Gene Transfer in Intracellular Bacterial Parasites

Trafficking for intracellular bacteria can be categorized into intraphagosomal and intracytoplasmic pathways, in which different relationships with the host cell compartment may involve different modes ofgene transfer and different types ofacquired genes. Intraphagosomal pathogens include Afipia, Brucella, Burkholderia, Chlamydia, Coxiella, Ehrlichia, Francisella, Legionella, Mycobacterium, Rhodococcus, Nocardia and Salmonella, while intracytoplasmic pathogens include Listeria, Rickettsia and Shigella [6, 9, 35]. Specific genes are major targets for gene transfer [6]. These include those encoding proteins for type III and IV secretion systems, which are responsible for infection/virulence, and polymorphic surface proteins, which are responsible for attachment and/or antigenic variation. As expected, genes involved in housekeeping functions have been documented to undergo gene transfer less frequently [6].

Using sequences from various prokaryotes and eukaryotes in 2001, Koonin et al. [3] identified several horizontally transferred genes such as DNA gyrase A and B subunits among intracellular bacteria, including C. trachomatis, C. pneumoniae, R. prowazekii, M. thermoautotrophicum and M. tuberculosis, and S-adenosylmethionine (SAM)-dependent methyltransferase genes for M. tuberculosis. The authors calculated the number of domain transfer events among these different prokaryotic genomes and reported interdomain transfer to be less frequent in parasitic bacteria, except for the families Chlamydiaceae and Rickettsiales, compared with free-living bacteria, in which approximately 3% of the genomes were predicted to be involved.

Several transposons and foreign genes, such as the 14 tra genes that are usually involved in pilus formation and conjugal DNA transfer, have recently been discovered in the genomes of R. prowazekii, Rickettsia massiliae and Rickettsia bellii, representing both conjugation and transformation events in these organisms [6, 7]. Using quantitative assessments [3] and score-based identification of genomic islands [19], the numbers of horizontal gene transfer events, which include transfers from prokaryotes, archaea and eukaryotes, were highest in R. prowazekii (3.6% of the genome), C. pneumoniae (3.1%), Ehrlichia ruminantium (3.0%) and M. tuberculosis (3.0%). Ehrlichia is a member in the order Rickettsials [19]. The high gene transfer rate in rickettsias may relate to the multiple copies of genes involved in facilitating transfer, including the tra genes. Whereas R. prowazekii (2.8% of the genome) and C. pneumoniae (2.2%) appear to have a predilection for paralogous and xenologous gene transfers, M. tuberculosis (1.7%) has an abundance of new gene transfers [3]. These findings support characteristic frequencies for gene transfer in different microorganisms. Nonetheless, the actual number of transferred genes may be substantially higher as some genes may not have sufficient similarity scores with prokaryotes, archaea or eukaryotic genes, especially for those that have not recently been transferred because they have become part ofthe core genes for those lineages and have undergone the same directional mutational pressures as the rest of the genome. Examples of core genes include those involved in antibiotic-resistance, type III secretion and antigenic surface proteins [3, 4].

Pathogenicity islands, also called genomic islands or virulence cassettes, refer to an area where there is excess genetic variation within the species. The islands often occur at tRNA and tRNA-like loci, which are frequent sites for integration of foreign sequences [23, 27]. The short direct target or inverted repeat sequences flanking the islands are hallmarks of insertion sequences [23]. Consequently, genomic islands generally serve as an indicator for horizontal gene transfer via conjugation. Pathogenicity islands tend to encode numerous important physiologic functions such as iron uptake and type III secretion, and are promiscuously transferred, facilitating a similar pathogenic lifestyle even for quite disparate bacterial taxa [1, 23, 27, 36, 37].

Genetic exchange among intracellular microorganisms is biologically feasible, as mixed infections do occur in vivo. For example, up to 57% of C. trachomatis sexually transmitted infections represent co-infections with different strains of C. trachomatis [38-43], or with Neisseria gonorrhoeae, Trichomonas vaginalis and human papillomavirus [42, 44-46]. In in vitro studies, different C. trachomatis strains can infect the same cell, leading to fusion of the phagosomes with the opportunity for transfer and recombination [47]. Cohabitation and vacuole fusion of Coxiella within the host cell with other intracellular pathogens, including Mycobacteria, Salmonella and Leishmania, has also been described [48], although evidence for horizontal transfer in Coxiella remains minimal.

Chlamydiaceae species contain DNA elements that support transformation, conjugation and transduction, including genes involved in DNA repair and recombination such as recA and yqgF [5, 36, 37, 47, 49-51]. Phylogenetic, statistical and modeling analyses using membrane-encoding genes such as ompA and pmpA to pmpI, and the tryptophan synthesis operon trpRBA, for example, support horizontal transfer between genera and among different chlamydial species and indicate phenotypic differences, including niche specificity [52], tissue tropism [15, 17], differential host immune responses [15] and persistence of the organism [15]. Intraspecies recombinations of these genes have also been detected, which also likely assist the bacteria in surviving under host immune pressure and in establishing infection in new cell types [5, 15-18]. Figure 2.1 shows examples of intraspecies recombination among recent clinical strains of C. trachomatis and examples of recombinants created by in vitro experimentation [53] that may represent transformation or conjugation [54]. Interestingly, consistent with the clinical findings of C. trachomatis strain Da, which infects both the eye and genital tract, there is demonstrated evidence for intraspecies recombination whereby this strain has acquired genetic components from both ocular and urogenital strains [17]. Furthermore, although genetic transfer between strains of the same species may be subtle compared with interspecies transfer, these variations may be critical, depending on the physiochemical type and structural location of the amino acid substitution [52, 55]. The evidence for conjugation includes the acquisition by C. suis of tetracycline-resistant genomic islands and the insertion element, IS605, which is homologous to another gut pathogen, Helicobactorpylori, inserted into the chlamydial invasion (inv)-like gene [37]. Transduction involving different phages is a possibility for certain species of Chlamydiaceae, including Chlamydophila caviae, Chlamydophila abortus, Chlamydophila pneumoniae, Chlamydophila psittaci and Chlamydophila pecorum [36, 52, 56-59].

Chlamydophila Trachomatis

Figure 2.1 Diagram of Chlamydia trachomatis recombinationtypes based on in vivo observation of genomic sequences and in vitro experimentation. (a) Six different recombination types were detected among 10 recent clinical isolates [5]. The number of clinical isolates for each type is shown in parentheses. The genes and intergenic regions (IGRs) that were sequenced are labeled on top of each arrow.

The CT designation followed by numbers represents the gene number within the C. trachomatis genome. The arrows denote crossover regions. (b) Four main types were noted for in vitro recombinants of reference strains D and L1 [53]. The shaded gray groupings of crossovers represent regions that may be similar or identical to those observed in vivo or, vice versa, in vitro.

Figure 2.1 Diagram of Chlamydia trachomatis recombinationtypes based on in vivo observation of genomic sequences and in vitro experimentation. (a) Six different recombination types were detected among 10 recent clinical isolates [5]. The number of clinical isolates for each type is shown in parentheses. The genes and intergenic regions (IGRs) that were sequenced are labeled on top of each arrow.

The CT designation followed by numbers represents the gene number within the C. trachomatis genome. The arrows denote crossover regions. (b) Four main types were noted for in vitro recombinants of reference strains D and L1 [53]. The shaded gray groupings of crossovers represent regions that may be similar or identical to those observed in vivo or, vice versa, in vitro.

Horizontal transfer is also important in the evolution of food-borne pathogens including Listeria and Salmonella [60]. In Listeria, genomic differences between pathogenic Listeria monocytogenes and non-pathogenic Listeria innocua have resulted from transformation, conjugation and transduction [61]. Six virulence genes (prfA, plcA, hly, mpl, actA and pclB) are present in all Listeria, clustered in a region known as the LAPI-1 (Listeria pathogenicity island 1) [60] while the non-pathogenic Listeria strains have lost this cluster during evolution [60]. Interestingly, this genomic region appears to have evolved by vertical transfer from an ancestral strain and not by horizontal gene transfer as there is no evidence of insertion sequence-like elements or transposons [60, 63]. However, the plasmid pLM80 and transposons containing tetracycline-resistant determinants have been identified in L. monocytogenes [60]. Bertrand and colleagues [62] suggested that tetracycline resistance might be horizontally transferred from other Gram-positive bacteria, such as Enterococcusfaecalis. While bacteriophages have been reported in L. monocytogenes and L. innocua, transduction has not been considered a predominant mechanism in the acquisition of virulence genes due to the narrow host range of its bacteriophages, resulting inlow cross-species genetic changes [60, 61]. In contrast, the genomes of many species and strains of Salmonella exhibit several mobile genetic elements, virulence cassettes, plasmids and bacterio-phages. Examples of transformation and conjugation in Salmonella are genes encoding for the type III secretion system and lipopolysaccharide, and the pef fimbrial operon located on the plasmid, respectively [60]. Gene transfer by all three methods has been authenticated as the main mechanism for pathogenic evolution in Salmonella [60, 64].

Evidence for gene transfer has also been discovered in other intracellular pathogens, including Afipia, Brucella, Burkholderia, Francisella, Nocardia, Rhodococcus, Coxiella and Legionella [65-69], which were previously thought to undergo minimal if any transformation or conjugation. Rhodococcus and Mycobacterium, although phylogenetically distinct based on 16S rRNA analyses, share a highly homologous haloalkane dehalo-genase gene. This gene has recently evolved and functions to degrade an environmental pollutant known as synthetic haloalkane [69]. Synthetic haloalkanes are used as refrigerants, solvents and fire extinguishing agents. Extensive gene transfer events, including recombination and rearrangement involving the methionyl-tRNA synthetase and methyl-directed mismatch repair (mutS) genes, have also been described for Brucella suis and Salmonella enterica SARB strains [8, 68]. Interestingly, mutS also functions to block foreign DNA insertion, serving as a barrier for horizontal gene transfer [8]. Hence, mutations in mutS may affect its function by facilitating DNA insertion and gene shuffling. Horizontal gene transfer has also been responsible for genetic diversity among Burkholderia, especially within pathogenicity islands [70]. Several homologs between Pseudomonas aeruginosa and Burkholderia cepacia that function in the pathogenesis of cystic fibrosis suggest frequent genetic exchange [71]. Legionellapneumophila, ahumanpathogencausinglegionnaires' disease (pneumoniae) and Pontiac fever (respiratory illness), is also competent in DNA transformation. In vitro, many L. pneumophila loci, especially those regions that are responsible for surface pili proteins, were shown to uptake extrachromosomal DNA, demonstrating pilus variants that correlated with the transformed DNA [72].

In contrast to transferred genes from prokaryotes that may often resemble paralogs, gene transfer from eukaryotes may result in new or xenologous genes that reveal key host-parasite associations and adaptive virulence mechanisms [23]. Due to the acquisition of several plant genes, such as ATP/ADP translocases, by members of the Chlamydiaceae family, these organisms have been hypothesized to have originally shared a symbiotic relationship with plants, later becoming parasites of humans and animals through evolution [3]. As human parasites, chlamydiae acquired eukaryotic domains and genes, such as the actin-dependent regulator of chromatin (SWIB) domain [3], aminoacyl-tRNA synthetase genes [30] and ATP/ADP translocase genes [3, 73], to facilitate their in vivo lifestyle.

Plasmids have also been identified in the intracellular bacteria listed above, and were expected to confer infectivity and/or virulence characteristics to the host pathogens. Examples of plasmids with these characteristics include the pFNL10 in Francisella [74] and the pXT107 in many Nocardia and Rhodococcus species [75]. In contrast, the 7.5 kb cryptic plasmid of C. trachomatis has no known function [76]. Supportive data for conjugation also comes from experimental studies by Voeykova et al. [77]. The frequency of conjugation for the E. coli plasmid pT01 into Nocardia and Rhodococcus was reported to range from 1 x 10~3 to 1 x 10~5.

Bacteriophages have been identified in Brucella [78], Burkholderia [79], C. caviae [52], C. abortus [36, 56], C. pneumoniae [80], C. psittaci [59], C. pecorum [57], Legionella [81], Mycobacterium [82], Rhodococcus [83], Salmonella [60], Listeria [60] and Shigella [79]. Surprisingly, while phage have been documented in many Chlamydiaceae genera and species, none have been detected in C. trachomatis [36] nor among Rickettsia [84]. The absence of phages in C. trachomatis and Rickettsia suggest a preference for transformation and conjugation by these microorganisms. Many genes involved in pathogenicity, including niche-specific and virulence factors, and antibiotic resistance exhibit sequences reminiscent of bacteriophages, suggesting horizontal acquisition of virulence genes by the phages [27, 83]. For instance, transduction was believed to be a major gene acquisition method for Burkholderia evolution in order to survive in diverse niches [79]. Indeed, genetic exchange by bacteriophages has been a central mechanism in genetic communication for shiga-like toxins and antibiotic-resistant genes in Shigella and Salmonella species [85]. Furthermore, while vaccine and drug development for intracellular bacteria are difficult, understanding of the interrelationship between phage and host genomes may promote development of phage-directed therapy [82].

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