In contrast to Charles Darwin's theory, assuming that evolution of species progresses slowly by very small steps (Darwin, 1859), the concept of PAI acquisition allows ‘quantum leaps’ in genetic variation, and therefore in the evolution of bacterial species (Groisman and Ochman, 1996). Bacterial pathogens fitness can be defined as how well a bacterial strain can infect a host, persist, proliferate, and be transmitted to a new host in a specific niche (i.e. reproduce). In the light of this definition, an increased fitness could be achieved by a simultaneous acquisition of many genes by HGT that allow the bacteria to rapidly gain complex virulence functions and to exploit a new environmental niche (Ochman et al., 2000). In some cases, introduction of a new PAI can result in a dramatic or even total change of the phenotype or lifestyle of a bacterium. The ancestor of S. enterica, for example, was likely an intestinal-dwelling bacterium which was not capable of invading epithelial cells. An acquisition of fully functional PAIs known as SPI 1 and subsequently SPI 2 provided Salmonella new physiological capabilities and was an effective strategy to make a transition towards adaptation to a new intracellular environment.
Often an acquired PAI contains an entire operon(s) that acts as a functional unit conferring new virulence traits. According to the ‘selfish operon’ theory (Lawrence and Roth, 1996), an ongoing selective pressure leads to the clustered organization of genes whose products contribute to a single function in order to facilitate their HGT and their propagation in the population. This kind of selection shapes gene organization and actually drives the ability of PAIs to transfer large numbers of genes in a single event.
In order to demonstrate how PAIs specifically contribute to the virulence of pathogens we examine in more detail three representative PAIs including the LEE of pathogenic E. coli and related species; the cag PAI of H. pylori; and the PAI encoding toxic shock syndrome toxins (TSST) of the Gram-positive pathogen S. aureus (SaPI).
The LEE PAI
The locus of enterocyte effacement (LEE) was initially described in an EPEC strain, the causative agent of infant diarrhoea (McDaniel et al., 1995). EPEC is an attaching and effacing (A/E) pathogen that is able to attach to host intestinal epithelium and efface brush border microvilli. All the genes necessary for this phenotype are located on a 35 kb PAI, termed LEE, which is absent from laboratory E. coli strains (Elliott et al., 1998). Cloning the LEE into E. coli K-12 strain confers the complete A/E phenotype, reinforcing the notion that avirulent bacteria can be transformed into pathogenic ones through a single genetic step (McDaniel and Kaper, 1997). The LEE contains 41 ORFs (Fig. 2A) and is organized as five polycistronic operons (LEE1–LEE5). Analysis of the G + C content of the LEE (38%) showed that it is strikingly lower than that of the rest of the chromosome (50.8%). The chromosomal integration site of LEE in the EPEC reference strain E2348/69 is the selC tRNA gene, but other sites are found in different EPEC strains. The LEE PAI consists of functionally different modules including: (i) a T3SS, which is used as a molecular syringe to translocate effector proteins into host cells, (ii) the secreted translocator proteins (EspA, EspD and EspB) required for translocating effectors into host cells, (iii) the adhesin (intimin, EAE), which mediates intimate attachment to Tir on the host cell cytoplasmic membrane and (iv) the secreted effector proteins EspF, EspG, EspZ, EspH, Map and Tir, the intimin receptor, chaperoned by CesT. Translocation of these molecules by T3SS into the host cells results in changes of the host cell cytoskeleton arrangement leading to the formation of actin-rich pedestals in which the Tir effector is located at their tip. This structure allows the direct interaction of Tir with the bacterial outer membrane protein intimin, as well as the host cytoskeleton (reviewed in Zaharik et al., 2002).
Figure 2. The genetic organization of three representative PAIs. A schematic illustration of the LEE PAI from EPEC (A), the cag PAI of H. pylori (B), and the SaPI1 of S. aureus (C) is shown. The genetic nomenclature of the EPEC LEE is based on the suggested terminology by Pallen et al. (2005). The organization of the cag PAI is according to Fischer et al. (2001), and the organization of the SaPI1 is based on Novick (2003).
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LEE genes are controlled in a complex manner by different regulators encoded within the PAI, on a plasmid, and on the core genome. These regulators include the Ler (LEE-encoded regulator), GrlA (global regulator of LEE-activator), GrlR (global regulator of LEE-repressor), the plasmid-encoded regulator (Per), the DNA-binding protein H-NS and the integration host factor (IHF) (Clarke et al., 2003; Deng et al., 2004).
Similarly to EPEC, the LEE PAI was also found in EHEC strains. The LEE PAI of EHEC encodes proteins also involved in the A/E phenotype and is inserted in the selC site as well. The LEE region of EHEC contains 54 ORFs, of which 41 are common with the EPEC LEE. The remaining 13 ORFs belong to a putative P4-like prophage element, designated 933 L that is located close to the selC locus and, probably, has been acquired at a later time point. It is interesting to note that despite EPEC and EHEC sharing virtually the same LEE PAI, their primary host, the colonization sites, and the disease they cause are different. EPEC strains are classically associated with diarrhoea in young children and humans are considered their primary host. In contrast, EHEC infections are known to originate from particular ruminants. EHEC colonization of ruminants is generally asymptomatic while in humans it can cause a spectrum of diseases ranging from uncomplicated watery diarrhoea to bloody diarrhoea with abdominal cramps. These differences likely result from the evolution of EHEC from EPEC through the acquisition of phage-encoded Shiga toxins (Stx) (Reid et al., 2000).
LEE has also been identified in C. rodentium, the causative agent of transmissible murine colonic hyperplasia in suckling mice. Although the C. rodentium LEE shares 41 ORFs with EPEC and EHEC LEE, it is unique in the rorf1 and espG gene location, and the presence of several ISs. As opposed to the EHEC and EPEC LEE, C. rodentium LEE is not integrated into the selC locus and contains on one side an ABC transport system and an IS element on the other side. Based on this, it has been suggested that the LEE PAI may have been acquired several times during the evolution of different A/E pathogens.
Particular E. coli strains are associated with diarrhoea and other enteric infections in rabbits, pigs, calves, lambs and dogs. These EPEC strains contain LEE PAIs inserted in selC, pheV or pheU tRNA loci. The rabbit diarrhoeagenic E. coli strain RDEC-1 contains a LEE that is flanked by an IS2 element and the lifA toxin gene (Zhu et al., 2001). LEE has also been characterized in a bovine Shiga toxin-producing E. coli (STEC) O103:H2 strain (Jores et al., 2001).
Besides the secreted effector proteins encoded within the LEE PAI, recent work in our and others labs led to the identification of six non-LEE-encoded, conserved effector proteins (NleA, NleB, NleC, NleD, NleE and NleF). In EHEC, these effectors are organized in three additional distinct PAIs (Deng et al., 2004; reviewed in Garmendia et al., 2005). Recent analyses showed that nleB and nleE, encoded within a PAI known as O-Island 122, are associated with outbreaks and haemolytic–uraemic syndrome of non-O157:H7 STEC; and that NleA and NleB are absolutely necessary to cause mortality in the mouse model (Wickham et al., 2006). These observations indicate that diseases mediated by A/E pathogens require co-ordinated and regulated action of effectors encoded by the LEE and other PAIs (Deng et al., 2004).
The cag PAI of H. pylori
Since its isolation from human stomach biopsies in 1983 (Marshall and Warren, 1984), H. pylori has been the focus of intense research. As a human gastric pathogen, H. pylori colonizes over half of the world's population. While many of H. pylori infected individuals are clinically asymptomatic, most will exhibit some degree of gastritis; approximately 10% of the infected subjects will develop more severe gastric pathologies like peptic ulcer disease, atrophic gastritis; and approximately 1% of infected individuals will develop gastric cancer. One of the defined H. pylori virulence factors is the cag PAI. Strains of H. pylori associated with severe gastric disease, such as peptic ulcer disease, possess the cag PAI, which is absent from strains isolated from patients with uncomplicated gastritis (Censini et al., 1996). Similar correlation was also found in the infection model of Mongolian gerbils demonstrating that H. pylori strains with an intact cag PAI induced strong inflammation and ulceration in the stomach (Ogura et al., 2000). Interestingly, studies with a mouse model have shown an association between cag PAI-negative H. pylori strains and strains that are mouse adapted, less virulent, and can better colonize mice, indicating that the cag PAI may become lost during colonization of animals (Philpott et al., 2002).
The cag PAI is a 37–40 kb chromosomal region that was acquired by horizontal transfer and inserted at the distal end of the glutamate racemase gene (glr). cag has a distinct G + C content, and is flanked by DRs of 31 bp that probably function as sites for recombination and deletion of the locus. Sequence analysis of the cag PAI predicted 27 ORFs and an additional element which is not present in all of the cag positive strains (hp548/cagΩ; Fig. 2B). A large portion of the cag PAI genes encode a functional T4SS and eight of them are homologues to components of the prototype T4SS represented by the A. tumefaciens virB operon. In addition to the T4SS, the cag PAI encodes CagA, the only effector protein of the H. pylori T4SS currently known (Segal et al., 1999). Studies of CagA's cellular activities reveal that CagA interacts with a large number of host proteins and has multiple effects on host signal transduction pathways, the cytoskeleton and cell junctions (for a recent review see Bourzac and Guillemin, 2005). After translocation of CagA into host cells, it becomes phosphorylated by Src kinases and is recruited to the plasma membrane, where it interacts with a number of host proteins. The best studied of these interactions is with the SRC-homology 2 (SH2) domain-containing tyrosine phosphatase (SHP-2). Interaction of SHP-2 and CagA activates particular pathways and leads to actin polymerization, cell elongation, pedestal formation as well as growth factor-like response and abnormal proliferation of gastric epithelial cells. Besides SHP-2, other substrates which CagA interacts with include ZO-1, Grb2 and C-Met (reviewed in Naumann, 2005). In tissue culture cells and in the mouse model, it has been shown that the cag PAI induces expression of proinflammatory cytokines, such as interleukin-8 (IL-8), which is thought to contribute to H. pylori-induced inflammation in the stomach (Crabtree et al., 1995; Philpott et al., 2002). A recent study also showed an interaction between CagA and another cag protein, namely CagF, suggesting that CagF might function as a chaperone-like protein for CagA (Couturier et al., 2006).
Systematic mutagenesis approaches to analyse the function of the 27 genes in the cag PAI identified a subset of 17 genes that are absolutely required for the translocation of CagA and a subset of 14 genes that are required for the stimulation of IL-8 synthesis in host cells (Fischer et al., 2001). Although the assembly of the T4SS is not understood in full detail, these observations indicate that the majority of the cag PAI genes are required for the formation of a functional T4SS which is used to: (i) translocate the bacterial effector protein CagA into host cells, and (ii) induce the synthesis and secretion of chemokines, such as IL-8.
Cumulatively, these studies show a pivotal role of the cag PAI in the virulence of H. pylori and clearly demonstrate the way by which a single locus contributes to the pathogenic lifestyle of a bacterium.
Staphylococcus PAI encoding TSST
Chromosomal regions with the typical features of PAIs as in Gram-negative bacteria are apparently less abundant in Gram-positive pathogens, although some of the characteristics of PAIs have also been identified in these microorganisms. Genome comparative analyses between related Gram-positive bacteria have demonstrated that acquisitions of genomic islands are indeed the main source of pathogenicity and resistance profile differences and therefore play a similar role as in Gram-negative bacteria (Gill et al., 2005).
Staphylococcus aureus is a common commensal bacterium found on human skin and respiratory tract mucosal surfaces. However, it is also a pathogen causing a range of acute and pyogenic infections, including abscesses, bacteraemia, central nervous system infections, endocarditis, osteomyelitis, pneumonia, urinary tract infections, chronic lung infections associated with cystic fibrosis and several syndromes caused by a variety of toxins. These toxins, including haemolysins, staphylococcal exotoxins (Set) and superantigens (SAgs), are major virulence factors of S. aureus. Staphylococcal SAgs are a group of high molecular-weight proteins that are potent stimulatory agents for CD4+ T lymphocytes. As such, they have profound effects on the immune system, leading to non-specific activation of a large proportion of T cells, resulting in the release of various cytokines. Certain S. aureus strains possess secreted virulence factors known as TSST that function as superantigens toxins (Bachert et al., 2002). The consequences of these TSST may include high fever, rash, vomiting, diarrhoea, renal and hepatic dysfunction and desquamation.
The chromosomal tst gene, encoding TSST-1, is located on a series of discrete 15–20 kb chromosomal elements that are mobilized at high frequencies by certain staphylococcal phages. These elements are referred to as staphylococcal pathogenicity islands (SaPIs) and were the first clearly defined PAI characterized in Gram-positive bacteria (reviewed in Novick, 2003). The prototype of this family is the SaPI1 that was the first characterized SaPI. SaPI1 is 15.2 kb long, carries a tst gene, flanked by 17 bp DR sequences, and is inserted in an attc site close to the tyrB gene (Fig. 2C). The integration into the chromosome is facilitated by the presence of a functional integrase (int) gene encoded in the island. Remarkable features of SaPI1 are therefore its mobility and instability. The excision of SaPI1 from the chromosome and its presence as episomal DNA have been observed (Ruzin et al., 2001).
In addition to SaPI1, other SaPIs, which carry tst and different SAgs genes, have been characterized. A PAI related to SaPI1, termed SaPIbov, was identified in a bovine isolate of S. aureus. SaPIbov is 15.9 kb long and is inserted at the 3′ end of the GMP synthase gene (gmps), in an att integration site. SaPIbov is flanked by 74 bp DR sequences and harbours, in addition to tst, two other enterotoxins, encoded by sec and sel genes (Fitzgerald et al., 2001). SaPI3 has been shown to contain two novel enterotoxins encoded by the sek and seq, as well as the enterotoxin B (SEB). SaPI3 is flanked as well by att sites and displays an overall structure similar to SaPI1; however, a tst gene is absent in SaPI3 (Yarwood et al., 2002). Interestingly, the presence of int genes and att sites in the SaPIs suggests that they have been acquired from phage genomes.
A recent analysis of several S. aureus isolates has led to the identification (and renaming) of seven conserved PAI families in the S. aureus genome designated vSa1 (including SaPI1 and SaPI3); vSa2 (including SaPIbov); vSa3; vSa4 (including SaPI2); vSaα; vSaβ; and vSaγ (Gill et al., 2005). Besides the TSST encoded PAIs, another important PAI is the Staphylococcus cassette chromosome mec (SCCmec), which encodes the methicillin resistance determinants MetI MetR and MetA (Daum et al., 2002).
In summary, various PAIs in the S. aureus genome carry approximately one-half of its toxins or virulence factors, and allelic variation of these genes, along with the presence or absence of individual islands, contributes to the pathogenic profile of S. aureus species (Gill et al., 2005).