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Probing the microenvironment of intracellular bacterial pathogens
Microbes and Infection, 1, 1999, 445−453 © Elsevier, Paris
Review
Probing the microenvironment of intracellular bacterial pathogens Omar S. Harb, Yousef Abu Kwaik* Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, KY 40536-0084, USA
ABSTRACT – The identification of bacterial genes regulated in response to the intracellular environment is crucial to the understanding of host-pathogen interactions. Several techniques have been developed to identify and characterize bacterial genes that are induced during the intracellular infection and, potentially, may play a role in pathogenesis. This review discusses the strategies that have been utilized to examine differential gene expression by bacterial pathogens during the intracellular infection. Furthermore, a number of the differentially expressed genes are described. © Elsevier, Paris Salmonella / Legionella / phagosome / in vivo expression technology / DD-PCR
1. Introduction There are two major obstacles that intracellular bacterial pathogens must overcome in order to survive within their eukaryotic host cells. First, the pathogen needs to be able to avoid the natural killing mechanisms of the host cell. Second, the pathogen needs to be able to tap into the nutritional reservoirs of the host cell. Several intracellular bacterial pathogens have evolved elegant mechanisms to survive within the host cell. In general, intracellular bacterial pathogens may be divided into three classes, based on the mechanisms they use to avoid being killed by the host’s lysosomal degradation machinery. A) Bacterial pathogens such as Listeria monocytogenes and Shigella flexneri escape from the acidified compartment, thereby escaping from the destructive lysosomal environment. In fact, these bacteria require the acidification of their phagosome in order to exit into the nutritionally rich environment of the host cell cytosol. B) Pathogens such as Coxiella burnettii have evolved to survive and flourish in the harsh environment of the phagolysosome. C) Pathogens that act on host cell components to alter the maturation of their phagosome through the endosomal-lysosomal pathway include, but are not limited to, Mycobacterium tuberculosis, Chlamydia trachomatis, Salmonella typhimurium, and Legionella pneumophila. The latter two classes of pathogens are able to derive nutrition from the host cell through unknown mechanisms, most likely involving specific and complex means of obtaining nutrients. It is fascinating that the pathogens belonging to each of these classes have evolved a plethora of mechanisms to * Correspondence and reprints Microbes and Infection 1999, 445-453
modify host cell processes and to utilize host cell resources. All of these mechanisms are geared to ensure the survival and replication of the bacterial pathogen. L. pneumophila recruits host cell organelles to its endosomematuration-blocked phagosome, a process necessary for its intracellular survival [1–4]. Mycobacterium avium is able to block the acidification of its vacuole by excluding the host vacuolar ATPase proton-pump, thus, blocking the maturation of its vacuole into a phagolysosome [5–8]. L. monocytogenes and S. flexneri escape from their phagosomes and utilize host cell actin to propel themselves within the cytosol and from one cell to another [9]. Several of these mechanisms have been recently reviewed [10]. The ability of these bacterial pathogens to dictate the nature of their intracellular environments is dependent on their ability to regulate the expression of a bank of genes to adapt to their intracellular niche. In turn, the regulation of these genes is dependent on the ability of these pathogens to sense environmental cues that, in essence, tell them where they are. Thus, it becomes obvious that the identification and characterization of these genes is at the crux of understanding how intracellular pathogens can outsmart our own cells, and indeed, may prove useful in the elucidation of cell biology as a whole. In this review we describe several techniques that have been used in different intracellular bacterial pathogens for the identification of genes expressed by these pathogens during the course of infection (table I). It is important to note here that there are many modifications of the described procedures and that this review is merely an attempt at describing the basic concepts of commonly used techniques for the purpose of identification of bacte445
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Table I. Summary of commonly used techniques. Technique
Example of gene induced in vivo
References
β-Galactosidase
lisA (L. monocytogenes). rpoS, iroA, cadA, mgtB (S. typhimurium). gspA, ppa (L. pneumophila). ivi genes such as rfb, carAB (S. typhimurium). ivi genes such as recD and mgtA/B (S. typhimurium). yopE (Y. pseudotuberculosis). mig genes (S. typhimurium). dnaK, groE (S. typhimurium). gspA, ppa, Hsp60 (L. pneumophila). hsp65, hsp70 (M. tuberculosis). 34 MI proteins (S. typhimurium). 44 kDa protein (L. pneumophila). dev (M. tuberculosis). mig (M. avium). eml (L. pneumophila). Useful to search for common themes in pathogenic DNA such as Pais
[11, 13, 14, 16, 17]
In vivo expression technology Cat reporter Luciferases Green fluorescence protein 2-D SDS-PAGE DAP labeling of proteins Immunologic detection PCR (RSH and DD-PCR) Computer-based genetics
[18–20] [18] [22, 23] [24–27] [28–31, 33] [34] [37] [38–41, 43–48] [3, 52, 53]
ivi, in vivo-induced gene; MI, macrophage-induced; Pais, pathogenicity island.
rial genes differentially regulated in response to the intracellular microenvironment. Most techniques used for the identification of genes regulated in response to the intracellular environment have been developed based on the idea that bacteria are, in general, conservative in their gene expression. In other words, they will only express genes that they require in a particular environment. Thus, intracellular bacteria should have an array of genes that are differentially expressed in the intracellular environment. Examination of bacterial gene expression during intracellular infection may allow for the elucidation of the nature of the microenvironment the organism inhabits.
2. β-Galactosidase β-Galactosidase (lacZ) gene fusions have been widely used for the detection of genes expressed in response to the intracellular environment. Generally, this technique has been used to detect the expression of genes in response to laboratory conditions that are thought to mimic the intracellular environment that a pathogen may encounter, such as low pH or changes in temperature upon bacterial entry into the host. However, this method has also been adapted for the detection of gene expression in the intracellular environment of the host cell. lacZ-gene fusions have been used successfully in several intracellular pathogens, including L. monocytogenes, S. typhimurium, Mycobacteria spp., and L. pneumophila. In vitro studies have shown that lacZ-listeriolysin (lisA) gene fusions and lacZ fusions to other prfA-regulated virulence genes of L. monocytogenes are thermoregulated, suggesting that virulence may be triggered by thermal cues, such as entry into the host [11]. In mycobacterium this method has been used to show that the gene encoding the Mycobacterium leprae 18-kDa antigen is differentially activated in response to the intracellular environment of macrophages [12]. Chen and colleagues have used lacZ-reporter fusions to rpoS and rpoSregulated genes of S. typhimurium to show that they are regulated in response to the intracellular environment of both macrophages and epithelial cells [13]. Using highly 446
fluorogenic substrate derivatives, Garcia-del Portillo and colleagues have followed the regulation of a number of housekeeping genes in the microenvironment of the S. typhimurium-containing vacuole. They show that iroA (induced in response to low iron), mgtB (induced in response to low magnesium), and cadA (induced in response to pH 6.0, low oxygen, and lysine) were all induced in the S. typhimurium-containing vacuole [14]. The regulation of these genes has given clues about the microenvironment of this vacuole, indicating that it may be low in free iron and magnesium. Subsequently it was shown that the phoP/phoQ two-component system which controls many pathogenic features of S. typhimurium is regulated by magnesium concentrations [15]. Thus, under conditions of low magnesium within the phagosome, the phoP/phoQ system is turned on, which in turn regulates the phoP-activated or -repressed genes (pag and prg, respectively) [15]. In L. pneumophila, our laboratory has utilized lacZpromoter fusions to show the differential expression of two genes, the global stress protein (gspA) and the inorganic pyrophosphatase (ppa) gene, in response to the intracellular environment [16, 17]. The data indicated that gspA is a stress-induced gene that is expressed in high levels throughout the intracellular infection. Similarly, the expression of ppa is induced in the intracellular environment. However, unlike gspA, the induction of ppa gene expression is unique to the intracellular environment [16, 17]. This observation indicates that the induction of ppa expression is a response to some unique stimulus within the intracellular niche of L. pneumophila. Thus, lacZreporter fusions can be used in a variety of bacterial pathogens to examine the regulation of gene expression in response to both intracellular and extracellular environmental cues.
3. In vivo expression technology In order to specifically examine S. typhimurium genes that are induced in vivo, the in vivo expression technology was developed (figure 1) [18–20]. A major advantage of this technique is that the induced genes are isolated on the Microbes and Infection 1999, 445-453
Gene expression by intracellular pathogens
Review
Injection into host animal or tissue culture cells Followed by recovery of bacterial cells and in vitro screening for EG– strains bases of their expression within the host tissue. The in vivo expression technology system uses an S. typhimurium auxotroph for adenylosuccinate synthase (∆purA). In this technique, random chromosomal promoter fusions to a purA-lacZ gene fusion are made in the ∆purA S. typhimurium strain, followed by the selection of the clones in mice (figure 1). Thus, only strains with promoter-purA fusions that are active in vivo survive. A second selection is performed on bacteria recovered from the infected mice on laboratory media, to select for lacZ-negative strains. In other words, strains that are purA + lacZ + in vivo and purA–lacZ– in vitro contain promoter fusions that are transcriptionally active in vivo (figure 1) [19]. Using this approach, several in vivo-induced genes have been cloned and characterized [20]. Analysis of these genes has shown them to be mainly housekeeping genes (such as the rfb operon, involved in O-antigen synthesis, and the carAB operon, involved in carbamoyl-phosphate synthesis) or genes which had no homology to sequences in genetic databases [20]. A disadvantage of the this system is that promoter fusions need to be expressed throughout the infection. Thus, it is possible that certain genes required for only one stage of the infection process may be missed. In addition, since bacteria are harvested from whole organs, the microenvironmental stimulus (intracellular versus extracellular) that triggers the expression of a gene is not known. Furthermore, the genes identified thus far have been involved in housekeeping functions as opposed to being unique ‘virulence genes’ directly associated with a unique mechanism of bacterial pathogenesis.
4. Chloramphenicol acyltransferase This method is similar to the above-described reporter systems in that gene fusions to the chloramphenicol acylMicrobes and Infection 1999, 445-453
Figure 1. The in vivo expression technology system for the identification of differentially expressed genes of intracellular pathogens. 1) Digested genomic DNA fragments are ligated to a vector containing an essential metabolic gene (EG) such as purA and a reporter gene (RG) such as lacZ or cat. 2) The construct is then transferred into the host bacterium (auxotrophic for the EG) and allowed to recombine into the chromosome. 3) Transformed bacteria are used for animal or tissue culture infections followed by recovery of viable bacteria and screening for in vitro RG activity.
transferase gene (cat) are utilized to detect gene transcription. This method can be used to select for genes differentially expressed in the intracellular environment on the basis of resistance to chloramphenicol. Furthermore, the levels of gene expression can be determined by assaying the enzymatic activity of the acyltransferase. This method was used in conjunction with the in vivo expression technology described above [18]. In this system, random promoter-cat-lacZY fusion strains of S. typhimurium are made and are used to infect mice [18]. The mice are then treated with chloramphenicol to enrich for strains that contain promoter fusions that are active in vivo. A second screening is done in vitro to select for lacZY inactive strains. These strains contain promoter fusions that are differentially active in vivo. Analysis of several of these genes has enabled Heithoff and colleagues to divide the in vivo-induced genes of S. typhimurium into groups based on function and role in pathogenesis: regulatory, rpoS-regulated, metabolic, systemic adhesion/invasionlike, and macrophage survival genes [21]. Although all of the genes described are induced in vivo, many of them perform housekeeping functions, some are directly involved in macrophage survival, and others have unknown functions [18, 21].
5. Luciferaces This method depends on the emission of photons by luciferase. Luciferase genes (lux) can be fused to any gene/promoter of interest, and the expression of that gene can be monitored. The advantage of this technique is that luciferases exhibit a relatively short half-life. This fact ensures that the gene product of interest is actually expressed at or close to the time of observation. One disadvantage, however, is that luciferases are highly susceptible 447
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Figure 2. The use of green fluorescence protein (GFP) in the differential fluorescence induction technique (DFI).
to concentrations of molecular oxygen, which prevents making strong conclusions about the level of gene transcription. This method has been successfully used in Yersinia pseudotuberculosis [22]. Y. pseudotuberculosis harboring a yopE-luxAB operon fusion has been used to show that Yop proteins are expressed exclusively during contact with the host cell. A novel approach using bioluminescence has been utilized in S. typhimurium to follow the course of infection in mice, since it is possible to detect photonic emissions through the mouse’s tissue [23]. Thus, constitutive expression of a plasmid-encoded lux operon has enabled the identification of infected tissue in the mouse. Although gene fusions were not used in this study, lux-gene/promoter fusions may be used to monitor the possible temporal regulation of virulence genes during the course of an infection.
using FACS. Individual bacterial clones are further separated by FACS into low or no fluorescence in the absence of the host cell (figure 2). Characterization of the 14 mig loci has revealed a wide spectrum of genes that are induced in the intracellular environment. These include heat-shock proteins such as mig-29 (homologous to hslU, which encodes a stress-induced protease) and genes of unknown function or homology. Also included were genes homologous to yscF, mxiH, and prgI, which are essential components of type III secretion systems associated with virulence. Thus, DFI is useful in identifying differentially expressed genes, some of which may be directly involved in virulence. Furthermore, the combination of DFI with FACS certainly enhances the possibility of identifying differentially expressed genes that are unique ‘virulence’ genes.
6. Green fluorescence protein
7. Two-dimensional gel electrophoresis
The use of green fluorescence protein (gfp)-promoter fusions for the identification of intracellularly transcribed genes in bacteria is a relatively new technique [24–27]. This technique, termed differential fluorescence induction (DFI), is similar to other reporter systems in that bacterial clones are isolated on the bases of differential gene expression, here measured as differential fluorescence within the host cell (figure 2). One major advantage of using DFI is the fact that fluorescent clones can be isolated using a fluorescent-activated cell sorter (FACS) [24, 26]. Therefore, thousands of clones can be screened, with high sensitivity for gene expression at low levels. Valdivia et al. have used DFI to isolate fourteen S. typhimurium promoters that were differentially active in the intracellular environment (mig for macrophage-inducible genes) [24]. This is accomplished by generating a plasmid library of S. typhimurium containing random DNA fragments inserted upstream of a promoterless gfp gene (figure 2). Infected cells with active GFP are then isolated
This method relies on the selective labeling of bacterial proteins expressed in the intracellular environment of the host cell. The host cell is treated with cyclohexamide, an inhibitor of eukaryotic protein synthesis, during labeling of intracellular bacterial proteins with 35S-methionine. Proteins can then be visualized using two-dimensional gel electrophoresis and compared with 35S-methioninelabeled proteins from extracellularly grown bacteria. Thus, bacterial proteins specifically expressed in response to the intracellular environment of the host cell can be identified. This method has been used for numerous intracellular pathogens, including S. typhimurium and M. tuberculosis [28–30]. We and others have used this method to identify L. pneumophila proteins induced in response to the intracellular environment of macrophages. Several macrophage-induced proteins have been identified [17, 31]. These include heat-shock protein (Hsp60), global stress protein (GspA), and inorganic pyrophosphatase
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Figure 3. The use of DAP auxotrophs for the specific labeling of bacterial proteins. Infections are carried out with an intracellular pathogen that is auxotrophic for DAP in the presence of a tritiated DAP supplement. Bacteria decarboxylate DAP into lysine, which can then be incorporated into bacterial proteins.
(PPase) [16, 17, 31–33]. One disadvantage of this method is the possible adverse effects of cyclohexamide treatment on the intracellular life-cycle of the bacteria. We have observed that short-term treatments with cyclohexamide (< 6 hours) have relatively little effect on the L. pneumophila replicative phagosome at the ultrastructural level. However, long treatments with cyclohexamide result in deterioration of the host cell and the replicative phagosome [17]. Other studies, such as those done in S. typhimurium and M. tuberculosis, that used long periods of treatment with cyclohexamide have not investigated the adverse effect this drug may have on the host cell [28–30]. Thus, caution should be taken when using this method, since many bacterial proteins may be expressed as a secondary response to the effects of cyclohexamide on the host cell. Another difficulty that arises when using twodimensional gel electrophoresis is the process of determining which induced protein should be further studied. Most proteins that have been shown to be intracellularly induced using this method have also been shown to be induced in response to different stresses such as low pH, heat shock, and H2O2 [16, 28–32]. Thus, the efficacy of this technique in the identification of unique bacterial ‘virulence’ genes is limited (besides being time consuming) by the tremendous number of housekeeping genes that are up- or downregulated.
8. Specific labeling of intracellular bacterial proteins In order to specifically label bacterial proteins without inhibiting host protein synthesis, bacterial auxotrophs for diaminopimelic acid (DAP) can be used (figure 3). DAP is a major component of the bacterial peptidoglycan, it falls in the biosynthetic pathway of lysine, and is not produced or metabolized by eukaryotic cells. Thus, supplying a DAP auxotroph with radiolabeled DAP should result in the generation of radiolabeled lysine in the bacteria, and in turn, specific and differential labeling of bacterial proteins. Microbes and Infection 1999, 445-453
This method has been utilized for the detection of 57 S. typhimurium proteins expressed in the intracellular environment of Int-407 intestinal epithelial cells [34]. In addition to being time consuming, this method has several disadvantages, such as the variability of DAP uptake by different cell lines and the lack of different forms of radiolabeled DAP (currently available as a tritiated compound requiring prolonged exposure of gels to X-ray film for visualization). The broad-range application of this strategy may be limited, since the phagosomes of different intracellular bacteria may vary in their ability to transport DAP across the phagosomal membrane. For example, a DAP auxotroph of L. pneumophila does not grow intracellularly in the presence of a DAP supplement [35].
9. Immunologic detection of differentially expressed genes This method relies on the use of antibodies generated against a pathogen for the detection of proteins that may play critical roles in the infection. This method has been used to detect Borrelia burgdorferi proteins expressed during an infection. Antibodies from mouse serum infected with either virulent or heat-killed B. burgdorferi are used to screen a B. burgdorferi expression library [36]. A major disadvantage of this technique, however, is that the protein of interest needs to be sufficiently immunogenic for its detection to be possible. In addition, the generated antibody against the native protein may not detect denatured antigens in immunoblots. Susa and colleagues have used antibodies to in vitrogrown L. pneumophila to identify proteins differentially expressed in the intracellular environment [37]. This method relies on the general radiolabeling of proteins during an infection, followed by immunoprecipitation of bacterial proteins by using L. pneumophila-specific antisera. With this technique, a 44-kDa L. pneumophila protein expressed exclusively in the intracellular environment has been identified. Amino-terminal sequencing of the protein revealed no homology to known sequences in genetic 449
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databases. Furthermore, by using antibodies that recognized the 44-kDa protein, the kinetics of intracellular expression of this protein were determined in various host cell types. The results have shown that the onset of expression of this protein is different in each cell line tested [37]. Thus, antisera to bacterial pathogens may be used to detect differentially expressed proteins in various host cells. Once again, however, this technique is limited by the immunogenicity of the proteins of interest. In addition, the antiserum used was raised against in vitro-grown bacteria, eliminating the possibility of detecting proteins that are exclusively expressed in vivo.
10. Polymerase chain reaction techniques The polymerase chain reaction (PCR) has become a useful tool in bacterial genetics. Various techniques utilizing PCR have been developed for the purpose of identifying differentially transcribed bacterial genes. These techniques include RNA subtractive hybridization (RSH) and differential display PCR (DD-PCR). RSH was developed for the identification of differentially transcribed genes under defined conditions [38–41]. There are several variations of RSH, but all methods rely on the hybridization of cDNAs from bacterial populations exposed to different conditions (one of which is expected to be transcribing a certain gene and the other is not). The cDNAs found under both conditions are subtracted, and the unique cDNAs are detected and studied further [39–43]. This method has been used to identify differentially transcribed genes in M. tuberculosis [38, 43]. Two strains of M. tuberculosis, one virulent and the other avirulent (derived by repeated in vitro culture), have been compared by RSH [38, 43]. Genes differentially expressed in the virulent strain (dev genes) have been cloned, but the function and nature of these genes has not been studied further [38]. Plum and Clarck-Curtiss have used RSH to identify M. avium genes that are differentially transcribed in the intracellular environment of macrophages [44]. In this study, cDNAs from in vitro-grown bacteria are used to subtract cDNAs of macrophage-grown M. avium. This has permitted the identification of a macrophage-induced gene (mig) that is maximally transcribed between one and five days postinfection and has no homology to sequences in the genetic databases [44]. However, the mig gene seems to encode a secreted protein that is induced by a shift from pH 6.0 to pH 7.0 [45]. The role that the mig gene may play in ‘virulence’ has not been clearly established, although, its expression in saprophytic mycobacterium enhances resistance to intracellular killing by macrophages, and the mig gene seems to be common among M. avium strains that cause clinical disease [45, 46]. RSH is rather inefficient, since only one gene was found to be highly induced intracellularly. Additionally, it is important to screen cDNAs of intracellularly grown bacteria harvested at different time points postinfection, since bacterial gene transcription is likely to follow a temporal pattern of regulation in response to a continuously changing intracellular environment. 450
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The DD-PCR technique was originally developed for the detection of differentially expressed eukaryotic genes [47]. This method has been modified by our laboratory for the identification of prokaryotic cDNA fragments that are differentially expressed within the intracellular environment (figure 4) [48]. DD-PCR is useful for the detection of transcribed genes at the early stages of an infection, since low amounts of mRNA can be used as templates. DD-PCR utilizes reverse transcribed mRNA from intracellularly grown bacteria, which is then subjected to PCR amplification using random oligonucleotide primers. Comparison of the cDNA profiles of intracellular and extracellular bacteria allows for the identification of differentially transcribed genes [42]. Several cDNA fragments from L. pneumophila that are induced 4 h postinfection of U937 macrophage-like cells have been identified [48]. One of the fragments was cloned and has led to the identification of a novel locus, termed the early macrophage-induced locus (eml). This locus has been shown to be required for early survival in both macrophages and the environmental host of L. pneumophila, Hartmannella vermiformis [48]. DD-PCR is limited by the ability of the random primers to anneal to the cDNA [48]. Furthermore, the detection of false positives and the irreproducibility of detection of some transcribed fragments may occur [48].
11. Computer-based comparative genetics The recent advancements in microchip array technology which allow for the visualization of changes in gene expression in hundreds of genes should be of great use to the bacteriologist [49, 50]. This technology consists of a microchip that carries different arrays of DNA fragments that serve as probes to screen complementary nucleotide sequences [49]. Therefore, the expression of a whole bank of known genes can be studied simultaneously in one cell. Although currently this technology is used for eukaryotic gene expression, it should be possible to apply this technology to prokaryotes. This technology would enable the study of coordinate gene expression of hundreds of genes under any desirable condition, including the intracellular microenvironment. Thus, it is likely that, in the near future, snippets of entire genomes of pathogenic organisms can be displayed on a microchip and overall gene expression visualized in response to specific environmental changes. Major advancements in computer science and engineering have enabled the development of computer bioinformatics for comparing genes and genomes of various pathogens or different species of the same pathogen. Furthermore, the increased computer speed and tremendous communication network generated by the internet have made it easier for ‘database mining’ and computer-based comparative genetics to be performed; one example is the comparison of the Neisseria meningitidis genome with that of other nonpathogenic Neisseria using such techniques as representational difference analysis. This technique identified DNA sequences specific for pathogenic Neisseria [3]. Using such approaches, one can concenMicrobes and Infection 1999, 445-453
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Figure 4. DD-PCR adapted to identification of bacterial genes differentially expressed in the intracellular environment. Comparison of cDNAs from intracellular bacteria with in vitrogrown cDNAs allows for the identification of differentially transcribed intracellular genes.
trate on certain genomic regions of a pathogenic bacterium in order to elucidate their roles in pathogenesis as unique ‘virulence’ genes. These genes can be studied further for potential regulation in vivo. Pathogenicity islands of S. typhimurium have been shown to be induced during intracellular infection of macrophages [51]. Computer-based comparative genetics can be useful in the search for pathogenicity islands [52] -large genetic regions harboring several virulence genes that are found exclusively on the chromosome or virulence plasmid of pathogenic bacteria [52, 53]. Several criteria define a DNA region as a pathogenicity island. These include a different G + C content from the host’s DNA, which is indicative of horizontal transfer of genetic information; insertion near tRNA genes; and/or flanking by insertion sequence elements [52]. Therefore, with the sequencing of several genomes of bacterial pathogens, computer-based comparative genetics can be used for analysis of these sequences according to the criteria that describe chromosomal fragments associated with virulence.
12. Conclusions This review describes several techniques used to identify differentially transcribed bacterial genes. The induction of expression of certain genes during the intracellular infection is thought to be a global coordinated response by the bacteria to the intracellular microenvironment. This response is thought to allow the bacterium to adapt to and establish its niche within the host cell. These adaptations involve metabolic, environmental (i.e., pH and oxidative stress), and ‘virulence’ genes that perform unique functions exclusively involved in exploiting host cell processes. Examination of bacterial gene expression during the intracellular infection has been used as a strategy with the hope of discovering unique ‘virulence’ genes. It is Microbes and Infection 1999, 445-453
thought that some ‘virulence’ genes with unique functions should be expressed at high levels within the host cell compared with in vitro growth conditions. However, in most cases the results have been disappointing, since many of the induced genes have been found to be housekeeping genes (i.e., heat-shock proteins and several metabolic genes). The reasons for this may lie in the complex nature of bacterial gene regulation. It is likely that bacterial virulence genes are transcribed over a short period of time when absolutely needed, and the level of such transcription may be very low. This would greatly hamper the process of identifying these genes from among the many transcribed housekeeping genes. L. pneumophila is a good example of the tight regulation of ‘virulence’ gene expression and induction during stressful environmental conditions. Byrne and Swanson showed that several virulence traits of L. pneumophila that are induced in the intracellular environment are turned on only upon exiting the exponential phase of growth [54]. This finding stresses the complexity of bacterial gene regulation and accentuates the role of global gene regulation on the expression of virulence traits in a pathogen. Several studies utilizing the techniques described in this review revealed the presence of a battery of downregulated genes. Studies on S. typhimurium, L. pneumophila, and M. tuberculosis showed the repression of as many as 100 proteins during intracellular growth [28, 30, 31]. What is the function of these proteins? And is their repression important for virulence? Is there a relation between the down- and upregulated genes? The answers to these questions have been overlooked and may prove useful in the elucidation of the role and identification of other differentially transcribed ‘virulence’ genes. The identification of differentially transcribed genes constitutes merely the beginning of our quest to understand the basic question of how intracellular bacterial pathogens survive and thrive within our cells. The mode of action of the gene products and the regulation of these 451
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genes are of utmost importance to the understanding of host-pathogen interactions. New laboratory techniques and computer-based technologies should be of great use, in the future prospects of enhancing our understanding of the pathogenesis of intracellular bacteria.
Acknowledgments The authors thank Mr Gopi Shankar, Mr Lian-Yong Gao, and Dr Barbara J. Stone for their critical review of this manuscript. OSH is supported by a predoctoral National Research Service Award #TA09509. YA is supported by Public Health Service Award #R29A138410.
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[12] Dellagostin O.A., Esposito G., Eales L.J., Dale J.W., McFadden J., Activity of mycobacterial promoters during intracellular and extracellular growth, Microbiology 141 (1995) 1785–1792. [13] Chen C.Y., Eckmann L., Libby S.J., Fang F.C., Okamoto S., Kagnoff M.F., Fierer J., Guiney D.G., Expression of Salmonella typhimurium rpoS and rpoS-dependent genes in the intracellular environment of eukaryotic cells, Infect Immun. 64 (1996) 4739–4743. [14] Garcia-DelPortillo F., Foster J.W., Maguire M.E., Finlay B.B., Characterization of the micro-environment of Salmonella typhimurium- containing vacuoles within MDCK epithelial cells, Mol. Microbiol. 6 (1992) 3289–3297. [15] GarciaVescovi E., Soncini F.C., Groisman E.A., Mg2+ as an extracellular signal: environmental regulation of Salmonella virulence, Cell 84 (1996) 165–174. [16] AbuKwaik Y., Gao L.Y., Harb O.S., Stone B.J., Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant, Mol. Microbiol. 24 (1997) 629–642. [17] AbuKwaik Y., Induced expression of the Legionella pneumophila gene encoding a 20-kilodalton protein during intracellular infection, Infect. Immun. 66 (1998) 203–212. [18] Mahan M.J., Tobias J.W., Slauch J.M., Hanna P.C., Collier R.J., Mekalanos J.J., Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host, Proc. Natl. Acad. Sci. USA 92 (1995) 669–673. [19] Slauch J.M., Mahan M.J., Mekalanos J.J., In vivo expression technology for selection of bacterial genes specifically induced in host tissues, Methods Enzymol. 235 (1994) 481–492. [20] Mahan M.J., Slauch J.M., Hanna P.C., Camilli A., Tobias J.W., Waldor M.K., Mekalanos J.J., Selection for bacterial genes that are specifically induced in host tissues: the hunt for virulence factors, Infect. Agents Dis. 2 (1993) 263–268. [21] Heithoff D.M., Conner C.P., Hanna P.C., Julio S.M., Hentschel U., Mahan M.J., Bacterial infection as assessed by in vivo gene expression, Proc. Natl. Acad. Sci. USA 94 (1997) 934–939. [22] Pettersson J., Nordfelth R., Dubinina E., Bergman T., Gustafsson M., Magnusson K.E., Wolf-Watz H., Modulation of virulence factor expression by pathogen target cell contact, Science 273 (1996) 1231–1233. [23] Contag C.H., Contag P.R., Mullins J.I., Spilman S.D., Stevenson D.K., Benaron D.A., Photonic detection of bacterial pathogens in living hosts, Mol. Microbiol. 18 (1995) 593–603. [24] Valdivia R.H., Falkow S., Fluorescence-based isolation of bacterial genes expressed within host cells, Science 277 (1997) 2007–2011. [25] Valdivia R.H., Falkow S., Probing bacterial gene expression within host cells, Trends Microbiol. 5 (1997) 360–363. [26] Valdivia R.H., Falkow S., Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acidinducible promoters by differential fluorescence induction, Mol. Microbiol. 22 (1996) 367–378. [27] Valdivia R.H., Hromockyj A.E., Monack D., Ramakrishnan L., Falkow S., Applications for green fluorescent protein (GFP) in the study of host- pathogen interactions, Gene 173 (1996) 47–52. Microbes and Infection 1999, 445-453
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[28] Lee B.Y., Horwitz M.A., Identification of macrophage and stress-induced proteins of Mycobacterium tuberculosis, J. Clin. Invest. 96 (1995) 245–249. [29] Buchmeier N.A., Heffron F., Induction of Salmonella stress proteins upon infection of macrophages. Science 248 (1990) 730–732. [30] Abshire K.Z., Neidhardt F.C., Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage, J. Bacteriol. 175 (1993) 3734–3743. [31] AbuKwaik Y., Eisenstein B.I., Engleberg N.C., Phenotypic modulation by Legionella pneumophila upon infection of macrophages, Infect. Immun. 61 (1993) 1320–1329. [32] AbuKwaik Y., Engleberg N.C., Cloning and molecular characterization of a Legionella pneumophila gene induced by intracellular infection and by various in vitro stress conditions, Mol. Microbiol. 13 (1994) 243–251. [33] Fernandez R.C., Logan S.M., Lee S.H., Hoffman P.S., Elevated levels of Legionella pneumophila stress protein Hsp60 early in infection of human monocytes and L929 cells correlate with virulence, Infect. Immun. 64 (1996) 1968–1976. [34] Burns-Keliher L.L., Portteus A., Curtiss R., Specific detection of Salmonella typhimunium proteins synthesized intracellularly, J. Bacteriol. 179 (1997) 3604–3612. [35] Harb O.S., AbuKwaik Y., Identification of the aspartate-βsemialdehyde dehydrogenase gene of Legionella pneumophila and characterization of a null mutant, Infect. Immun. 66 (1998) 1898–1903. [36] Suk K., Das S., Sun W., Jwang B., Barthold S.W., Flavell R.A., Fikrig E., Borrelia burgdorferi genes selectively expressed in the infected host, Proc. Natl. Acad. Sci. USA 92 (1995) 4269–4273. [37] Susa M., Hacker J., Marre R., De novo synthesis of Legionella pneumophila antigens during intracellular growth in phagocytic cells, Infect. Immun. 64 (1996) 1679–1684. [38] Kinger A.K., Tyagi J.S., Identification and cloning of genes differentially expressed in the virulent strain of Mycobacterium tuberculosis, Gene 131 (1993) 113–117. [39] Straus D., Ausubel F.M., Genomic subtraction for cloning DNA corresponding to deletion mutations, Proc. Natl. Acad. Sci. USA 87 (1990) 1889–1893. [40] Lisitsyn N., Wigler M., Cloning the differences between two complex genomes, Science 259 (1993) 946–951.
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[41] Timblin C., Battey J., Kuehl W.M., Application for PCR technology to subtractive cDNA cloning: identification of genes expressed specifically in murine plasmacytoma cells, Nucleic Acids Res. 18 (1990) 1587–1593. [42] Quinn F.D., Newman G.W., King C.H., In search of virulence factors of human bacterial disease, Trends Microbiol. 5 (1997) 20–26. [43] Kikuta-Oshima L.C., King C.H., Shinnick T.M., Quinn F.D., Methods for the identification of virulence genes expressed in Mycobacterium tuberculosis strain H37RvAnn. NY Acad. Sci. 730 (1994) 263–265. [44] Plum G., Clark-Curtiss J.E., Induction of Mycobacterium avium gene expression following phagocytosis by human macrophages, Infect. Immun. 62 (1994) 476–483. [45] Plum G., Brenden M., Clark-Curtiss J.E., Pulverer G., Cloning sequencing and expression of the mig gene of Mycobacterium avium which codes for a secreted macrophage-induced protein, Infect. Immun. 65 (1997) 4548–4557. [46] Meyer M., VvonGrunberg P.W., Knoop T., Hartmann P., Plum G., The macrophage-induced gene mig as a marker for clinical pathogenicity and in vitro virulence of Mycobacterium avium complex strains, Infect. Immun. 66 (1998) 4549–4552. [47] Liang P., Pardee A.B., Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction, Science 257 (1992) 967–971. [48] AbuKwaik Y., Pederson L.L., The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages, Mol. Microbiol. 21 (1996) 543–556. [49] Service R.F., DNA chips survey an entire genome, Science 281 (1998) 1122. [50] Service R.F., Microship arrays put DNA on the spot, Science 282 (1998) 596–400. [51] Groisman E.A., Ochman H., Pathogenicity islands: bacterial evolution in quantum leaps, Cell 87 (1996) 791–794. [52] Hacker J., Blum-Oehler G., Muhldorfer I., Tschape H., Pathogenicity islands of virulent bacteria: structure function and impact on microbial evolution, Mol. Microbiol. 23 (1997) 1089–1097. [53] Frosch M., Reidl J., Vogel U., Genomics in infectious diseases: approaching the pathogens, Trends Microbiol. 6 (1998) 346–349. [54] Byrne B., Swanson M.S., Expression of Legionella pneumophila virulence traits in response to growth conditions, Infect. Immun. 66 (1998) 3029–3034.
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Probing the microenvironment of intracellular bacterial pathogens Omar S. Harb, Yousef Abu Kwaik* Department of Microbiology and Immunology, University of Kentucky Chandler Medical Center, Lexington, KY 40536-0084, USA
ABSTRACT – The identification of bacterial genes regulated in response to the intracellular environment is crucial to the understanding of host-pathogen interactions. Several techniques have been developed to identify and characterize bacterial genes that are induced during the intracellular infection and, potentially, may play a role in pathogenesis. This review discusses the strategies that have been utilized to examine differential gene expression by bacterial pathogens during the intracellular infection. Furthermore, a number of the differentially expressed genes are described. © Elsevier, Paris Salmonella / Legionella / phagosome / in vivo expression technology / DD-PCR
1. Introduction There are two major obstacles that intracellular bacterial pathogens must overcome in order to survive within their eukaryotic host cells. First, the pathogen needs to be able to avoid the natural killing mechanisms of the host cell. Second, the pathogen needs to be able to tap into the nutritional reservoirs of the host cell. Several intracellular bacterial pathogens have evolved elegant mechanisms to survive within the host cell. In general, intracellular bacterial pathogens may be divided into three classes, based on the mechanisms they use to avoid being killed by the host’s lysosomal degradation machinery. A) Bacterial pathogens such as Listeria monocytogenes and Shigella flexneri escape from the acidified compartment, thereby escaping from the destructive lysosomal environment. In fact, these bacteria require the acidification of their phagosome in order to exit into the nutritionally rich environment of the host cell cytosol. B) Pathogens such as Coxiella burnettii have evolved to survive and flourish in the harsh environment of the phagolysosome. C) Pathogens that act on host cell components to alter the maturation of their phagosome through the endosomal-lysosomal pathway include, but are not limited to, Mycobacterium tuberculosis, Chlamydia trachomatis, Salmonella typhimurium, and Legionella pneumophila. The latter two classes of pathogens are able to derive nutrition from the host cell through unknown mechanisms, most likely involving specific and complex means of obtaining nutrients. It is fascinating that the pathogens belonging to each of these classes have evolved a plethora of mechanisms to * Correspondence and reprints Microbes and Infection 1999, 445-453
modify host cell processes and to utilize host cell resources. All of these mechanisms are geared to ensure the survival and replication of the bacterial pathogen. L. pneumophila recruits host cell organelles to its endosomematuration-blocked phagosome, a process necessary for its intracellular survival [1–4]. Mycobacterium avium is able to block the acidification of its vacuole by excluding the host vacuolar ATPase proton-pump, thus, blocking the maturation of its vacuole into a phagolysosome [5–8]. L. monocytogenes and S. flexneri escape from their phagosomes and utilize host cell actin to propel themselves within the cytosol and from one cell to another [9]. Several of these mechanisms have been recently reviewed [10]. The ability of these bacterial pathogens to dictate the nature of their intracellular environments is dependent on their ability to regulate the expression of a bank of genes to adapt to their intracellular niche. In turn, the regulation of these genes is dependent on the ability of these pathogens to sense environmental cues that, in essence, tell them where they are. Thus, it becomes obvious that the identification and characterization of these genes is at the crux of understanding how intracellular pathogens can outsmart our own cells, and indeed, may prove useful in the elucidation of cell biology as a whole. In this review we describe several techniques that have been used in different intracellular bacterial pathogens for the identification of genes expressed by these pathogens during the course of infection (table I). It is important to note here that there are many modifications of the described procedures and that this review is merely an attempt at describing the basic concepts of commonly used techniques for the purpose of identification of bacte445
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Table I. Summary of commonly used techniques. Technique
Example of gene induced in vivo
References
β-Galactosidase
lisA (L. monocytogenes). rpoS, iroA, cadA, mgtB (S. typhimurium). gspA, ppa (L. pneumophila). ivi genes such as rfb, carAB (S. typhimurium). ivi genes such as recD and mgtA/B (S. typhimurium). yopE (Y. pseudotuberculosis). mig genes (S. typhimurium). dnaK, groE (S. typhimurium). gspA, ppa, Hsp60 (L. pneumophila). hsp65, hsp70 (M. tuberculosis). 34 MI proteins (S. typhimurium). 44 kDa protein (L. pneumophila). dev (M. tuberculosis). mig (M. avium). eml (L. pneumophila). Useful to search for common themes in pathogenic DNA such as Pais
[11, 13, 14, 16, 17]
In vivo expression technology Cat reporter Luciferases Green fluorescence protein 2-D SDS-PAGE DAP labeling of proteins Immunologic detection PCR (RSH and DD-PCR) Computer-based genetics
[18–20] [18] [22, 23] [24–27] [28–31, 33] [34] [37] [38–41, 43–48] [3, 52, 53]
ivi, in vivo-induced gene; MI, macrophage-induced; Pais, pathogenicity island.
rial genes differentially regulated in response to the intracellular microenvironment. Most techniques used for the identification of genes regulated in response to the intracellular environment have been developed based on the idea that bacteria are, in general, conservative in their gene expression. In other words, they will only express genes that they require in a particular environment. Thus, intracellular bacteria should have an array of genes that are differentially expressed in the intracellular environment. Examination of bacterial gene expression during intracellular infection may allow for the elucidation of the nature of the microenvironment the organism inhabits.
2. β-Galactosidase β-Galactosidase (lacZ) gene fusions have been widely used for the detection of genes expressed in response to the intracellular environment. Generally, this technique has been used to detect the expression of genes in response to laboratory conditions that are thought to mimic the intracellular environment that a pathogen may encounter, such as low pH or changes in temperature upon bacterial entry into the host. However, this method has also been adapted for the detection of gene expression in the intracellular environment of the host cell. lacZ-gene fusions have been used successfully in several intracellular pathogens, including L. monocytogenes, S. typhimurium, Mycobacteria spp., and L. pneumophila. In vitro studies have shown that lacZ-listeriolysin (lisA) gene fusions and lacZ fusions to other prfA-regulated virulence genes of L. monocytogenes are thermoregulated, suggesting that virulence may be triggered by thermal cues, such as entry into the host [11]. In mycobacterium this method has been used to show that the gene encoding the Mycobacterium leprae 18-kDa antigen is differentially activated in response to the intracellular environment of macrophages [12]. Chen and colleagues have used lacZ-reporter fusions to rpoS and rpoSregulated genes of S. typhimurium to show that they are regulated in response to the intracellular environment of both macrophages and epithelial cells [13]. Using highly 446
fluorogenic substrate derivatives, Garcia-del Portillo and colleagues have followed the regulation of a number of housekeeping genes in the microenvironment of the S. typhimurium-containing vacuole. They show that iroA (induced in response to low iron), mgtB (induced in response to low magnesium), and cadA (induced in response to pH 6.0, low oxygen, and lysine) were all induced in the S. typhimurium-containing vacuole [14]. The regulation of these genes has given clues about the microenvironment of this vacuole, indicating that it may be low in free iron and magnesium. Subsequently it was shown that the phoP/phoQ two-component system which controls many pathogenic features of S. typhimurium is regulated by magnesium concentrations [15]. Thus, under conditions of low magnesium within the phagosome, the phoP/phoQ system is turned on, which in turn regulates the phoP-activated or -repressed genes (pag and prg, respectively) [15]. In L. pneumophila, our laboratory has utilized lacZpromoter fusions to show the differential expression of two genes, the global stress protein (gspA) and the inorganic pyrophosphatase (ppa) gene, in response to the intracellular environment [16, 17]. The data indicated that gspA is a stress-induced gene that is expressed in high levels throughout the intracellular infection. Similarly, the expression of ppa is induced in the intracellular environment. However, unlike gspA, the induction of ppa gene expression is unique to the intracellular environment [16, 17]. This observation indicates that the induction of ppa expression is a response to some unique stimulus within the intracellular niche of L. pneumophila. Thus, lacZreporter fusions can be used in a variety of bacterial pathogens to examine the regulation of gene expression in response to both intracellular and extracellular environmental cues.
3. In vivo expression technology In order to specifically examine S. typhimurium genes that are induced in vivo, the in vivo expression technology was developed (figure 1) [18–20]. A major advantage of this technique is that the induced genes are isolated on the Microbes and Infection 1999, 445-453
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Injection into host animal or tissue culture cells Followed by recovery of bacterial cells and in vitro screening for EG– strains bases of their expression within the host tissue. The in vivo expression technology system uses an S. typhimurium auxotroph for adenylosuccinate synthase (∆purA). In this technique, random chromosomal promoter fusions to a purA-lacZ gene fusion are made in the ∆purA S. typhimurium strain, followed by the selection of the clones in mice (figure 1). Thus, only strains with promoter-purA fusions that are active in vivo survive. A second selection is performed on bacteria recovered from the infected mice on laboratory media, to select for lacZ-negative strains. In other words, strains that are purA + lacZ + in vivo and purA–lacZ– in vitro contain promoter fusions that are transcriptionally active in vivo (figure 1) [19]. Using this approach, several in vivo-induced genes have been cloned and characterized [20]. Analysis of these genes has shown them to be mainly housekeeping genes (such as the rfb operon, involved in O-antigen synthesis, and the carAB operon, involved in carbamoyl-phosphate synthesis) or genes which had no homology to sequences in genetic databases [20]. A disadvantage of the this system is that promoter fusions need to be expressed throughout the infection. Thus, it is possible that certain genes required for only one stage of the infection process may be missed. In addition, since bacteria are harvested from whole organs, the microenvironmental stimulus (intracellular versus extracellular) that triggers the expression of a gene is not known. Furthermore, the genes identified thus far have been involved in housekeeping functions as opposed to being unique ‘virulence genes’ directly associated with a unique mechanism of bacterial pathogenesis.
4. Chloramphenicol acyltransferase This method is similar to the above-described reporter systems in that gene fusions to the chloramphenicol acylMicrobes and Infection 1999, 445-453
Figure 1. The in vivo expression technology system for the identification of differentially expressed genes of intracellular pathogens. 1) Digested genomic DNA fragments are ligated to a vector containing an essential metabolic gene (EG) such as purA and a reporter gene (RG) such as lacZ or cat. 2) The construct is then transferred into the host bacterium (auxotrophic for the EG) and allowed to recombine into the chromosome. 3) Transformed bacteria are used for animal or tissue culture infections followed by recovery of viable bacteria and screening for in vitro RG activity.
transferase gene (cat) are utilized to detect gene transcription. This method can be used to select for genes differentially expressed in the intracellular environment on the basis of resistance to chloramphenicol. Furthermore, the levels of gene expression can be determined by assaying the enzymatic activity of the acyltransferase. This method was used in conjunction with the in vivo expression technology described above [18]. In this system, random promoter-cat-lacZY fusion strains of S. typhimurium are made and are used to infect mice [18]. The mice are then treated with chloramphenicol to enrich for strains that contain promoter fusions that are active in vivo. A second screening is done in vitro to select for lacZY inactive strains. These strains contain promoter fusions that are differentially active in vivo. Analysis of several of these genes has enabled Heithoff and colleagues to divide the in vivo-induced genes of S. typhimurium into groups based on function and role in pathogenesis: regulatory, rpoS-regulated, metabolic, systemic adhesion/invasionlike, and macrophage survival genes [21]. Although all of the genes described are induced in vivo, many of them perform housekeeping functions, some are directly involved in macrophage survival, and others have unknown functions [18, 21].
5. Luciferaces This method depends on the emission of photons by luciferase. Luciferase genes (lux) can be fused to any gene/promoter of interest, and the expression of that gene can be monitored. The advantage of this technique is that luciferases exhibit a relatively short half-life. This fact ensures that the gene product of interest is actually expressed at or close to the time of observation. One disadvantage, however, is that luciferases are highly susceptible 447
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Figure 2. The use of green fluorescence protein (GFP) in the differential fluorescence induction technique (DFI).
to concentrations of molecular oxygen, which prevents making strong conclusions about the level of gene transcription. This method has been successfully used in Yersinia pseudotuberculosis [22]. Y. pseudotuberculosis harboring a yopE-luxAB operon fusion has been used to show that Yop proteins are expressed exclusively during contact with the host cell. A novel approach using bioluminescence has been utilized in S. typhimurium to follow the course of infection in mice, since it is possible to detect photonic emissions through the mouse’s tissue [23]. Thus, constitutive expression of a plasmid-encoded lux operon has enabled the identification of infected tissue in the mouse. Although gene fusions were not used in this study, lux-gene/promoter fusions may be used to monitor the possible temporal regulation of virulence genes during the course of an infection.
using FACS. Individual bacterial clones are further separated by FACS into low or no fluorescence in the absence of the host cell (figure 2). Characterization of the 14 mig loci has revealed a wide spectrum of genes that are induced in the intracellular environment. These include heat-shock proteins such as mig-29 (homologous to hslU, which encodes a stress-induced protease) and genes of unknown function or homology. Also included were genes homologous to yscF, mxiH, and prgI, which are essential components of type III secretion systems associated with virulence. Thus, DFI is useful in identifying differentially expressed genes, some of which may be directly involved in virulence. Furthermore, the combination of DFI with FACS certainly enhances the possibility of identifying differentially expressed genes that are unique ‘virulence’ genes.
6. Green fluorescence protein
7. Two-dimensional gel electrophoresis
The use of green fluorescence protein (gfp)-promoter fusions for the identification of intracellularly transcribed genes in bacteria is a relatively new technique [24–27]. This technique, termed differential fluorescence induction (DFI), is similar to other reporter systems in that bacterial clones are isolated on the bases of differential gene expression, here measured as differential fluorescence within the host cell (figure 2). One major advantage of using DFI is the fact that fluorescent clones can be isolated using a fluorescent-activated cell sorter (FACS) [24, 26]. Therefore, thousands of clones can be screened, with high sensitivity for gene expression at low levels. Valdivia et al. have used DFI to isolate fourteen S. typhimurium promoters that were differentially active in the intracellular environment (mig for macrophage-inducible genes) [24]. This is accomplished by generating a plasmid library of S. typhimurium containing random DNA fragments inserted upstream of a promoterless gfp gene (figure 2). Infected cells with active GFP are then isolated
This method relies on the selective labeling of bacterial proteins expressed in the intracellular environment of the host cell. The host cell is treated with cyclohexamide, an inhibitor of eukaryotic protein synthesis, during labeling of intracellular bacterial proteins with 35S-methionine. Proteins can then be visualized using two-dimensional gel electrophoresis and compared with 35S-methioninelabeled proteins from extracellularly grown bacteria. Thus, bacterial proteins specifically expressed in response to the intracellular environment of the host cell can be identified. This method has been used for numerous intracellular pathogens, including S. typhimurium and M. tuberculosis [28–30]. We and others have used this method to identify L. pneumophila proteins induced in response to the intracellular environment of macrophages. Several macrophage-induced proteins have been identified [17, 31]. These include heat-shock protein (Hsp60), global stress protein (GspA), and inorganic pyrophosphatase
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Figure 3. The use of DAP auxotrophs for the specific labeling of bacterial proteins. Infections are carried out with an intracellular pathogen that is auxotrophic for DAP in the presence of a tritiated DAP supplement. Bacteria decarboxylate DAP into lysine, which can then be incorporated into bacterial proteins.
(PPase) [16, 17, 31–33]. One disadvantage of this method is the possible adverse effects of cyclohexamide treatment on the intracellular life-cycle of the bacteria. We have observed that short-term treatments with cyclohexamide (< 6 hours) have relatively little effect on the L. pneumophila replicative phagosome at the ultrastructural level. However, long treatments with cyclohexamide result in deterioration of the host cell and the replicative phagosome [17]. Other studies, such as those done in S. typhimurium and M. tuberculosis, that used long periods of treatment with cyclohexamide have not investigated the adverse effect this drug may have on the host cell [28–30]. Thus, caution should be taken when using this method, since many bacterial proteins may be expressed as a secondary response to the effects of cyclohexamide on the host cell. Another difficulty that arises when using twodimensional gel electrophoresis is the process of determining which induced protein should be further studied. Most proteins that have been shown to be intracellularly induced using this method have also been shown to be induced in response to different stresses such as low pH, heat shock, and H2O2 [16, 28–32]. Thus, the efficacy of this technique in the identification of unique bacterial ‘virulence’ genes is limited (besides being time consuming) by the tremendous number of housekeeping genes that are up- or downregulated.
8. Specific labeling of intracellular bacterial proteins In order to specifically label bacterial proteins without inhibiting host protein synthesis, bacterial auxotrophs for diaminopimelic acid (DAP) can be used (figure 3). DAP is a major component of the bacterial peptidoglycan, it falls in the biosynthetic pathway of lysine, and is not produced or metabolized by eukaryotic cells. Thus, supplying a DAP auxotroph with radiolabeled DAP should result in the generation of radiolabeled lysine in the bacteria, and in turn, specific and differential labeling of bacterial proteins. Microbes and Infection 1999, 445-453
This method has been utilized for the detection of 57 S. typhimurium proteins expressed in the intracellular environment of Int-407 intestinal epithelial cells [34]. In addition to being time consuming, this method has several disadvantages, such as the variability of DAP uptake by different cell lines and the lack of different forms of radiolabeled DAP (currently available as a tritiated compound requiring prolonged exposure of gels to X-ray film for visualization). The broad-range application of this strategy may be limited, since the phagosomes of different intracellular bacteria may vary in their ability to transport DAP across the phagosomal membrane. For example, a DAP auxotroph of L. pneumophila does not grow intracellularly in the presence of a DAP supplement [35].
9. Immunologic detection of differentially expressed genes This method relies on the use of antibodies generated against a pathogen for the detection of proteins that may play critical roles in the infection. This method has been used to detect Borrelia burgdorferi proteins expressed during an infection. Antibodies from mouse serum infected with either virulent or heat-killed B. burgdorferi are used to screen a B. burgdorferi expression library [36]. A major disadvantage of this technique, however, is that the protein of interest needs to be sufficiently immunogenic for its detection to be possible. In addition, the generated antibody against the native protein may not detect denatured antigens in immunoblots. Susa and colleagues have used antibodies to in vitrogrown L. pneumophila to identify proteins differentially expressed in the intracellular environment [37]. This method relies on the general radiolabeling of proteins during an infection, followed by immunoprecipitation of bacterial proteins by using L. pneumophila-specific antisera. With this technique, a 44-kDa L. pneumophila protein expressed exclusively in the intracellular environment has been identified. Amino-terminal sequencing of the protein revealed no homology to known sequences in genetic 449
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databases. Furthermore, by using antibodies that recognized the 44-kDa protein, the kinetics of intracellular expression of this protein were determined in various host cell types. The results have shown that the onset of expression of this protein is different in each cell line tested [37]. Thus, antisera to bacterial pathogens may be used to detect differentially expressed proteins in various host cells. Once again, however, this technique is limited by the immunogenicity of the proteins of interest. In addition, the antiserum used was raised against in vitro-grown bacteria, eliminating the possibility of detecting proteins that are exclusively expressed in vivo.
10. Polymerase chain reaction techniques The polymerase chain reaction (PCR) has become a useful tool in bacterial genetics. Various techniques utilizing PCR have been developed for the purpose of identifying differentially transcribed bacterial genes. These techniques include RNA subtractive hybridization (RSH) and differential display PCR (DD-PCR). RSH was developed for the identification of differentially transcribed genes under defined conditions [38–41]. There are several variations of RSH, but all methods rely on the hybridization of cDNAs from bacterial populations exposed to different conditions (one of which is expected to be transcribing a certain gene and the other is not). The cDNAs found under both conditions are subtracted, and the unique cDNAs are detected and studied further [39–43]. This method has been used to identify differentially transcribed genes in M. tuberculosis [38, 43]. Two strains of M. tuberculosis, one virulent and the other avirulent (derived by repeated in vitro culture), have been compared by RSH [38, 43]. Genes differentially expressed in the virulent strain (dev genes) have been cloned, but the function and nature of these genes has not been studied further [38]. Plum and Clarck-Curtiss have used RSH to identify M. avium genes that are differentially transcribed in the intracellular environment of macrophages [44]. In this study, cDNAs from in vitro-grown bacteria are used to subtract cDNAs of macrophage-grown M. avium. This has permitted the identification of a macrophage-induced gene (mig) that is maximally transcribed between one and five days postinfection and has no homology to sequences in the genetic databases [44]. However, the mig gene seems to encode a secreted protein that is induced by a shift from pH 6.0 to pH 7.0 [45]. The role that the mig gene may play in ‘virulence’ has not been clearly established, although, its expression in saprophytic mycobacterium enhances resistance to intracellular killing by macrophages, and the mig gene seems to be common among M. avium strains that cause clinical disease [45, 46]. RSH is rather inefficient, since only one gene was found to be highly induced intracellularly. Additionally, it is important to screen cDNAs of intracellularly grown bacteria harvested at different time points postinfection, since bacterial gene transcription is likely to follow a temporal pattern of regulation in response to a continuously changing intracellular environment. 450
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The DD-PCR technique was originally developed for the detection of differentially expressed eukaryotic genes [47]. This method has been modified by our laboratory for the identification of prokaryotic cDNA fragments that are differentially expressed within the intracellular environment (figure 4) [48]. DD-PCR is useful for the detection of transcribed genes at the early stages of an infection, since low amounts of mRNA can be used as templates. DD-PCR utilizes reverse transcribed mRNA from intracellularly grown bacteria, which is then subjected to PCR amplification using random oligonucleotide primers. Comparison of the cDNA profiles of intracellular and extracellular bacteria allows for the identification of differentially transcribed genes [42]. Several cDNA fragments from L. pneumophila that are induced 4 h postinfection of U937 macrophage-like cells have been identified [48]. One of the fragments was cloned and has led to the identification of a novel locus, termed the early macrophage-induced locus (eml). This locus has been shown to be required for early survival in both macrophages and the environmental host of L. pneumophila, Hartmannella vermiformis [48]. DD-PCR is limited by the ability of the random primers to anneal to the cDNA [48]. Furthermore, the detection of false positives and the irreproducibility of detection of some transcribed fragments may occur [48].
11. Computer-based comparative genetics The recent advancements in microchip array technology which allow for the visualization of changes in gene expression in hundreds of genes should be of great use to the bacteriologist [49, 50]. This technology consists of a microchip that carries different arrays of DNA fragments that serve as probes to screen complementary nucleotide sequences [49]. Therefore, the expression of a whole bank of known genes can be studied simultaneously in one cell. Although currently this technology is used for eukaryotic gene expression, it should be possible to apply this technology to prokaryotes. This technology would enable the study of coordinate gene expression of hundreds of genes under any desirable condition, including the intracellular microenvironment. Thus, it is likely that, in the near future, snippets of entire genomes of pathogenic organisms can be displayed on a microchip and overall gene expression visualized in response to specific environmental changes. Major advancements in computer science and engineering have enabled the development of computer bioinformatics for comparing genes and genomes of various pathogens or different species of the same pathogen. Furthermore, the increased computer speed and tremendous communication network generated by the internet have made it easier for ‘database mining’ and computer-based comparative genetics to be performed; one example is the comparison of the Neisseria meningitidis genome with that of other nonpathogenic Neisseria using such techniques as representational difference analysis. This technique identified DNA sequences specific for pathogenic Neisseria [3]. Using such approaches, one can concenMicrobes and Infection 1999, 445-453
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Figure 4. DD-PCR adapted to identification of bacterial genes differentially expressed in the intracellular environment. Comparison of cDNAs from intracellular bacteria with in vitrogrown cDNAs allows for the identification of differentially transcribed intracellular genes.
trate on certain genomic regions of a pathogenic bacterium in order to elucidate their roles in pathogenesis as unique ‘virulence’ genes. These genes can be studied further for potential regulation in vivo. Pathogenicity islands of S. typhimurium have been shown to be induced during intracellular infection of macrophages [51]. Computer-based comparative genetics can be useful in the search for pathogenicity islands [52] -large genetic regions harboring several virulence genes that are found exclusively on the chromosome or virulence plasmid of pathogenic bacteria [52, 53]. Several criteria define a DNA region as a pathogenicity island. These include a different G + C content from the host’s DNA, which is indicative of horizontal transfer of genetic information; insertion near tRNA genes; and/or flanking by insertion sequence elements [52]. Therefore, with the sequencing of several genomes of bacterial pathogens, computer-based comparative genetics can be used for analysis of these sequences according to the criteria that describe chromosomal fragments associated with virulence.
12. Conclusions This review describes several techniques used to identify differentially transcribed bacterial genes. The induction of expression of certain genes during the intracellular infection is thought to be a global coordinated response by the bacteria to the intracellular microenvironment. This response is thought to allow the bacterium to adapt to and establish its niche within the host cell. These adaptations involve metabolic, environmental (i.e., pH and oxidative stress), and ‘virulence’ genes that perform unique functions exclusively involved in exploiting host cell processes. Examination of bacterial gene expression during the intracellular infection has been used as a strategy with the hope of discovering unique ‘virulence’ genes. It is Microbes and Infection 1999, 445-453
thought that some ‘virulence’ genes with unique functions should be expressed at high levels within the host cell compared with in vitro growth conditions. However, in most cases the results have been disappointing, since many of the induced genes have been found to be housekeeping genes (i.e., heat-shock proteins and several metabolic genes). The reasons for this may lie in the complex nature of bacterial gene regulation. It is likely that bacterial virulence genes are transcribed over a short period of time when absolutely needed, and the level of such transcription may be very low. This would greatly hamper the process of identifying these genes from among the many transcribed housekeeping genes. L. pneumophila is a good example of the tight regulation of ‘virulence’ gene expression and induction during stressful environmental conditions. Byrne and Swanson showed that several virulence traits of L. pneumophila that are induced in the intracellular environment are turned on only upon exiting the exponential phase of growth [54]. This finding stresses the complexity of bacterial gene regulation and accentuates the role of global gene regulation on the expression of virulence traits in a pathogen. Several studies utilizing the techniques described in this review revealed the presence of a battery of downregulated genes. Studies on S. typhimurium, L. pneumophila, and M. tuberculosis showed the repression of as many as 100 proteins during intracellular growth [28, 30, 31]. What is the function of these proteins? And is their repression important for virulence? Is there a relation between the down- and upregulated genes? The answers to these questions have been overlooked and may prove useful in the elucidation of the role and identification of other differentially transcribed ‘virulence’ genes. The identification of differentially transcribed genes constitutes merely the beginning of our quest to understand the basic question of how intracellular bacterial pathogens survive and thrive within our cells. The mode of action of the gene products and the regulation of these 451
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genes are of utmost importance to the understanding of host-pathogen interactions. New laboratory techniques and computer-based technologies should be of great use, in the future prospects of enhancing our understanding of the pathogenesis of intracellular bacteria.
Acknowledgments The authors thank Mr Gopi Shankar, Mr Lian-Yong Gao, and Dr Barbara J. Stone for their critical review of this manuscript. OSH is supported by a predoctoral National Research Service Award #TA09509. YA is supported by Public Health Service Award #R29A138410.
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