virulence plasmids code for a conserved type III protein secretion system (T3SS) [80,81]. All pathogenic species of Yersinia (Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica) harbor a virulence plasmid (pYV) of about 70 kb in size. This plasmid encodes the Yop virulon that organizes the secretion of several proteins termed Yersinia outer proteins (Yops), which are essential for virulence [82–85]. Several T3SS effectors (YopH, YopE, YopT, YopO, YopJ, and YopM) counteract innate immunity by directly interacting with host proteins and inhibiting signaling pathways [86].

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Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

A second T3SS, the Yersinia secretion apparatus (Ysa), was described in biotype IB Y. enterocolitica strains. It is different from the chromosome-encoded TTSS of Y. pestis but is instead closely related to the Mxi-Spa TTSS of Shigella and to the SPI-1 encoded TTSS of Salmonella enterica. Phylogenetic analyses based on the nucleotide sequence of the different TTSSs indicated that TTSSs have been distributed by HGT [87]. All virulent Shigellae and enteroinvasive E. coli strains harbor a 220-kb pInv plasmid that encodes all genes that are essential for epithelial cell invasion [88]. The effectors of the invasion process, termed invasion plasmid antigens (Ipas), are encoded on this plasmid [89–91], as well as the invasin IpaB, which has homology to pore-forming toxins [92]. In addition, the invasion plasmid contains the sen gene encoding the Shigella enterotoxin 2, which is also called the EIEC enterotoxin. The sen gene from Shigella flexneri 2a and EIEC share 99% identity [44].

Gram-positive bacteria The Clostridium tetani structural gene tent encoding the tetanus neurotoxin (TeNT) is located on a 74-kb plasmid such as pE88 [93–96]. Five tetanus toxin-encoding plasmids have been sequenced. Whereas they may differ with regard to their gene content, the tent sequences located on these plasmids are highly conserved [95]. Botulinum neurotoxin (BoNT)–encoding genes can be located on different mobile genetic elements, including plasmids and bacteriophages or on the bacterial chromosome [97–99,189]. The bont/A, /B, /E, and /F-encoding genes have previously been shown to be located on the bacterial chromosome or on plasmids in C. botulinum strains of groups I and II [98,100,101], whereas the BoNT/C and /D-encoding genes are located within bacteriophage genomes in C. botulinum group III strains [97,102]. In group IV C. botulinum, the bont/G gene was found to be located on a plasmid [103–105]. Sequence analysis of bont/B1-, /B2-, /B5-, and /A3-encoding plasmids demonstrated that they share significant parts of their sequence [101,106]. The neurotoxin genes are arranged in two different clusters in different C. botulinum types: Whereas in subtype A1 and type B strains, the bont genes are part of the so-called ha cluster (ha70-ha17-ha33-botR-ntnh-bont), the orfX cluster (orfX3-orfX2-orfX1-[botR]p47-ntnh-bont) is present in C. botulinum subtypes A2, A3, and A4, type F, subtypes E1 and E3, and BoNT/E [107]. Interestingly, the bont/B1, /B2 and/B5 genes are flanked by homologous DNA regions, including a truncated IS256 element. This suggests that the different bont variants derive from a common ancestor plasmid [101]. Several toxin genes of Clostridium perfringens are located on plasmids, such as the genes coding for the necrotic enteritis toxin B (netB), the TpeL toxin (tpeL), and the beta (cpb, cbp2), epsilon (etx), and iota toxins (ia, ib). The toxin genes have been detected on various plasmids, which differ in size and gene content and thus contribute to the marked diversity of toxinotypes [99,108,305]. Virulent Bacillus anthracis strains contain two large plasmids, pXO1 and pXO2, which are both required for virulence [109]. Generally, the species-specific pathogenicity of members of the Bacillus cereus group (which includes, among others, B. anthracis) depends on their plasmid content. The genes for the three-component anthrax exotoxin are located on the 182-kb plasmid pXO1 [110,111], whereas the 96-kb

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

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plasmid pXO2 contains genes coding for another virulence factor of B. anthracis, the D-glutamic acid–composed capsule [112,113]. The “pathogenicity region” of pXO1 comprises the structural genes pagA coding for the protective antigen (PA), lef coding for the lethal factor (LF), and cya encoding the edema factor (EF). The “pathogenicity region” is flanked by mobile genetic elements that are supposed to be involved in the acquisition of this region by pXO1 [114]. The plasmids pXO1 and pXO2 are considered to be B. anthracis–specific, but similar plasmids have been detected in some B. cereus strains isolated from humans and animals with anthraxlike disease [115,301,302]. Comparative analysis of the complete genome sequence of B. anthracis strain Ames and of its nonpathogenic relative B. cereus strain ATCC10987 demonstrated that pXO1 is similar to plasmid pBc10987 (about 208 kb) of B. cereus. Roughly 65% of the encoded proteins were homologous, and about 50% of them were in a syntenic location. Interestingly, the “pathogenicity region” of pXO1 is absent from pBc10987. Instead, a B. cereus–specific region can be found that is not flanked by mobile genetic elements [116]. Comparative genomics of pXO1- and pXO2-like plasmids isolated from other members of the B. cereus group revealed a great overall nucleotide homology and synteny relative to pXO1 and pXO2 and confirmed that the plasmids from non–B. anthracis isolates lack the “pathogenicity region” [117,301]. These data underline that variability in plasmid gene content and the acquisition of plasmid-encoded virulence factors play an important role in evolution of pathogenic bacteria. Cytolytic strains of Enterococcus faecalis produce a cytolysin with homology to lantibiotics. The cytolysin determinant (cyl) consists of the genes cylR1R2LLLSLMBAI. These are encoded in one operon on large (60-kb), transmissible, and pheromoneresponsive plasmids, such as pAD1 [118,119]. Cytolysin determinants have also been located on the chromosome of Enterococcus [120–122]. Different Staphylococcus aureus enterotoxins can be plasmid-encoded. The bestknown example is the enterotoxin D-encoding gene sed, which has been initially described on the 27.6-kb penicillinase plasmid pIB485 [123]. Meanwhile, a lot of genomic and DNA sequence data has been accumulated, indicating that also other staphylococcal enterotoxin determinants, including those coding for enterotoxin B (seb), R (ser), S (ses), and T (set), as well as for the enterotoxin-like protein J (selJ) can be located on plasmids [124,125]. The two exfoliative toxin determinants responsible for the staphylococcal scalded skin syndrome have been assigned to different genetic loci. The gene for exfoliative toxin A (eta) has been mapped on the chromosome and that for the exfoliative toxin B (etb) on a plasmid [126–128]. Recently, this plasmid type was described with an additional insertion of a 16-kb region coding for macrolide and beta-lactam resistance. This finding supports the continous evolution of mobile genetic elements and the combination of virulence and resistance determinants on mobile genetic elements [129].

Protein toxins encoded by bacteriophages Bacteriophages encode a variety of toxin genes of pathogenic bacteria. The toxin genes are frequently located next to the bacteriophage attachment site, which argues

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Table 2.2 

Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

Protein toxins encoded by bacteriophages

Host organism

Bacteriophageencoded toxin

Gene symbol

Phage designation

V. cholerae E. coli (EHEC) E. coli P. aeruginosa P. multocida C. diphtheriae, C. ulcerans C. botulinum

CT Stx CDT CTX PMT DT

ctx stx cdt ctx toxA tox

BoNT types C1 and D (BoNT/C1, BoNT/D) Pyrogenic exotoxin (SPE) A, C, G, H, I, M, L, and K SSA Enterotoxin A (SEA)

bont/C, bont/D speA, -C, -I, -H, -M, -L, -K ssa sea

CTXϕ H19, 933 – ϕCTX1 − β, ω2, ϕCULC0102-I c-st

Enterotoxin E (SEE) Enterotoxin K (SElK)

see selk

Enterotoxin P (SElP) Exfoliative toxin A (ETA) PVL Staphylokinase (SAK)

selp eta lukSF-PV sak

S. pyogenes

S. aureus

SPE-phage, CS112, T123 ϕSa3ms, ϕNM3, ϕMu50A ϕSa ϕSa3ms, ϕSa3ms, ϕSa3mw ϕN315, ϕMu3A ϕN315 ϕPVL ϕ13, ϕSa3ms

The chromosomal attachment site (attB) of ϕCTX has been mapped to the 3′-end of a tRNASer-encoding gene [130]. The chromosomal attachment sites (attB1 and attB2) of β and ω overlap with a duplicate tRNA gene encoding the tRNA2Arg [131]. 3 The chromosomal attachment site (attB) of T12 has been mapped to the 3′-end of a tRNASer-encoding gene [132]. 1 2

for acquisition by mechanism of transduction. The bacteriophage-encoded toxins are given in Table 2.2.

Gram-negative bacteria Full virulence of Vibrio cholerae depends on two coordinately expressed factors: the cholera toxin (CT) and toxin-coregulated pili (TCP). The CT encoding genes (ctxAB) are located on the CTX element, which ranges from 7 to 9.7 kb in size and occurs frequently in multiple, tandemly arranged copies. This element has been identified as a lysogenic filamentous bacteriophage designated CTXϕ [133,134] and is restricted to toxigenic strains [135–137]. Structurally, the CTX element resembles a compound transposon [138]. The core region of the CTX element encodes several toxins, such as CT (ctxAB), zonula occludens toxin (zot), and the accessory cholera toxin (ace). The complete CTX element is self-transmissible and can replicate as a plasmid, as

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

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well as leading to the production of extracellular virions [134]. Transmission of CTXϕ requires the expression of TCP pili. The tcp genes are located on a separate PAI [135,139]. Shiga toxin (Vero toxin) is the major virulence factor of EHEC strains. Two immunologically noncross-reactive groups of Stx can be distinguished (Stx1 and Stx2). One EHEC strain expresses Stx1 only, Stx2 only, both toxin types, or multiple forms of Stx2 [140,141]. The Stx1 of EHEC is essentially identical to Shiga toxin from Shigella dysenteriae [142]. Sequence variation exists among the members of the Stx1 and Stx2 groups [141,143–146]. The identically organized structural genes for Stx1 and Stx2 are located on lysogenic lambdoid phages, whereas those for Stx2v are encoded on the chromosome [147,148]. In some cases, these lysogenic bacteriophages may be defective and lost the ability to enter the lytic cycle [149,150]. In some Citrobacter freundii and Enterobacter spp., the stx2 gene has been detected as well [151]. The mitogenic Pasteurella multocida toxin (PMT) is also an example of a bacteriophage-encoded toxin. The coding gene toxA is located within a prophage genome that is chromosomally inserted at a tRNA-Leu gene in P. multocida strain LFB3 [152]. Certain Pseudomonas aeruginosa strains produce a cytotoxin (Ctx). The corresponding structural gene (ctx) is carried by a temperate bacteriophage (ϕCTX), which is able to convert nontoxigenic into toxigenic strains. The chromosomal attachment site (attB) of ϕCTX has been mapped to the 3′-end of the tRNASer [130,153].

Gram-positive bacteria The determinant encoding diphtheria toxin (tox) is part of the genome of corynephage β, which converts nontoxigenic C. diphtheriae strains into toxinogenic strains [154,155]. Various different corynephages are known, including some nonconverting phages. The corynephages ω and β are able to form polylysogens by inserting into two different bacterial attachment sites, attB1 and attB2. As a result, the level of toxigenicity is increased as the gene dose also influences the toxin production [156,157]. Both attachment sites overlap with a duplicate tRNA gene encoding the tRNA2Arg [131]. C. ulcerans can also be lysogenized by a bacteriophage and acquire the tox gene. As a result, the lysogenized C. ulcerans can cause a diphterialike disease. Analysis of such a tox-positive prophage from C. ulcerans, ϕCULC0102-I, demonstrated that although the tox genes were similar, the prophage structural genes in C. diphteriae and C. ulcerans were not syntenous. This, together with the fact that an integrase gene is located downstream of tox in both prophages, suggests that the tox gene was subsequently acquired by ϕCULC0102-I [158]. The earlier assumption that the tox gene has been initially incorporated into a nontoxinogenic ancestor of prophage ω and β by illegitimate recombination [159] is in accordance with this observation. Although many different C. botulinum strains carry bacteriophages, only in the case of the structural genes of BoNT/C1 and BoNT/D (bont/C and bont/D) has it been proven that they are part of the genomes of bacteriophages [97,102,160]. The BoNT/C1-encoding bacteriophage c-st has been shown to exist as a circular plasmid

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Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

prophage rather than as a lysogenic prophage. This can explain the unstable lysogeny of such isolates that has been described before. The BoNT/C1 and D bacteriophages represent a structurally heterogenous family of phages [102]. Streptococcal pyrogenic exotoxins and related exotoxins of S. aureus belong to a family of molecules that is also designated as pyrogenic toxin superantigens (PTSAgs) [161]. The genome sequences of five group A Streptococcus (GAS) strains have been analyzed and indicate that each strain harbors four to eight prophages or prophagelike elements, most of which encode PTSAgs. The structural genes of the streptococcal pyrogenic exotoxins (SPE) type A, C, H, I, L, M, and K, as well as that of the streptococcal superantigen Ssa, have been shown to be phage-encoded. The prophages, which represent a small fraction of the complete GAS genome, account for up to 74% of the gene content variation among different strains. Only 3 out of 21 GAS prophage elements currently sequenced do not encode virulence-associated factors. Accordingly, they have been important for evolution and genome diversification of GAS genomes and may also be responsible for the spread of related toxins among related species [162–167]. The description of the S. dysgalactiae mitogen (SDM) underlines that similar toxins exist in other streptococci as well [168]. Furthermore, two pyrogenic superantigens have been detected in S. equi (SePE-H and SePE-I), which are closely related to SPE H and I of S. pyogenes and the S. aureus superantigens SEL, SEI, and SEM, respectively [169]. The staphylococcal enterotoxins A–Q also belong to the PTSAg family. The majority of them are encoded by formerly mobile genetic elements, but only the sea, see, sek, selp, and selq genes are located on bacteriophages [125]. The structural gene for the staphylokinase (sak) is encoded on a bacteriophage [170], as well as those coding for the exfoliating toxin A and Panton-Valentine leukocidin (PVL; [171–176]).

Protein toxin genes and other mobile genetic elements Plasmids and bacteriophages are elements that increase the genetic flexibility of bacteria. They contribute to the evolution of pathogens upon HGT, followed by integration into the chromosome. The fact that toxin genes have the capacity to spread among bacterial strains and even between species is underlined by the occurrence of identical toxin determinants or those with similar functions on different genetic elements, such as chromosomes, phages, and plasmids (see also Table 2.3). Thus, the E. coli type I heat-labile enterotoxin (LT-I) genes (elt) are located on plasmids [140], while the related cholera toxin (CT) structural gene (ctxAB) of toxigenic V. cholerae strains is phage-associated [134]. The genes coding for the type II heatlabile enterotoxin of E. coli (LT-II) have so far been described only as chromosomally located. Their nucleotide sequences is more diverse than that of LT-I determinants. Interestingly, the LT-II genes are flanked by phage-related genes that may belong to defective prophages inserted into the E. coli chromosome [177]. A similar situation is reported for the Shiga toxin structural genes of S. dysenteriae (chromosomally encoded) [178] and E. coli (phage-encoded). The genes encoding α-hemolysin and CNF-toxins of pathogenic E. coli may be located either on plasmids [62,71,179] or

Table 2.3  The

location of protein toxin–encoding genes and their homologues1

Toxin

Location

Organism

Homologue

Location

Organism

CT α-hemolysin Cytolysin CNF 1 CDT-I, -IV, -V EAST1 Vat autotransporter serine protease Subtilase SubAB(1) ShET1 Toxin complex

Phage Chromosome, PAI Chromosome, PAI Chromosome, PAI Phage Chromosome Chromosome, PAI

V. cholerae E. coli E. faecalis E. coli E. coli E. coli E. coli E. coli S. flexneri P. luminescens

Plasmid, chromosome Plasmid Plasmid Plasmid Plasmid Plasmid Plasmid, chromosome, PAIs Chromosome, SE-PAI Plasmid Plasmid

E. coli E. coli E. faecalis E. coli E. coli E. coli E. coli

Plasmid pO113 Chromosome, PAI Chromosome, PAI

LT α-hemolysin Cytolysin CNF 2 CDT-III EAST1 Autotransporters, serine proteases Subtilase SubAB(2) ShET2 Toxin complex– like toxins

BoNT/A3, /A4, F (orfX cluster) BoNT/B (ha cluster) Enterotoxins EDIN-C

Plasmid

C. botulinum

Chromosome

Plasmid Phage Plasmid

C. botulinum S. aureus S. aureus

BoNT/A1, /A2, /E1, /E3, /4, /F BoNT/A1, /B Enterotoxins EDIN-B

1

Not all homologues are listed for each toxin.

Chromosome SaPIs, Plasmid Chromosome, PAI

E. coli E. coli S. entomophila, Xenorhabdus spp., Y. pestis, Ps. syringae C. botulinum, C. butyricum C. botulinum S. aureus S. aureus

52

Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

as part of PAIs on the chromosome [66,67,180–182]. Similar findings have been reported for clostridial neurotoxin genes [103,183,184], for some enterotoxins, the exfoliative toxins A and B, as well as for ADP-ribosyltransferases B and C (EDIN-B, EDIN-C) of staphylococci [123,128,171,185–191]. The CDT is produced by many pathogenic E. coli [192,193] and also expressed in other bacterial pathogens, such as Aggregibacter actinomycetemcomitans or Campylobacter spp. [194–196]. The CDT-I, CDT-IV, and CDT-V toxin variants are encoded on bacteriophages [197–199]. The cdt-III determinant of septicemic E. coli isolate 1404 has been described to be plasmid-encoded [65], highly similar genes have been detected in the E. coli O157:H− strain 493/89 chromosome, where they are flanked by genes with homology to late genes of lambdoid bacteriophages. This indicates that cdt-III may have been acquired by bacteriophage transduction [200]. The sequence diversity and presence of individual “morons” within the cdt-carrying prophages detected in E. coli suggest that they evolved independently as an adaptation to their hosts after they were integrated into the bacterial chromosome [198,201]. The location of identical or highly related toxin-encoding genes on different genetic elements raises the question of that whether toxin determinants may be part of transposons or other genetic structures that have the capacity to jump between different genetic elements. A transposon location was only reported for the ST enterotoxin structural genes of E. coli, which have been found on Tn1681 [24] and on Tn4521 [25–27]. Interestingly, both enterotoxin genes (sta and stb), earlier described as part of different transposons, have been reported to be linked within a 40-kb mobile DNA region of virulence plasmid pTC isolated from a porcine enterotoxigenic E. coli strain [202]. The Bacteroides fragilis pathogenicity islet carrying the fragilysin toxin (bft) gene has been described to be part of the conjugative transposons CTn86 and CTn9343. This may explain how this gene could be transferred from toxigenic to nontoxigenic B. fragilis isolates [203,204]. Many protein toxin genes, such as those encoding α-hemolysin, CNF1, LT enterotoxins, cholera toxin, and others, are located next to intact IS elements, which may form complex transposons similar to Tn5 or Tn10.

Toxins encoded by PAIs PAIs Virulence genes are frequently located on mobile or formerly mobile genetic elements, including PAIs [3,205]. PAIs can evolve from lysogenic bacteriophages and plasmids and are defined as large genomic regions present in pathogenic variants, but less frequently present in closely related nonpathogenic bacteria. PAIs are often unstable and contain mobility genes coding for integrases or transposases. Similar structures have been discovered in many Gram-negative and Gram-positive human, animal, and plant pathogens. However, several of these traits can be important for nonpathogenic, symbiotic, and environmental bacteria alike [7].

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

53

PAI-encoded toxins Enterobacteria Initially, PAIs were discovered in UPEC by Goebel, Hacker, and coworkers [206,207]. Typical PAI-encoded protein toxins of UPEC and other extraintestinal pathogenic E. coli (ExPEC) are α-hemolysin and the cytotoxic necrotizing factor 1 (CNF-1; see Table 2.4). In many ExPEC isolates, the corresponding hly and cnf1 determinants are closely associated, underlining a possible coevolution of these genes [67,208,209]. Table 2.4 

Examples of protein toxin–encoding PAIs

Organism

Designation

Encoded toxins

Other Important VirulenceAssociated Traits

ExPEC (e.g., UPEC strain 536)

PAI I536, PAI II536

α-hemolysin

ExPEC (e.g., UPEC strain 536)

PAI III536

ExPEC (e.g., UPEC strain J96)

PAI IIJ96

ExPEC (e.g., E. coli Ec222)

Vat-PAI

Rabbit enteropathogenic E. coli 83/39 (REPEC) EPECi E2348/69

LEE

Hemoglobin protease (autotransporter serine protease) α-hemolysin, cytotoxic necrotizing factor 1 (CNF1) Vat cytotoxin (autotransporter serine protease) Enterotoxin (Sen)

Adhesins, putative heat resistant agglutinin S. fimbrial adhesin (SfaI), salmochelin siderophore system P. fimbrial adhesin

LEE-negative Shiga toxin– expressing E. coli (e.g., STEC O113 strain 98NK2) S. flexneri

SE- PAI

K. pneumonia (e.g., strain Kp52.145) B. fragilis N. gonorrhoeae B. pertussis P. luminescens (e.g., P. luminescens strain W14)

EspC-PAI

EspC enterotoxin (autotransporter) Subtilase cytotoxin SubAB

SHI-1 (she)

–

T3SS, invasion, put. adhesin – –

Enterotoxin (Set), autotransporter serine protease (Pic) T6SS insertion Phospholipase D

–

B. fragilis PAI atlA locus ptx-ptl locus PAI 1

– Serum resistance T4SS –

PAI 2 PAI 3

B. fragilis toxin (BFT) Cytotoxin Pertussis toxin (Ptx) Toxin complex (Tc) proteins Mcf protein Cytonecrosis-like toxin (Cnt)

Sel1 lipoprotein

HecAB-like adhesin or hemolysin – (Continued)

54

Table 2.4  (Continued) Examples of

Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

protein toxin–encoding PAIs

Organism

Designation

Encoded toxins

Other Important VirulenceAssociated Traits

H. pylori Gram-negative proteobacteria (e.g., V. cholera, P. aeruginosa, S. marcescens, B. pseudomallei, and E. coli)

cag island

CagA oncoprotein Amidases, muraminidases, phospholipases

T4SS T6SS

S. aureus

S. aureus PAI (SaPI) Enterotoxin gene cluster (egc) SSCmec cassette etd-PAI

SEB, SEC, SElK, SElL, SElQ, TSST-1 SEG, SEI, SElM, SElN, SElO, SElU, SElV SElH

–

Exfoliative toxin ETD

E. faecalis

E. faecalis PAI

Cytolysin

L. monocytogenes, L. ivanovii

LIPI-1

Listeriolysin (Hly)

asc-dapE cluster Pathogenicity locus (PaLoc) Cdtloc

Internalins

Epidermal cell differentiation inhibitor-B (EDIN-B) Surface protein (Esp), aggregation substance PrfA-dependent virulence gene cluster (phospholipases, ActA) Sphingomyelinase C –

C. difficile

Cytotoxins A, B (TcdA, TcdB) Binary toxin CDT

–

–

–

Genes coding for autotransporters are usually located on mobile genetic elements, such as plasmids, bacteriophages, and PAIs. Autotransporters constitute a distinct protein family that represents virulence factors, such as adhesins, proteases, and toxins [210–212]. Vat, a serine protease autotransporter of Enterobacteriaceae (SPATE), is a vacuolating toxin and was initially isolated from avian pathogenic E. coli strain Ec222. This gene is located on a 22-kb PAI, chromosomally inserted at the transfer RNA (tRNA) locus thrW that also serves as a chromosomal insertion site for several

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

55

other PAIs and bacteriophages. The Vat toxin induces cytotoxic effects as known for the VacA toxin of Helicobacter pylori [213]. Vat shows high homology to other PAI-encoded autotransporters of pathogenic E. coli, such as the putative hemoglobin protease Hbp of UPEC O6 strains [214,215] or the hemagglutinin Tsh of an avian pathogenic E. coli isolate [216]. The enterotoxin EspC is also an autotransporter protein and is encoded by the espC-PAI in EPEC strains [217]. LEE-negative Shiga toxin–producing E. coli often express the subtilase cytotoxin SubAB. Two subAB alleles have been described, and the subAB(1) gene cluster is located on the pO113 virulence plasmid [79,218], whereas subAB(2) is part of a PAI inserted into the pheV tRNA locus designated subtilase-encoding PAI (SE-PAI) [219]. Another protein toxin–encoding gene is located on a PAI of Shigella flexneri, termed the she locus. The she locus of S. flexneri 2a codes for the protein ShMu, which has hemagglutinin and mucinase activities. ShMu shares homology with the virulence-related immunoglobulin A proteaselike family of secreted proteins. Within the she locus, the two Shigella enterotoxin 1 encoding genes set1B and set1A are tandemly located in opposite orientations. This element is part of a 51-kb deletable chromosomal element, which has been termed she PAI. The flanking regions of this PAI contain several IS elements like IS2, IS600, and a copy of IS629, which was disrupted by the insertion of a bacterial group II intron [220]. The Shigella enterotoxin gene (senA) has also been detected on a LEE-PAI variant of a rabbit EPEC isolate [221]. The genome sequence analysis of Klebsiella pneumoniae K2 reference strain Kp52.145 identified a putative cytotoxic outer membrane protein (cOMP) encoded on the GEI GI-II. Interestingly, a region comprising nine genes was found to be inserted in the type VI secretion system locus III of K. pneumoniae Kp52.145. This region includees among other three genes coding for putative phospholipase D family proteins which could contribute to host cell invasion, bacterial dissemination and disease progression. One of these phospholipase D family proteins, PLD1, contributes to in vivo virulence and is involved in cardiolipin metabolism [222].

Other Gram-negative bacteria Photorhabdus luminescens is pathogenic to a wide range of insects and has a complex symbiotic relationship with nematodes. The P. luminescens genome comprises more toxin genes than any other bacterial genome sequenced so far. Additionally, a large number of adhesins, proteases, and lipases, which also may be expressed during the pathogenic phase of its complex lifecycle, were predicted from the available complete genome sequences. A multitude of so-called toxin complex (tc) loci exist on the chromosome of P. luminescens strains. All of them code for different highmolecular-weight multi-subunit insecticidal Tc toxins, designated toxin complex (TC), that exhibit oral and injectable activity against several insects. Tc homologues are expressed in several other insect pathogens, including Xenorhabdus and Yersinia. It is anticipated that some of the Tc proteins may function to destroy the insect midgut [223,224]. The so-called tcd locus is part of the so-called PAI 1 of P. luminescens W14 [225]. This DNA region comprises multiple tandem repeats of the tcdAB genes

56

Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

interspersed with tcc-like sequences. The entire locus is chromosomally inserted at a tRNA-Asp gene (aspV) and is also associated with multiple enteric repetitive intergenic consensus (ERIC)–like sequences and with a tcdA-like pseudogene. This indicates that the tcdAB island represents an unstable DNA region [226,227]. This is supported by the fact that deletions within this locus have been observed in other Photorhabdus strains [228]. The genetic structure of “tc-island” sequences in several Photorhabdus strains is variable, and furthermore, tc-like genes are localized on the chromosome or a plasmid of pathogenic microorganisms, such as Xenorhabdus spp., Serratia entomophila, Yersinia pestis, Pseudomonas syringae pv. tomato, Fibrobacter succinogenes, Treponema denticola, several of which are insect-associated. The PAI 2 of P. luminescens W14 is chromosomally inserted at a tRNA-Phe locus and carries the mcf gene coding for the “Makes caterpillars floppy” (Mcf) pro-apoptotic protein, as well as a gene coding for a member of the Shl/HecA/FhaA hemagglutinin-like protein family [225]. The chromosomal insertion site as well as the number of mcf loci differs between individual Photorhabdus strains. The CNF-like toxin gene pnf is located on the P. luminescens W14 PAI 3, which seems not to be inserted at a specific tRNA-encoding gene. Nevertheless, this chromosomal regions exhibits a similarity to prophage genome regions and represents a distinct type of mobile genetic element, the so-called Photorhabdus virulence cassette (PVC) ([224–227]; see Table 2.3). H. pylori strains associated with the development of peptic ulcers and gastric cancer harbor the cytotoxin-associated gene A (cagA) PAI (cag-PAI). This 40-kb chromosomal region includes a type 4 secretion system (T4SS) determinant, as well as cagA coding for the CagA oncoprotein, which is secreted via the T4SS into eukaryotic cells and alters host cell signaling. As a result, cytoskeleton reorganization and cell elongation is induced and tight and adherent junctions are disrupted, inducing proinflammatory and mitogenic responses [229]. Bordetella pertussis, the causative agent of whooping cough, secretes several toxins implicated in this disease. The major virulence factor produced by B. pertussis is the pertussis toxin PTX. The five genes encoding the different subunits of the toxin (ptx) and the nine accessory genes required for the PTX (type IV) secretion system (ptl) are organized as a polycistronic operon [230–232], which has some characteristics of a PAI. In B. pertussis, the upstream region of the ptx/ptl locus was found to contain a truncated IS element similar to IS481 of B. pertussis. The last gene of ptx/ptl locus (ptlH) is followed by a tRNA gene coding for an asparagine-specific tRNA and by an inverted repeat. When the chromosomal insertion point of the ptx/ptl locus of B. pertussis is compared to that of B. parapertussis and B. bronchiseptica, this site in B. pertussis appears to be different from B. parapertussis and B. bronchiseptica [233]. Sequence comparison of this genomic region between B. pertussis, B. parapertussis, and B. bronchiseptica isolates revealed traces of HGT within the ptx/ptl gene cluster, indicating that parts of these genes have been acquired by members of the classical Bordetella subspecies. Their nucleotide sequences are divergent in different phylogenetic lineages or even absent [234]. Enterotoxigenic strains of B. fragilis produce a metalloprotease toxin designated fragilysin (FRA) or B. fragilis toxin (BFT). The encoding gene is located on a 6-kb pathogenicity islet, which contains an open reading frame coding for a second

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

57

metalloprotease (MPII) and another ORF that encodes a protein with homology to a snake cytotoxin. The fragilysin pathogenicity islet is 6033 bp in size, contains nearly perfect 12-bp direct repeats near both ends of the islet, and is not present in nontoxigenic strains of B. fragilis [235]. Three bft variants have been described, as well as the presence of a duplicated islet in clinical B. fragilis isolates [236]. The B. fragilis pathogenicity islet is part of a conjugative transposon (CTn) and therefore also could be transferred to nontoxigenic B. fragilis strains [204].

The bacterial T6SS Initially, a conserved gene cluster surrounding icmF in V. cholerae has been designated IcmF-associated homologous protein (IAHP) gene cluster. Later, these genes have been shown to be located on a GEI and to code for a secretion system in Gramnegative bacteria with similarity, the so-called type 6 secretion system (T6SS). A T6SS is a dynamic secretion apparatus expressed in human, animal, and plant pathogens, as well as in plant symbionts and several nonpathogenic bacteria, which injects toxic proteins directly into eukaryotic or prokaryotic cells [237–240], thereby mediating antagonistic behavior between bacteria or pathogenic or symbiotic interactions with eukaryotes [238,241]. T6SS-positive Gram-negative pathogens display marked interspecies but also intraspecies variability in the composition and arrangement of T6SS determinants. Within one strain, the T6SSs belong to different groups, have nonredundant functions, and are differentially regulated. This suggests that the T6SS islands have been acquired by HGT from distinct sources. It is speculated that HGT is the main mechanism of disseminating T6SS genes across species. The chromosomal organization of the T6SS gene clusters shows a high variability and the T6SS-positive bacterial species encode up to six T6SS-encoding islands [242–244]. Besides the presence of often multiple copies of T6SS gene clusters within a genome, T6SSpositive bacteria often possess several copies of genes coding for different structural components of the secretion apparatus, such as hcp, vgr, and PAAR-proteins, which are not part of the main T6SS gene clusters [245]. Toxic effectors can either be fused as toxin domains to structural proteins of the injection system or exist as independent proteins. The first described T6SS effector was VgrG1 from V. cholerae, which has a catalytic domain fused to the C-terminus. This domain was shown to cross-link actin in eukaryotic cells. Also, the VgrG3 of V. cholerae was shown to carry a C-terminal extension with peptidoglycan hydrolysis activity to outcompete other bacteria. VgrG proteins carrying a C-terminal extension with a catalytic function have been termed evolved VgrG. Further evolved VgrGs have been described in Aeromonas hydrophila and Burkholderia pseudomallei and Burkholderia thailandensis. VgrG1 of A. hydrophila possess a vegetative insecticidal protein (VIP-2) domain, which interferes with the cytoskeleton of eukaryotic cells by catalyzing ADP-ribosylation of actin. The conserved C-terminal extension of VgrG5 from B. pseudomallei and B. thailandensis was shown to be required for eukaryotic host cell fusion. Meanwhile, Hcp and PAAR-proteins fused to catalytic domains also have been identified by bioinformatic

58

Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

approaches. Hence, these proteins can have a dual function—that is, be essential functional components of the secretion apparatus and effectors [237,238,241]. The first truly secreted effectors have been identified in P. aeruginosa: the effectors Tse1 and Tse3 are peptidoglycan lysing enzymes, and Tse2 acts as a toxin. Similar toxic effectors to target competitor bacterial cells have been described in V. cholerae and S. marcescens. In contrast to P. aeruginosa, which can outcompete different other T6SS-positive species, the T6SS of S. marcescens seems to be exclusively directed against closely related but nonisogenic Serratia strains. T6SS effectors are often not encoded by T6SS determinants. In at least a few species, effectors have been found adjacent to orphan hcp-vgr genes [237]. To structure the growing number of effectors, a classification based on the catalytical activity has been proposed. Accordingly, T6SS effectors can be grouped into (i) peptidoglycan hydrolyzing effectors with amidase or muraminidase activity and (ii) phospholipases. Other activities have also been described, including nucleases, pore forming, ADP ribosylation, and actin crosslinking [237,238,241]. Interestingly, antibacterial effector genes are located adjacent to the gene encoding the cognate immunity protein to prevent suicide [246]. Beside the often reported anti-eukaryotic and prokaryotic functions, which have a negative effect on target cells, T6SSs are also involved in biofilm formation, stress response, and symbiosis with plants. As T6SSs are present in nonpathogenic and nonsymbiontic bacteria, an additional role in cell-to-cell communication has been suggested. Taken together, T6SSs provide a versatile and widespread tool to deliver a multitude of antieukaryotic and antibacterial toxic effectors directly into target cells.

Gram-positive bacteria Most of the known S. aureus enterotoxins are located on different S. aureus PAIs (SaPIs) [247,248]. Nine SaPIs have been identified in human and bovine isolates (see Table 2.4; [249–256]). SaPIs can use the phage reproduction cycle in order to promote their own transduction via phagelike infectious particles without reproduction of the helper phage [256]. They often carry the toxic shock syndrome toxin 1 (TSST-1)– encoding gene (tstH). Most staphylococcal enterotoxins are also SaPI-encoded [189,256] and most clinical S. aureus isolates possess at least two SaPIs with different gene content. Furthermore, the S. aureus exfoliative toxin D (ETD) and the ADPribosyltransferase EDIN-B are encoded on a PAI [187]. The cytolysin of E. faecalis contributes to the severity of enterococcal infection and is distantly related to lantibiotics [257]. The corresponding cyl genes can be located on either pheromone-responsive plasmids; e.g., pAD1 [258] or on the chromosome within a PAI. In E. faecalis strain MMH594, the cytolysin operon is part of a 150-kb PAI that also encodes other virulence-associated factors, such as the surface proteins Esp and aggregation substance [120,121]. An extensive nucleotide sequence identity between the cytolysin operons of pAD1 and strain MMH594 suggests that the PAI-located gene cluster resulted from a transfer of the operon from pAD1 into the chromosome of strain MMH594 [257]. A cytolysin- and Esp-positive E. faecalis strain can develop into a cytolysin- and Esp-negative variant at a high frequency, most

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

59

likely due to homologous recombination between IS905-like elements located within the cyl operon and downstream of the esp gene [121]. Intraspecies as well as interspecies transfer of this E. faecalis PAI have been observed [259]. The pathogenic species of Listeria (Listeria monocytogenes, Listeria ivanovii, as well as the nonpathogenic Listeria seeligeri), harbor a 10-kb virulence cluster containing the listeriolysin gene (hly), which is flanked on one side by the plcA-prfA region and on the other side by the lecithinase operon, which are required for intercellular spread during infection [260,261]. In L. seeligeri, this gene cluster is not properly expressed due to a reduced transcription of prfA, which encodes the transcriptional activator of Listeria virulence genes. The same genes that are located at both ends of the virulence cluster of Listeria (ldh, prs) are present in nonvirulent strains, but instead of the virulence gene cluster, housekeeping genes are located between ldh and prs [262]. Internalins have several functions as virulence-associated proteins of L. monocytogenes. The chromosomal asc-dapE internalin cluster comprises several inl alleles and exhibits a different organization in different lineages of L. monocytogenes, thus contributing to the different internalin repertoire of these bacteria [263–264]. The 19.6-kb Clostridium difficile pathogenicity locus (PaLoc), which exhibits several features of a PAI, is specific for virulent strains of C. difficile and comprises the genes encoding the cytotoxins A and B (tcdA, tcdB) of the large clostridial glucosylating toxin (LCGT) family, as well as the regulatory and accessory genes tcdR, tcdC, and E [265]. The integration site of the PaLoc was defined as a 115-bp fragment that forms a 20-bp hairpin loop. This stretch is found only in nontoxinogenic strains and is replaced by the PaLoc in toxinogenic strains [265,266]. Although the PaLoc genetic organization and its chromsomal insertion site are conserved among toxinogenic C. difficile isolates, the different toxinotypes differ by smaller genetic alterations present in the individual PaLocs, such as nucleotide insertions and deletions or SNPs. So far, deletions have only been detected in tcdA. This is probably due to the presence of longer repetitive regions at the 5′-end of tcdA compared to tcdB [267,268], which may promote recombination events. In contrast, SNPs occur frequently in those parts of tcdA and tcdB, which code for catalytic domains of the toxins. SNPs are more frequently present in tcdB than in tcdA, and tcdB is generally more variable than tcdA [266]. The tcdC gene also exhibits nucleotide sequence variability, which may affect the tcdAB gene expression [269–271,307]. The binary toxin CDT consists of the CDTa and CDTb proteins, which are encoded by a 4.2-kb chromosomal region designated Cdtloc, which also includes the regulatory gene cdtR [272].

Instability of PAIs The flanking regions of several PAIs are often characterized by the occurrence of direct repeats that can be involved in recombinational events leading to the deletion of the PAI (see Table 2.4). In the case of the PAIs of UPEC strain 536 and one PAI of strain J96, respectively, it has been shown that they can spontaneously delete from the chromosome at high frequencies upon site-specific recombination between short direct repeats [66,67,207,273]. IS elements or RS-sequences can also be responsible

60

Basic Genomic and Physiological Aspects of Bacterial Protein Toxins

for the excision and possible integration of genetic elements. In Y. pestis, homologous recombination between IS100 elements flanking the HPI leads to the deletion of this PAI [274]. PAI excision is accompanied by the loss of PAI-encoded determinants and reduced virulence of the resulting strains [66,67,207,274,275]. Such deletion events may represent a specific form of adaptation of these pathogens to certain niches, hosts, and tissues, or during transition of the pathogen from an acute state of infection to a chronic state where expression of highly antigenic factors may be disadvantageous. More generally, pathogens with a relatively high genetic flexibility are more competent in the colonization of new ecological niches and may have a selective advantage over organisms with less flexible genomes [18]. Genome plasticity and gene loss (e.g., in E. coli) can be detected in different nuances: in addition to the loss of complete PAIs, deletion of distinct regions within GEIs may frequently occur due to homologous recombination of IS elements and also results in phenotypic changes. Comparison of the genome content of nonpathogenic and UPEC O6 strains demonstrated that the gene content and the general genetic structure of the pheV-associated islands of UPEC strain CFT073 and nonpathogenic strain Nissle 1917 are highly similar. In the latter strain, however, the α-hemolysin and P fimbriae determinants, present on this island in UPEC strain CFT073, are deleted. This is supposed to be one of the reasons for this strain’s nonpathogenic phenotype relative to strain CFT073 [8]. Similarly, asymptomatic bacteriuria (ABU) E. coli isolates are attenuated and frequently lost the ability to express functional virulence factors, including toxins. A genomic analysis of nonhemolytic ABU E. coli isolate 83972 demonstrated that the α-hemolysin-encoding hlyCABD operon present in this strain was inactivated due to the presence of a premature stop codon within the hlyA gene [276].

Conclusion Evolution of new pathogenic variants caused by PAIs and mobile genetic elements Genes encoding toxins and other virulence factors can be located on PAIs and on mobilizable genetic elements such as plasmids, transposons, and bacteriophages. This is especially true for microorganisms, which lack the capacity to take up foreign DNA because of natural competence. The presence of so-called mobility sequences (e.g., direct repeats, sequences with homology to integrases or to plasmid origins of replication near the borders of PAIs) imply that PAIs are derived from integrated mobile elements such as bacteriophages and plasmids [3]. Plasmids and phages are able to form cointegrates. This has been found frequently in Streptomyces [277] and may be an explanation for the existence of features of plasmids and phages on PAIs. Therefore, integrated plasmids and bacteriophages have been considered as PAI precursors. This underlines the strong mutual dependence between plasmids, bacteriophages, and PAIs. This interdependence not only contributes to the genetic flexibility of bacteria carrying these genetic elements but to the fast evolution of new pathogenic variants as well (see also Figure 2.1; [7,18,278]). Mobile genetic elements, including

Mobile genetic elements and pathogenicity islands encoding bacterial toxins

attP

61

Site-specific chromosomal integration

vir

tra

ori

int

attL int int

attB

attP

ori

tra vir attR

vir Reductive evolution

Core genome

attL int

vir attR

Island/Lysogenic prophage attL int

IS vir

Tn

vir attR attL int

Recombination with other similar accessory elements in the genome

attL int

IS vir

vir Tn

vir

vir attR

Acquisition of functions (e.g., by insertion of mobile genetic elements)

attR

Figure 2.1  Model illustrating the impact of mobile genetic elements on the evolution of PAIs. A chromosomal integration of mobile genetic elements into the chromosome can occur upon recombination of homologous sites. A stabilization of the integrated DNA may occur by mutations in integrase genes (int), origins of replication (ori), or both. PAIs and other chromosomally inserted genetic elements can constantly evolve due to recombination, insertion, and deletion events, as well as the accumulation of point mutations.

plasmids, bacteriophages, GEIs and PAIs, as well as conjugative transposons, integrative, and conjugative elements, facilitate the exchange and acquisition of foreign DNA by HGT. They can be inserted into the chromosome or be excised by site-specific or homologous recombination [21,279–281]. The spread of specific genomic regions through lateral gene transfer, followed by integration into the bacterial genome, is a mechanism by which new variants of microbes could be generated. This can occur by lysogenic conversion, in which the prophage confers specific changes in the bacterial phenotype, or by the uptake of plasmids or DNA fragments by conjugation or transformation. A GEI or PAI may be the result of such transfer processes and subsequently generated point mutations. Spontaneous point mutations in mobility genes may lead to a fixation of laterally acquired genes in specific strains. Under specific selective pressure, a homing of new variants by inactivation of mobility genes could be advantageous, resulting in some of the structures of PAIs described in this chapter (see Figure 2.1). Another exception is the superantigen-encoding gene ypm (Y. pseudotuberculosis–derived mitogen) of Y. pseudotuberculosis that, unlike other superantigenic toxin genes, is not associated with mobile genetic elements but are localized in unstable regions of the chromosome. This explains why deletion of the ypm gene can occur with significantly increased frequency [282]. The analysis of different Shiga toxin–encoding phages led to the discovery that the Stx-encoding bacteriophage genome represents a moscaic of DNA regions of different origins. These bacteriophages are under constant evolution due to recombination

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events between the bacteriophage genome, fragments thereof, and the host genome. The prophage genome can also be frequently modified by gene deletions and insertion of IS elements, thereby negatively affecting the stx gene expression and prophage activation [283,284].

HGT and the evolution of toxin families The existence of toxin families with common properties and sequence homologies in the relevant genes argues that such a distribution of genetic information is a result of lateral gene transfer. The most prominent examples of genetically related toxin families are ADP-ribosylating toxins (diphtheria toxin, pertussis toxin, cholera toxin, E. coli LT, Pseudomonas exotoxin A and S, and others) and other AB toxins (Shiga toxins), pore-forming RTX toxins (i.e., various hemolysins, Pasteurella haemolytica leucotoxin, B. pertussis adenylate cyclase-hemolysin), clostridial neurotoxins, enterobacterial autotransporters, and proteins secreted by T3SSs. Many of them are encoded on plasmids, phages, or PAIs. There are various reasons why members of these toxin families can be transferred by mobile genetic elements or may have formerly been transferred by them. Other examples, such as the occurrence of many different proteins that share functional features of superantigens, may argue for convergent evolution of proteins with similar toxic characteristics in different bacteria. This is less probable in the case of toxin families than the acquisition by HGT because of the complexity of mutational and evolutionary events on the DNA level that would have to occur in order to result in functionally and structurally similar proteins. In S. aureus, the superantigenic toxins SEG- and SEI-encoding genes (seg, sei) are linked by a DNA region, the enterotoxin gene cluster (egc), that comprises three enterotoxinlike open reading frames sek, sel, and sem, as well as two pseudogenes. The egc gene cluster was detectable in many other S. aureus isolates, and phylogenetic analyses of staphylococcal enterotoxin-encoding genes implied that they all potentially derive from the egc cluster. It has been speculated that the existing great variety of superantigens in S. aureus may represent an enterotoxin nursery that enables the expression of a wide variety of toxin variants when needed [285]. Phylogenetic analysis of staphylococcal exotoxinlike protein determinants (set) localized on islandlike region RD13 within a variety of S. aureus isolates reveals that gene loss and recombination contributed to the diversification of this island [286]. The great similarity in the protein sequences of the E. coli LT and V. cholerae CT led to the assumption that CT, LT I, and LT II originally derived from the same ancestral gene. Other toxins immunologically related to CT can be found in Salmonella, Pseudomonas, Campylobacter, and Aeromonas. This may suggest that this is the result of different evolutionary pathways that this gene followed in ­different species [287]. The genetic organization of the bont genes located on the chromosome or on plasmids of C. botulinum shares similarity. Furthermore, the increasing genome

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63

sequence data indicate that bont gene sequences and their genetic structure also have been modified by recombination, insertion, and transfer events, leading to new toxin variants [107]. The pattern of distribution of genes involved in toxin expression on plasmids relative to the chromosome indicates that they may derive from a common ancestor, such as the T3SSs of various bacterial animal or plant pathogens (Yersinia spp., Shigella flexneri, Salmonella typhimurium, EPEC, Ps. aeruginosa, Chlamydia spp., Erwinia spp., Ps. syringae, Ralstonia solanacearum, and Xanthomonas campestris) [288–292]. Although the pathogenicity mechanisms (and therefore the secreted effector molecules of these bacteria) differ, the T3SS is conserved and effector molecules from one pathogen can be translocated by another system when the appropriate chaperons are present [289]. The ever-increasing wealth of whole genome sequence data from multiple isolates of individual pathogens allows us to comprehensively study the distribution, diversity, and variability of protein toxin determinant and, their sequence context. Analyses like this will help us to better understand differences in protein toxin function and expression levels between individual isolates. Comparative genomics of LEE-negative STEC, for example, supports previous findings that the Stx-encoding prophages have a mosic nature (i.e., the lambdoid prophages, which are inserted in the same chromosomal insertion site or which carry the same stx allele) can often harbor nonhomologous DNA regions [293,294]. This study also suggested that stx expression, and thus virulence, may correlate with the genetic structure of the stx sequence context and the presence of a particular Q antiterminator protein variant [293]. Comparative genomewide analysis, including protein toxin–encoding mobile elements, has been performed in order to gain insights into microepidemiology and evolution of toxin variants [95,99,264,265,294–296]. Taken together, virulence determinants in general and protein toxin genes in particular are frequently encoded by mobile genetic elements. They are encoded on large genomic regions as well, designated PAIs, which often contain clusters of virulence-associated genes. These genetic elements with the capacity to spread by HGT contribute to the rapid evolution of bacterial pathogens as the rearrangement, excision, and acquisition of large genomic regions constantly creates new variants. The occurrence of protein toxin–encoding genes on various interdepending mobile genetic elements, their ability to delete from and integrate into chromosomal DNA, and the existence of toxin families among a wide variety of bacterial species show that the continuing process of the evolution of new pathogens is significantly connected with the transfer of foreign DNA harboring toxin determinants. The large EHEC outbreak in May and June 2011 in central Europe, which was caused by an E. coli O104:H4 strain combining characteristics of EHEC and EAEC, demonstrated how new combinations of virulence genes can result in dangerous pathogenic variants [297–300]. Parallel evolution and the generation of new virulence gene combinations due to horizontal transfer of mobile genetic elements and recombination events will constantly lead to the emergence of new variants of already existing pathogenic microbes.

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Acknowledgments Our own work related to the subject of this chapter is supported by the DFG (International Graduate College GRK1409/3, project B01; Collaborative Research Center 1009, project B05) and the Federal Ministry of Research and Education (ERA-NET Pathogenomics III, FKZ 0315904A).

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