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Jonathan McLatchie

The Role of Mobile DNA in the Evolution of Bacterial Virulence


1. Introduction Evolution requires that genetic information be passed from one individual to other members of a population. There are two ways in which this can be accomplished vertical transmission of genes (from parent to offspring) and horizontal transmission of genes (between individuals of a population who are not of a parent-offspring relationship). In prokaryotes, the vertical transfer of genes takes place by virtue of replication of the bacterial chromosome and subsequent cell division by binary fission. Conversely, horizontal DNA transfer does not involve chromosomal replication and cell division. Horizontal DNA transfer occurs by several different means, including plasmid conjugation, bacteriophage transduction, and transformation. In eukaryotes, genetic variation is produced principally by virtue of chromosomal recombination during meiosis. Conversely, in prokaryotes, genomes have been shaped instead by other mechanisms, including frequent point mutations and genetic transfer. Exchange of genetic material between bacteria has resulted in very rapid evolution of microbial genomes (Ochman et al., 2000). There is considerable evidence that the mechanisms of genetic exchange have played an important part in the evolution of virulence genes. The purpose of the present paper is to examine this evidence and consider the role that lateral gene transfer has played in the evolution of pathogenicity.

2. Mechanisms of Lateral DNA Transfer

Jonathan McLatchie There are three main mechanisms by which bacteria accomplish lateral DNA transfer. The mechanism of transformation involves a bacterial cell taking up naked DNA from the environment or surrounding medium. Conjugation involves the direct transfer of genes between bacterial cells by means of direct cell-to-cell contact. In this case, a conjugative or mobile element (usually a transposon or plasmid) is supplied by a donor cell. Transduction involves the transfer of DNA mediated by bacteriophages.

Not all species of bacteria participate in all three of those modes of genetic exchange. Conjugation, for example, occurs more frequently in Gram-negative bacteria (Cruz et al., 2011), though it does take place in certain Gram-positive genera, such as Streptococcus and Streptomyces (Grohmann et al., 2003). In some bacterial taxa, cells only gain competence following artificial pre-treatment (though other species undergo natural transformation). For example, Escherichia coli has been shown to take up bacteriophage DNA following treatment with calcium chloride (Mandel and Higa, 1970), a technique which was subsequently improved on by Douglas Hanahan (Hanahan, 1983). Lets consider these modes of lateral DNA transfer in more detail.

2.1 Transformation The mechanism of transformation was first observed by Frederick Griffith in 1928 (Griffith, 1928). Griffith showed that a non-pathogenic strain of Streptococcus pneumonia could be made virulent by incubating it with an extract of heat-killed virulent cells. Avery, MacLeod and McCarty later showed that the so-called transforming principle was DNA by making an avirulent strain of Streptococcus pneumonia virulent by exposing it to the DNA of a virulent strain (Avery et al., 1944).

Jonathan McLatchie

Apart from the already-mentioned Streptococcus pneumonia, naturally occurring spontaneous transformation has been most extensively studied in Haemophilus influenza (Mell et al., 2011) and Bacillus subtilis (Nijland et al., 2010). Though it was once thought to be limited to these and related organisms, it is now recognised to be a much more widespread phenomenon than this. In Neisseria gonorrhoeae, for example, the transfer of pil genes (which code for the primary protein subunit of the Pili that allow attachment of the bacteria to epithelial cells) by means of transformation plays a key role in the production of antigenic variation (Chaussee et al., 1999; Baron et al., 1978).

Competence typically is acquired at a particular stage of bacterial growth, generally in the late logarithmic phase, as the cells are just entering into the stationary phase (Baltrus and Guillemin, 2005). This may constitute a response to population density by means of quorum sensing as opposed to being stimulated by growth phase. In Bacillus subtilis, for instance, there is overlap between the genes involved in the acquisition of competence and those involved in the formation of endospores (Piggot and Coote, 1976). In this case, the development of competence is linked to the accumulation of competence factors. These secreted products operate using a twocomponent regulatory circuit to trigger the expression of competence-relevant genes (Dubnau, 1991).

Once competence has been acquired, bacterial cell surface receptors are bound by double-stranded (ds) DNA fragments. Only one of those strands, however, will enter the cells interior. Bacterial transformation can be made specific to DNA of the same

Jonathan McLatchie species. This is true of, for example, Neisseria meningitides, the genome of which contains nearly 2000 copies of a specific 10 base pair uptake sequence (Ambur et al., 2007). This allows the meningococcus to make its uptake specific to DNA derived from bacterial cells of the same species. The setup in Haemophilus influenze is similar except that it has a 29 base pair uptake sequence which is found roughly 1500 times in its genome (Smith et al., 1995). Other bacterial species (such as Bacillus subtilis and Streptococcus pneumoniae), conversely, are able to take up linear DNA essentially indiscriminately.

In the case of linear chromosomal DNA fragments, following their acquisition by the bacterial cell, the linear DNA strand has to be recombined with the recipient chromosome in order to ensure a stable transformation. This necessitates some degree of homology between the DNA fragment and the recipient chromosome. This implies that the more closely related the DNA fragment is to the genome of the recipient bacterial species, the more likely it is that they will successfully recombine. A good instance of this is the evolution of penicillin resistance in Streptococcus pneumonia (Fani et al., 2011; Trzcinski et al., 2004). This seems to have developed by virtue of replacing part of the genes which code for the penicillin target enzymes with DNA sequences from oral streptococci (which is naturally resistant to the antibiotic).

2.2 Conjugation Bacterial conjugation refers to the transmission of DNA usually in the form of a plasmid or transposon from one cell to another by means of direct contact between the donor and recipient or by formation of a bridge-like structure, called a pilus, which connects the two cells (Llosa et al., 2002). Bacterial conjugation was first

Jonathan McLatchie discovered in 1946 by Edward Tatum and Joshua Lederberg (Lederberg and Tatum, 1946). Such mobile DNA may confer advantages such as resistance to antibiotics or the ability to utilise different metabolites.

The donor bacterial cell possesses an episomal fertility factor, referred to as an F plasmid, which can insert itself into the bacterial chromosome by virtue of homologous recombination. The F plasmid possesses its own origin of replication (OriV) and origin of transfer (OriT). Strains of bacteria in possession of an F plasmid are designated F+, and have gene necessary for synthesising the protein pilin (which is the subunit of the pili by means of which the DNA will be transferred). On the other hand, bacterial cells which dont have the F plasmid are designated F-. The pili allow the donor bacteria to make contact with the cell-surface receptors of the recipient.

After the pili have made contact with the cell-surface receptors of the recipient, they contract to bring the donor and recipient closer together, and a pore or channel is created through which the genetic material is transferred. An enzyme called relaxase subsequently creates a single-strand break (nick) at the origin of transfer (oriT). The plasmid DNA is then unwound by a helicase enzyme (itself encoded by the plasmid). The transfer of the DNA strand from host to recipient, in the 5-terminus to 3terminus direction, can then take place. The strand that remains is replicated to replace the transferred DNA. This can happen in a fashion resembling rolling circle replication or by vegetative replication commencing at the oriV.

Jonathan McLatchie Plasmids which are capable of making the transfer from one cell to another unaided are called conjugative plasmids. Other plasmids are non-conjugative, though on occasion their mobilisation can be promoted by other, conjugative, plasmids.

2.3 Transduction Transduction is phage-mediated lateral DNA transfer. During the lytic growth stage of the phage, DNA is packaged into the phage head. Occasionally, fragments of bacterial DNA can be mistakenly packaged. This transduced DNA segment will subsequently be incorporated into the genome of a different host cell as the phage attaches and injects its DNA into the recipient. If the transduced DNA is a fragment of chromosomal DNA, then it must be incorporated into the chromosome of the new host by homologous recombination. If, on the other hand, the transduced DNA is a bacterial plasmid, it is able to simply be replicated and inherited.

2.4 Recombination Except for the transfer of genetic elements which can replicate independently of the chromosome of the recipient cell, the transferred DNA needs to be integrated into the recipient cells chromosome. This is accomplished by breaking the two DNA molecules, crossing them over and re-joining them. Since this necessitates the presence of highly similar DNA regions between the two molecules, this process is classified as homologous recombination.

For a linear DNA element to be integrated into the host chromosome, two breakage/re-joining events must take place (double cross-over). This ensures that a portion of the linear DNA molecule will become incorporated into the circular

Jonathan McLatchie chromosome, thus replacing the corresponding chromosomal region. If, however, both the acquired mobile element and the DNA of the host are circular, then only one recombination event is necessary.

3. The Role of Transposons in Gene Transfer In some instances, a gene can transpose from one plasmid to another. One classic example of this relates to the ubiquitous nature of the penicillin-inhibiting enzyme lactamase (Uemura et al., 2010; Heritage et al., 1992; Rowland and Dyke, 1989; Weber and Goering, 1988). This enzyme imparts penicillin-resistance to bacteria, and is the most abundant variety of plasmid within the family of gram-negative bacteria called Enterobacteriaceae (which includes Salmonella and E. coli). TEM -lactamase is able to be transposed from one plasmid to another. The transposition of resistance genes can also occur between plasmids and chromosomes.

Some transposons called conjugative transposons are able to transpose not only between sites in the same cell, but also between cells by virtue of a conjugative mechanism (Salyers et al., 1995). This procedure involves excision of the transposable element to create a covalently closed circular intermediate, which is able to undergo conjugative transfer to another cell.

4. Mobile DNA and the Evolution of Virulence 4.1 Pathogenicity Islands Pathogenicity islands comprise a class of genomic islands which harbour virulence genes and which are involved in pathogenicity (Hacker and Carniel, 2001). They are acquired by virtue of horizontal transfer of genes. Pathogenicity islands contain genes

Jonathan McLatchie which code for various virulence factors. These include secretion systems, iron uptake systems, invasins, adhesins and toxins, as well as mechanisms involved in countermanding the hosts defence system. Pathogenicity islands are often integrated into tRNA genes, and are flanked by direct repeats which are homologous to phage attachment sites. These contain insertion sequences, and also code for proteins such as transposases and integrases, which facilitate recombination and integration into host DNA (Buchrieser et al., 1998; Hacker et al., 1997).

In Salmonella senftenberg, a conjugative transposon carrying sucrose-uptake genes is essential for the bacterias metabolic adaptation when infecting a host (Hochhut et al., 1997). Pathogenicity islands coding for iron-uptake systems are also found in many enterobacteria and also strains of the genus Pseudomonas which are found in the rhizosphere that is, soil which is in close proximity to plants and which is influenced by root secretion. Pseudomonas genomic islands may also carry genes which code for enzymes needed for phenolic compound degradation (Ravatn et al., 1998). Other genomic islands code for factors which confer antimicrobial resistance, for example in the mec-A DNA of Staphylococcus aureus (Ito et al., 1999).

Pathogenicity islands can also carry genes which code for Type III or IV secretion systems. Examples of this include Salmonella (Galan and Collmer, 1999) and Yersinia (Cornelis et al., 1998) strains.

4.2 Phage Conversion On occasion, defective remnants of bacteriophages can prove to be of adaptive benefit. In Pseudomonas aeruginosa, two gene clusters which originally coded for

Jonathan McLatchie phage tails have evolved to form toxic bacteriocins (Michel-briand and Baysse, 2002). Similarly of note is the defective prophage PBSX in Bacillus subtilis (Anderson and Bott, 1985). This defective prophage still has the capacity to produce a phage head into which bacterial DNA fragments can still be packaged. This aids in the process of DNA transfer. Phage conversion refers to the conversion of a non-pathogenic strain into a pathogenic one by means of bacteriophage infection.

Bacteriophages can be of great importance in the evolution of pathogenicity. Scarlet fever, for example, is caused by erythrogenic toxin which is released by the bacterium Streptococcus pyogenes (Weeks and Ferretti, 1984). The gene which specifies the erythrogenic toxin produced by the bacteria is carried by a bacteriophage. A similar scenario holds true with respect to the causative agent of diphtheria, Corynebacterium diphtheria (Maximescu et al., 1973; Gill et al., 1972). In the case of Vibrio cholerae, infection by VPI phage facilitates the evolution of pathogenicity (Karaolis et al., 1999). The pilus coded by the phage provides an attachment site for the filamentous phage CTX. CTX carries the genes for the cholera toxin subunits ctxA and ctxB. When the CTX DNA is integrated into the VPI prophage, the Vibrio cholera strain becomes pathogenic.

One well-documented example of phage conversion relates to the common foodborne pathogen Escherichia coli 0157:H7, the genome of which carries an extra 1.4 Mb of strain-specific DNA (Ogura et al., 2009). This sequence is contributed to by two dozen prophages, making up nearly half of this DNA sequence. Prophages have thus played an important role in the evolutionary trajectory taken by this pathogenic strain of Escherichia coli.

Jonathan McLatchie

4.3 Plasmids and Virulence Bacterial plasmids are known to carry genes conferring antibiotic resistance, colicins and bacteriocins, as well as virulence determinants. Plasmids often carry genes which code for proteins which have antimicrobial activity. In Escherichia coli, one particular group of these proteins which are called colicins are able to kill competing strains of the same organism (Cascales et al., 2007). In colicinogenic bacteria that is, those which carry genes coding for the production of colicins a second gene is carried on the same plasmid in order to confer immunity to the colicin. Colicins are able to bind to the outer membrane proteins of the target cell, before being translocated through the outer membrane and progressing through the periplasm by means of the TonB or Tol system. Upon reaching the target, Colicins subsequently employ their endonuclease activity on the target cells nucleic acid.

Plasmids are also known to frequently carry virulence determinants. While the gene which codes for cholera toxin in Vibrio cholerae is carried by a prophage, certain Escherichia coli strains are able to produce a very similar toxin, called labile toxin, which is carried on a plasmid (So et al., 1978). Another example is the pathogen Yersinia, the genus which includes the causative agent of bubonic plague, Yersinia pestis. Yersinia carries a 70kb virulence plasmid which enables the pathogen to inject its effector proteins into the immune-associated cells in order to disrupt their communication pathways and thereby disarm them (Cornelis et al., 1998).

In the case of the causative agent of anthrax, Bacillus anthracis, the genes coding for the anthrax toxin are carried on a plasmid called pXO1. Another plasmid, called

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Jonathan McLatchie pXO2, contains the genes necessary for the synthesis of a capsule which provides protection for the bacteria from the host organisms immune response (Pannucci et al., 2002). This allows the bacterium to induce septicaemia.

The plant pathogen Argobacterium tumefaciens, which causes crown gall tumours, carries a Ti (tumour-inducing) plasmid which is involved in its pathogenicity, except that, in this incidence, a specific part of plasmid DNA is transferred into the plant cell (Das et al., 1986).

5. Conclusion Mobile genetic elements have played an important role in the evolution of bacterial virulence and pathogenicity. The transfer of mobile DNA by means of conjugation, transformation, transduction and recombination routinely provides adaptive benefit, conferring an arsenal of virulence factors to their host.

Questions for future research endeavour include the role played by the environment and its corresponding resident organisms in determining the trajectories of pathogen evolution, as well as other potential drivers of pathogen evolution. Another interesting question is whether or not the processes of transformation and transduction induce an SOS response as they have been shown to do in the case of conjugation (Baharoglu et al., 2010), thus triggering the mobilisation of other mobile DNA. A better understanding of the triggers which underlie the mobilisation of transposable DNA will be important in ensuring that our activities do not promote the development and spread of virulence.

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