In Vivo Expression Technology Approaches

The original IVET approach involves a tandem set of two promoterless reporter genes, namely purA and lacZ, which were used to identify promoters that are specifically switched on in Salmonella typhimurium during infection (35). Purine auxotroph mutants (DpurA) of Salmonella typhimurium were only able to survive in a mouse model system when complemented in trans with a plasmid encoded purA copy. The promoterless purA gene was thereby utilized as a reporter for the identification of chromosomal fragments that are capable to complement the mutants, thereby strongly selecting for chromosomal fragments which harbor promoter elements that are active in the mouse model system. Subsequently, the in vivo active promoters are tested for the absence of promoter activity in vitro utilizing the second reporter gene (lacZ). The second variation of IVET is based on selection of an antibiotic resistance gene as selectable marker. One obvious disadvantage of this second variation of IVET is that the antibiotic must be administered to the host animal, which will certainly disturb the naturally occurring microflora in the GI tract. Therefore, the screening conditions assessed with this variant of IVET significantly differ from the native, in vivo situation. On the other hand, the addition of different levels of the selective antibiotic allows for selection of in vivo induced genes in a wider range of promoter activities. The third type of IVET selection uses a single gene as a dual reporter. The first example of such a dual reporter was hly, encoding the pore-forming haemolysin listeriolysin O (LLO) of Listeria monocytogenes (36). LLO mediates lysis of the phagosomal membrane in macrophages following infection. This reporter provides an in vivo selection for active fusions that allow for escape from the phagosomal compartment and subsequent multiplication. Moreover, a convenient screen on blood agar plates can be performed to identify inactive fusions in vitro, since clones harboring such fusions do not display haemolysis on these plates. The major drawback of the three aforementioned IVET variations is that the experimental set-up is designed in such a way that gene activity is required throughout the residence of the bacteria in the host. Hence, genes that are weakly expressed in the laboratory or transiently expressed only in a specific compartment of the host's GI tract slip through the selection procedure without being noticed. The fourth IVET variation circumvents this disadvantage by using the irreversible enzymatic activity of resolvases as reporter gene. Recombination-based IVET (R-IVET) is the only IVET approach that functions as a genetic screen. An antibiotic resistance marker flanked by two resolvase-recognition sites is integrated into the chromosome of the bacterium of interest. Subsequently, a promoterless copy of a resolvase-encoding gene, typically the tnpR gene from TngS, is introduced on a plasmid and used to trap transcriptional activation by monitoring changes in the antibiotic resistance phenotype. Importantly, this approach does not rely on selective pressure during the animal experiments, as promoter activations are irreversibly trapped by the excision of the antibiotic resistance marker and can be identified after recovery of the bacterium under investigation from the host.

In the first decade, (R-)IVET was extensively utilized for the identification of genes important during infection of at least 15 different pathogens, including Klebsiella pneumoniae, Salmonella enterica, and Listeria monocytogenes (29,37). Thereby, (R)-IVET is the most extensively applied screen for the identification of in vivo-induced genes during infection in animal models. The number of genes that are identified with an individual (R)-IVET screen varies strongly and ranges from 1 to approximately 100 genes (37).

Several of these screens identified genes that were already known to be involved in virulence and this observation was considered an intrinsic validation of these (R-)IVET screens (29). An exemplary finding along these lines is the identification of agrA using R-IVET in Staphylococcus aureus (38). This gene encodes a quorum-sensing transcriptional activator and agrA mutants constructed in this organism prior to the R-IVET screen had already been shown to display a virulence defective phenotype (39). In general, regulators are one of the predominant classes of genes identified with (R-)IVET (29). Another frequently encountered class of in vivo induced genes in pathogenic bacteria are involved in the uptake of divalent cations, including many examples of Fe2+ transporters (29). The harsh conditions these pathogens encounter when they transit from rich laboratory media to the host's GI tract apparently results in the induction of this group of genes. This suggestion is further supported by the observation that several in vivo induced genes were demonstrated to be similarly regulated under low Fe2+ concentrations in vitro (40-42). Other genes that frequently arise from (R-)IVET screens have functions in a variety of generally recognized functional categories, including cell metabolism, DNA repair and general stress response.

Recently, the first two reports appeared that describe the utilization of (R-)IVET strategies in food-grade or commensal micro-organisms in order to determine the specific induction of gene expression in these bacteria after introduction in the GI tract of animal models. In L. reuteri an IVET strategy based on in vivo selection of an antibiotic resistant phenotype (the aforementioned second variation of IVET) led to the identification of three genes important for this organism during colonization of the GI tract of Lactobacillus-free mice (43). One of these genes encodes a peptide methionine sulfoxide reductase (msrB) which has previously been identified using IVET in the non-food-associated Streptococcus gordonii during endocarditis (44). Although not noticed by the authors at that time, this was an important clue suggesting an overlap in the genetic response triggered in the pathogenic and non-pathogenic world following contact with the host. The second report dealing with in vivo induction of genes in food-associated microbes describes a R-IVET approach in L. plantarum (45). Previously, the resolvase-encoding tnpR-res system (46) has been applied to trap promoter activities in R-IVET experiments in several pathogenic bacteria. Therefore, initial attempts aimed at implementation of this system in L. plantarum. A res-ery-res cassette was successfully integrated into the chromosome of this bacterium and a promoterless copy of the tnpR gene was cloned on a low-copy plasmid. Despite the successful cloning of the endogenous, highly active ldhL1 promoter upstream of tnpR, excision of the ery gene from the chromosome of L. plantarum was never observed (Bron et al. unpublished data). These experiments strongly suggest that the tnpR resolvase is not functional in L. plantarum under the conditions applied during the experiments. Therefore, an alternative strategy was chosen to implement R-IVET in L. plantarum, which involved the cre-loxP system (47). This system was previously demonstrated to be functional in another lactic acid bacterium (LAB), Lactococcus lactis (48). Hence, a loxP-ery-loxP cassette was integrated into the chromosome of L. plantarum and a promoterless copy of cre was cloned on a low-copy vector. This system appeared to be functional in L. plantarum, as ldhL1 -promoter driven expression of the cre gene led to the irreversible excision of the loxP-ery-loxP cassette from the chromosome. Subsequently, a library containing L. plantarum chromosomal fragments upstream of cre was constructed and administered to mice. The library was recovered from fecal samples and analyzed for L. plantarum colonies that had lost their erythromycin resistant phenotype during passage through the animal model. These erythromycin sensitive colonies potentially harbor chromosomal fragments of which the expression was in vivo induced. Using this strategy, 72 L. plantarum genes were identified as being in vivo induced (ivi genes) during host GI-tract transit (45). The distribution over the generally recognized classes of main biological functions appeared to be random. A slight overrepresentation of R-IVET genes is observed around the origin of replication as compared to the rest of the genome (Fig. 3). However, the significance of the latter

Figure 3 Using R-IVET 72 L. plantarum genes could be identified as in vivo induced (ivi) during passage of the mouse GI tract. The chromosomal localization of these ivi genes is represented in the inner circle, while the outer two circles represent the ORFs on the positive (outer circle) and negative (middle circle) chromosomal DNA strand.

observation is unclear. Nine of the 72 ivi genes appeared to encode sugar-related functions, including genes involved in ribose, cellobiose, sucrose, and sorbitol transport. Another nine genes encode functions involved in acquisition and synthesis of amino acids, nucleotides, cofactors, and vitamins, indicating their limited availability in the GI tract. Four genes involved in stress-related functions were identified, reflecting the harsh conditions that L. plantarum encounters in the GI tract. Another four genes encoding extracellular proteins were identified that could mediate interactions with host GI-tract epithelial cells. Remarkably, the protein encoded by one of the hypothetical proteins identified in this study in L. plantarum is a homologue (32% identity) of the only conserved hypothetical protein that was identified with IVET in L. reuteri (43). Moreover, a large number of the functions and pathways identified in L. plantarum have previously been identified in pathogens as being important in vivo during infection (45). This striking amount of parallels between the pathogenic and non-pathogenic in vivo response suggests that survival rather than virulence is the explanation for the importance of these genes during host residence. Recently, nine of the L. plantarum ivi genes were selected, mainly focusing on genes that encode proteins with a predicted role in cell envelope functionality, stress response and regulation, for the construction of isogenic gene replacement mutants. Quantitative polymerase chain reaction (PCR) experiments were performed to monitor the relative population abundance of the group of L. plantarum replacement mutants in fecal samples after competitive passage through the GI tract of mice. These experiments revealed that after GI-tract passage the relative abundance of three of the ivi gene mutants was 100- to 1000-fold reduced as compared to other mutant strains, suggesting an important role for these three ivi genes, encoding the IIC transport component of a cellobiose phosphotransferase system (PTS), an extracellular protein that contains an LPQTNE motif, and a copper transporting ATPase, in the functionality of L. plantarum during passage of the GI tract (49).

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