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Genetic analysis of cell invasiveness by Yersinia pseudotuberculosis
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1er F O R U M D E M I C R O B I O L O G I E
GENETIC ANALYSIS OF CELL INVASIVENESS BY Y E R S I N I A P S E U D O T U B E R C U L O S I S b y R . R . Isberg a n d S. F a l k o w
Department o f Medical Microbiology, Stanford University Medical Center, Stanford, CA 94305 (USA) The ability to invade and survive within host cells is a property of many enteropathogenic bacteria. As a rule, these invasive enteropathogenic microorganisms establish an infectious niche by employing one of two strategies: either (a) to replicate within the host cell, as exemplified by the growth of Shigellae in intestinal epithelial cells, or (b) to pass through the cell in order to go from one locale in the host to another [1-4]. Among those microorganisms which do not undergo significant replication within a host cell once invasion has taken place, the intent is usually to cross intestinal epithelial barriers, find an entrance to the lymphatic system and become distributed throughout many tissues of the host [4]. A clear example o f this process is the interaction between intestinal epithelial cells and certain enteric pathogens, such as the Salmonella [3, 4] and the enteropathogenic Yersinia [5, 6]. Clearly, the establishment of an intracellular location is a c o m m o n motif in the infection process, yet the factors encoded by microorganisms which promote their successful internalization and survival in the host cell have not been identified [7, 81. We have been investigating the invasion process of enteropathogens which cause systemic illnesses. As a model pathogen for this group, Y. pseudotuberculosis is studied in order to gain detailed information on how these bacteria enter epithelial cells. In diseases caused by this facultative intracellular pathogen, the microorganism is found in the ceils of its host as well as in extracellular spaces [8, 9]. As is true of other enteropathogenic Yersinia, it probably
uses both of the tactics described above to establish itself intracellularly. Initially, it invades the host by entering intestinal epithelial cells after ingestion o f contaminated foodstuff, eventually gaining access to the lamina propria in a step that apparently takes place without significant replication [5]. Once it gains entry into the lymphatic system, it seems able to survive and replicate within macrophages [10], and becomes distributed in the liver, spleen, and mesenteric lymph nodes [10, 11]. To initially investigate the ability of this microorganism to enter animal cells, we wanted to identify the genetic loci encoded by this bacterium which allow it to enter a cultured epithelial cell line [12]. Our approach was to construct a cosmid clone bank [13, 14] of Y. pseudotuberculosis and select sequences that were capable of conferring tissue invasion on the normally innocuous E. coli K12 strain. To this end, the entire bank was introduced into E. coli, pooled, grown in broth culture and used to infect a monolayer of cultured HEp-2 cells [15]. After several hours' incubation, bacteria that were unable to associate stably with the cell monolayer were removed by washing. Bacteria that remained associated with the HEp-2 cells were isolated on an appropriate growth medium following treatment of the monolayer with Triton-X100. We found that approximately 50 % of the bacterial clones which survived such an enrichment harboured cosmids that conferred on Escherichia coli the ability to penetrate cultured epithelial cells [12]. Most strikingly, the E. coli strains isolated in this manner behaved identically to the parental Y. pseudotuberculosis
MICROBIAL strain with respect to its efficiency of entering the HEp-2 monolayer cells. We refer to the locus that confers this property as inv. Figure 1 depicts the interaction between a HEp-2 cell and an E. coli K12 strain harbouring the inv locus. E. coli K12 is normally unable to enter animal cells [12]. In contrast, the same bacterial strain harbouring an intact inv locus yields a large number of bacteria associated with the animal cell. These bacteria are both bound to the outside of the cells as well as present within large endocytic vacuoles. Micrographs such as these provide strong evidence that the invasive phenotype can be successfully mimicked by introducing DNA from a Yersinia species into E. coli. To perform a detailed analysis of the inv locus, a series of Tn5 and TnlO00
insertion mutations were isolated [17] which eliminated the ability of E. coli strains harbouring this locus to invade. It was found that all the mutations mapped in a contiguous 3.2-kilobase-pair region of DNA, and each mutation which eliminated invasion of the bacterium also eliminated its ability to bind the surface of the HEp-2 cell. A plas-
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309
mid containing only the 4.6-Kb region o f Y. pseudotuberculosis D N A encompassing this locus was sufficient to permit E. coli K12 to enter HEp-2 ceils. Further genetic and biochemical analysis o f the gene product produced from this locus indicate that inv is a single gene encoding a large outer membrane protein ( > 100,000 daltons). Apparently, synthesis of this protein is sufficient to convert E. coli into an invader of animal cells. We were surprised to find that so little was needed to permit E. coli to enter animal cells. Apparently, this phenotype is the result of a much simpler set of factors than had been assumed from the work on other invasive pathogens, especially the Shigella. The work o f Sansonetti, Formal and co-workers [7, 16] indicates that many genes are required to reconstruct the invasive phenotype of the Shigella. It seems likely that these two invasive enteropathogens use different strategies for entry into host cells (P. Small and S. Falkow, manuscript in preparation). We believe these different strategies reflect the fact that these two enteropathogenic organisms have different rationales for ente-
FIG. 1. - - Thin-section electron microscopy of cultured cells incubated with an E. coli strain that harbours plasmM containing Y. pseudotuberculosis invasion region. Thin section of HEp-2 cells infected with E. coli K12 strain HB101 harbouring plasmid pRI203, a high copy number plasmid containing the inv locus. Fixation, sectioning and microscopy of sample was performed as described [12].
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ring intestinal epithelial cells, as noted above. Yersinia species enter epithelial cells as a prelude to establishing a systemic infection; the Shigella enter with the intent of replicating and establishing
infection within the epithelial cell itself. As more becomes known about the nature of microbial invasiveness, it will be interesting to see if bacterial pathogens follow these two model systems.
References.
[1] FORMAL, S.B., HALE, T.L. & SANSONETTI,P.J., Rev. infect. Dis., 1983, 5, 2702-2707.
[2] MOULDER,J.W., Microbiol. Rev., 1985, 49, 298-337. [3] LABREc, E.H., SCHNEIDER, H., MAGNANI, T.J. & FORMAL, S., Jr. Bact., 1964, 88, 1503-1518. [4] TAKEUCHI,A., Amer. J. Path., 1967, 50, 109-136. [5] DEVENISH,J.A. & SHIEMANN,D.A., Infect. Immun., 1981, 36, 48-55. [6] UNE, T., Microbiol. Immunol., 1977, 21, 349-36. [7] SANSONETTI,P.J., HALE, T.L., DAMMIN, G.J., KAPEER, C., COLLINS, H.A. & FORMAL, S.B., Infect. Immun., 1983, 39, 1392-1402. [8] BOVALLIUS,A. & NILSSON, G., Canad. J. Microbiol., 1975, 21, 1997-2007. [9] BOLIN, I., NOREANDER, L. 8r WOLE-WATz, H., Infect. Immun., 1982, 37, 506-512. [10] UNE, T., Microbiol. Immunol., 1977, 21, 505-506. [11] CARTER,P.B., Infect. Immun., 1975, 11, 164-170. [12] ISBERG,R.R. & FALKOW,S., Nature (Lond.), 1985, 317, 262-264. [13] HOHN, B., Meth. Enzymol., 1979, 68, 299-309. [14] KOOMEY,J.M., GILL, R.E. & FALKOW,S., Proc. nat. Acad. Sci. (Wash.), 1982, 79, 7881-7885. [15] MOORE, A.E., SABACHEWSKY,L. & TOOLAN, H.W., Cancer Res., 1959, 15, 598. [16] MAURELLI, A.T., BAUDRY, B., D'HAUTEVILLE, H., HALE, T.L. & SANSONETTI, P.J., Infect. lmmun., 1985, 49, 164-171. [17] GUVER, M., J. mol. Biol., 1978, 126, 347-365.
R.I. was supported by the Jane Coffin Childs Memorial Fund for Medical Research. This research was supported, in part, by NSF grant PCM 83-06654 and contract DAMD 17-82-C-2002from the US Army Medical Research Acquisitions Agency to S.F.
1er F O R U M D E M I C R O B I O L O G I E
GENETIC ANALYSIS OF CELL INVASIVENESS BY Y E R S I N I A P S E U D O T U B E R C U L O S I S b y R . R . Isberg a n d S. F a l k o w
Department o f Medical Microbiology, Stanford University Medical Center, Stanford, CA 94305 (USA) The ability to invade and survive within host cells is a property of many enteropathogenic bacteria. As a rule, these invasive enteropathogenic microorganisms establish an infectious niche by employing one of two strategies: either (a) to replicate within the host cell, as exemplified by the growth of Shigellae in intestinal epithelial cells, or (b) to pass through the cell in order to go from one locale in the host to another [1-4]. Among those microorganisms which do not undergo significant replication within a host cell once invasion has taken place, the intent is usually to cross intestinal epithelial barriers, find an entrance to the lymphatic system and become distributed throughout many tissues of the host [4]. A clear example o f this process is the interaction between intestinal epithelial cells and certain enteric pathogens, such as the Salmonella [3, 4] and the enteropathogenic Yersinia [5, 6]. Clearly, the establishment of an intracellular location is a c o m m o n motif in the infection process, yet the factors encoded by microorganisms which promote their successful internalization and survival in the host cell have not been identified [7, 81. We have been investigating the invasion process of enteropathogens which cause systemic illnesses. As a model pathogen for this group, Y. pseudotuberculosis is studied in order to gain detailed information on how these bacteria enter epithelial cells. In diseases caused by this facultative intracellular pathogen, the microorganism is found in the ceils of its host as well as in extracellular spaces [8, 9]. As is true of other enteropathogenic Yersinia, it probably
uses both of the tactics described above to establish itself intracellularly. Initially, it invades the host by entering intestinal epithelial cells after ingestion o f contaminated foodstuff, eventually gaining access to the lamina propria in a step that apparently takes place without significant replication [5]. Once it gains entry into the lymphatic system, it seems able to survive and replicate within macrophages [10], and becomes distributed in the liver, spleen, and mesenteric lymph nodes [10, 11]. To initially investigate the ability of this microorganism to enter animal cells, we wanted to identify the genetic loci encoded by this bacterium which allow it to enter a cultured epithelial cell line [12]. Our approach was to construct a cosmid clone bank [13, 14] of Y. pseudotuberculosis and select sequences that were capable of conferring tissue invasion on the normally innocuous E. coli K12 strain. To this end, the entire bank was introduced into E. coli, pooled, grown in broth culture and used to infect a monolayer of cultured HEp-2 cells [15]. After several hours' incubation, bacteria that were unable to associate stably with the cell monolayer were removed by washing. Bacteria that remained associated with the HEp-2 cells were isolated on an appropriate growth medium following treatment of the monolayer with Triton-X100. We found that approximately 50 % of the bacterial clones which survived such an enrichment harboured cosmids that conferred on Escherichia coli the ability to penetrate cultured epithelial cells [12]. Most strikingly, the E. coli strains isolated in this manner behaved identically to the parental Y. pseudotuberculosis
MICROBIAL strain with respect to its efficiency of entering the HEp-2 monolayer cells. We refer to the locus that confers this property as inv. Figure 1 depicts the interaction between a HEp-2 cell and an E. coli K12 strain harbouring the inv locus. E. coli K12 is normally unable to enter animal cells [12]. In contrast, the same bacterial strain harbouring an intact inv locus yields a large number of bacteria associated with the animal cell. These bacteria are both bound to the outside of the cells as well as present within large endocytic vacuoles. Micrographs such as these provide strong evidence that the invasive phenotype can be successfully mimicked by introducing DNA from a Yersinia species into E. coli. To perform a detailed analysis of the inv locus, a series of Tn5 and TnlO00
insertion mutations were isolated [17] which eliminated the ability of E. coli strains harbouring this locus to invade. It was found that all the mutations mapped in a contiguous 3.2-kilobase-pair region of DNA, and each mutation which eliminated invasion of the bacterium also eliminated its ability to bind the surface of the HEp-2 cell. A plas-
INVASION
309
mid containing only the 4.6-Kb region o f Y. pseudotuberculosis D N A encompassing this locus was sufficient to permit E. coli K12 to enter HEp-2 ceils. Further genetic and biochemical analysis o f the gene product produced from this locus indicate that inv is a single gene encoding a large outer membrane protein ( > 100,000 daltons). Apparently, synthesis of this protein is sufficient to convert E. coli into an invader of animal cells. We were surprised to find that so little was needed to permit E. coli to enter animal cells. Apparently, this phenotype is the result of a much simpler set of factors than had been assumed from the work on other invasive pathogens, especially the Shigella. The work o f Sansonetti, Formal and co-workers [7, 16] indicates that many genes are required to reconstruct the invasive phenotype of the Shigella. It seems likely that these two invasive enteropathogens use different strategies for entry into host cells (P. Small and S. Falkow, manuscript in preparation). We believe these different strategies reflect the fact that these two enteropathogenic organisms have different rationales for ente-
FIG. 1. - - Thin-section electron microscopy of cultured cells incubated with an E. coli strain that harbours plasmM containing Y. pseudotuberculosis invasion region. Thin section of HEp-2 cells infected with E. coli K12 strain HB101 harbouring plasmid pRI203, a high copy number plasmid containing the inv locus. Fixation, sectioning and microscopy of sample was performed as described [12].
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ring intestinal epithelial cells, as noted above. Yersinia species enter epithelial cells as a prelude to establishing a systemic infection; the Shigella enter with the intent of replicating and establishing
infection within the epithelial cell itself. As more becomes known about the nature of microbial invasiveness, it will be interesting to see if bacterial pathogens follow these two model systems.
References.
[1] FORMAL, S.B., HALE, T.L. & SANSONETTI,P.J., Rev. infect. Dis., 1983, 5, 2702-2707.
[2] MOULDER,J.W., Microbiol. Rev., 1985, 49, 298-337. [3] LABREc, E.H., SCHNEIDER, H., MAGNANI, T.J. & FORMAL, S., Jr. Bact., 1964, 88, 1503-1518. [4] TAKEUCHI,A., Amer. J. Path., 1967, 50, 109-136. [5] DEVENISH,J.A. & SHIEMANN,D.A., Infect. Immun., 1981, 36, 48-55. [6] UNE, T., Microbiol. Immunol., 1977, 21, 349-36. [7] SANSONETTI,P.J., HALE, T.L., DAMMIN, G.J., KAPEER, C., COLLINS, H.A. & FORMAL, S.B., Infect. Immun., 1983, 39, 1392-1402. [8] BOVALLIUS,A. & NILSSON, G., Canad. J. Microbiol., 1975, 21, 1997-2007. [9] BOLIN, I., NOREANDER, L. 8r WOLE-WATz, H., Infect. Immun., 1982, 37, 506-512. [10] UNE, T., Microbiol. Immunol., 1977, 21, 505-506. [11] CARTER,P.B., Infect. Immun., 1975, 11, 164-170. [12] ISBERG,R.R. & FALKOW,S., Nature (Lond.), 1985, 317, 262-264. [13] HOHN, B., Meth. Enzymol., 1979, 68, 299-309. [14] KOOMEY,J.M., GILL, R.E. & FALKOW,S., Proc. nat. Acad. Sci. (Wash.), 1982, 79, 7881-7885. [15] MOORE, A.E., SABACHEWSKY,L. & TOOLAN, H.W., Cancer Res., 1959, 15, 598. [16] MAURELLI, A.T., BAUDRY, B., D'HAUTEVILLE, H., HALE, T.L. & SANSONETTI, P.J., Infect. lmmun., 1985, 49, 164-171. [17] GUVER, M., J. mol. Biol., 1978, 126, 347-365.
R.I. was supported by the Jane Coffin Childs Memorial Fund for Medical Research. This research was supported, in part, by NSF grant PCM 83-06654 and contract DAMD 17-82-C-2002from the US Army Medical Research Acquisitions Agency to S.F.