Microbial Pathogenesis 1990 ; 8 : 3-11
Mini-review Virulence plasmids of Salmonella typhimurium and other salmonellae Paul A . Gulig Department of Immunology and Medical Microbiology, University of Florida College of Medicine, Box J-266, Gainesville, FL 32610, U .S.A .
Introduction In the 1970s several groups identified a large plasmid in strains of Salmonella typhimurium ;' -7 however, with few exceptions, no phenotype could be ascribed to the plasmid . Hence, the plasmid was called a 'cryptic plasmid' . The cryptic plasmid was able to suppress the fertility of the F plasmid,` and the plasmid could integrate into the chromosome of dnaA mutants of S. typhimurium to drive chromosomal replication .' In 1982 Jones et al.' described the large 'cryptic' plasmid of S . typhimurium as being associated with virulence . Since then, considerable work has been published concerning the pathogenesis associated with and genetics of the high molecular weight virulence plasmids of S. typhimurium and other serotypes and species of Salmonella . Although a consensus exists as to the genetic loci involved with plasmidmediated virulence of salmonellae, much is yet to be learned about how the virulence plasmids contribute to pathogenicity . This review examines these two subjects concerning the S. typhimurium virulence plasmid in addition to correlating results with those obtained with other salmonellae including S . dublin, S. enteritidis, S . choleraesuis, S . gallinarum and S . pullorum .
Pathogenesis associated with the virulence plasmid Jones et al.' initially described the virulence phenotype conferred on S . typhimurium by the cryptic plasmid . In studies including infection of mice and cultured animal cells, they concluded that the virulence plasmid was involved in the mannose-resistant adherence to and invasion of mammalian cells by salmonellae . Jones and co-workers also reported that the virulence plasmid could integrate into the salmonella chromosome resulting in lack of expression of the virulence phenotype . The plasmid could later excise and again confer virulence . Subsequent investigations by other laboratories confirmed the role of the plasmid in virulence, but failed to identify a relationship between the virulence plasmid and interactions of S . typhimurium with cultured animal cells ."' As discussed below, the adherence-invasion deficient phenotype is the result of the integration of the plasmid into the chromosome in the strain of Jones et al.' and not loss of the plasmid by curing . This integration is an artifact of the use of Tn 10 to label the plasmid with tetracycline resistance . Others have since observed an integration event mediated by Tn 10 but not by other transposons ." The S . typhimurium virulence plasmid is primarily responsible for spreading infection 0882-4010/90/010003+09 $03 .00/0
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1990 Academic Press Limited
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P . A . Gulig
beyond the intestines . Hackett et a/. 9 determined that virulence plasmid-cured S . typhimurium was proficient at infecting the Peyer's patches of orally inoculated mice . Pardon et al." and Gulig and Curtiss 10 subsequently identified the principal virulence attribute of the plasmid as the spreading of infection beyond the small intestine to deeper tissues such as the mesenteric lymph nodes or spleen since plasmid-cured derivatives were attenuated in infection of these sites . Gulig and Curtiss 10 reported that parenterally administered cured S . typhimurium, although detectably attenuated, retained considerable virulence as measured by growth within spleens and LD 50 . These results suggested that the plasmid primarily affects early infection between the intestines and mesenteric lymph nodes, as opposed to later events such as growth within the reticuloendothelial system . In contrast, Pardon et a/. 12 observed greater attenuation of plasmid-cured S . typhimurium after parenteral inoculation . The exact mechanism by which the virulence plasmid mediates the spreading of infection beyond the intestines to deeper tissues is not known . Interestingly, Hoertt et al."' reported evidence suggesting that the S . typhimurium virulence plasmid may encode factors mediating splenomegaly and non-specific immunosuppression . Until recently, a controversial issue has been the role of the virulence plasmid in resistance of S . typhimurium to complement-mediated bacteriolysis of normal serum . Helmuth et al." originally reported a correlation of the presence of high molecular weight plasmids among isolates of S . typhimurium and serum resistance in unrelated strains . Gulig and Curtiss 10 found no role for the virulence plasmid in serum resistance by constructing plasmid-cured derivatives of three different strains of S . typhimurium, all of which retained serum resistance . Hackett et a/. 9 ' 14 constructed a plasmid-cured S. typhimurium derivative that was serum sensitive, suggesting that the virulence plasmid was necessary for serum resistance . Hackett et al. also cloned from the S . typhimurium virulence plasmid a gene encoding an 11 000 molecular weight (11 k) protein which conferred increased, but not complete, serum resistance to E. co/i K-12 and their plasmid-cured S . typhimurium, J42 . 14 However, during construction, the plasmid-cured S . typhimurium strain also acquired a rough lipopolysaccharide phenotype . This result was not reproduced by others in the -construction of isogenic sets of wild-type and plasmid-cured strains ."" Because the virulence plasmid was not reintroduced into cured S . typhimurium J42 to demonstrate restoration of wildtype virulence and lipopolysaccharide phenotypes, 14 it is possible that the rough lipopolysaccharide and serum sensitivity of strain J42 were caused by a secondary mutation selected during curing . In this regard, Gulig and Curtiss (unpublished results) isolated at least one plasmid-cured S . typhimurium derivative that did not regain virulence upon re-introduction of the virulence plasmid, indicating that unidentified secondary mutations had occurred . Since complete, smooth lipopolysaccharide is essential for serum resistance of S. typhimurium, the serum sensitivity of J42 of Hackett et a/. 14 could have been caused by rough Iipopolysaccharide . The exact role of the 11 k protein in serum resistance has not been defined in wild-type S . typhimurium . A traT-like gene is present on the S . typhimurium virulence plasmid ."" This gene is functionally homologous to the traT gene of the F and other plasmids in terms of conferring limited serum resistance under specific conditions .""' For example, Rhen and Sukupolvi 16 observed increased serum resistance contributed by TraT in plasmidcured S . typhimurium with a rough lipopolysaccharide chemotype ; the relevance of these observations to smooth salmonellae is unclear since others have found that smooth, virulence plasmid-cured S . typhimurium is serum resistant .' 0,15 It is important to note that the TraT protein from S. typhimurium has a molecular weight of 27 k, 16 and that the restriction map of the loci encoding the 11 k protein 14 and TraT are different . Therefore, the 11 k protein is probably not related to TraT . It is possible that
Salmonella virulence plasmids
5
the 1 1 k and TraT proteins play secondary roles in serum resistance of S . typhimurium, but the serum resistance of plasmid-cured S . typhimurium precludes an essential role . VandenBosch et al. determined that S . typhimurium containing a TOO-mediated, chromosomally integrated virulence plasmid was serum sensitive 20 and hypothesized that the plasmid was therefore involved with serum resistance . VandenBosch et al. 21 subsequently cloned from the virulence plasmid a locus termed rsk (regulation of serum killing) which restored serum resistance to plasmid-integrated salmonellae . However, VandenBosch et al. 15 observed that S. typhimurium completely lacking the virulence plasmid, as examined by Gulig and Curtiss, 70 is serum resistant and proficient in infection of cultured animal cells . Furthermore, cloned rsk exerts no effects on virulence of plasmid-cured S . typhimurium ." The cloned rsk gene therefore appears to be involved in regulation of several chromosomally encoded virulence functions that, in an undetermined manner, are affected by the integration of the virulence plasmid into the salmonella chromosome . VandenBosch et al. suggest that rsk could bind and titrate a regulatory factor ." In summary, the virulence plasmid is not necessary for S. typhimurium to be resistant to killing by complement of normal serum . However, the plasmid does encode factors such as the 11 k and TraT proteins and rsk, which may have minor or indirect roles in serum resistance . Several groups have hypothesized that the virulence plasmid affects the resistance to killing by macrophages or bacterial growth within these host cells . However, direct proof of these hypotheses is lacking . S. typhimurium probably first encounters macrophages at the Peyer's patches and subsequently at the mesenteric lymph nodes and spleen ." S . typhimurium is considered to be a facultative intracellular pathogen and is able to at least survive within murine macrophages . 23 Contrary to the macrophage resistance hypotheses, Gulig and Curtiss 10 examined salmonella-macrophage interactions under a variety of conditions in vitro and in vivo and did not detect differences in phagocytosis and killing of wild-type and plasmid-cured S. typhimurium . For example, after intraperitoneal injection of mice with wild-type and cured salmonellae, equal numbers of each strain were associated with macrophages at 1 h post-injection (i .e . the plasmid did not affect phagocytosis) . Furthermore, equal numbers of each strain survived within peritoneal macrophages, as determined by culturing infected host cells from lavages in the presence of gentamicin for 2 h . To determine whether macrophages were responsible for preventing spreading infection by plasmid-cured S. typhimurium (i .e . the plasmid encoded macrophage-resistance), mice were treated with intravenous silica to deplete functional macrophages . 24 In silica-treated mice, wild-type S . typhimurium remained 100-fold more virulent than a plasmid-cured derivative in terms of spreading to the spleen after oral inoculation (Gulig and Curtiss, unpublished results) . Therefore, a host factor(s) other than macrophages was responsible for preventing splenic infection by cured salmonellae . These data must be considered with the contrasting results of Pardon et al." who observed decreased splenic infection by plasmid-cured S. typhimurium in intravenously inoculated mice . In summary, the virulence plasmid does not seem to directly affect a salmonellaemacrophage interaction at the level of phagocytosis or killing by macrophages . Many different groups have determined that related high molecular weight plasmids are involved with virulence of other serotypes of Salmonella . In an epidemiological investigation of diverse Salmonella isolates, Helmuth et al." identified high molecular weight plasmids in strains of S . typhimurium, S . enteritidis, S. dublin and S. choleraesuis . Terakado et al. 25 initially correlated the loss of a 75 kb plasmid of S . with a decrease in virulence in terms of intraperitoneal LD 50 . The requisite
dub/in
experiment to confirm this plasmid-pathogenesis relationship was performed by Chikami et a/. 26 by reintroducing a transposon-labeled virulence plasmid back into a
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P . A. Gulig
plasmid-cured S. dub/in derivative and demonstrating that virulence was regained . Chikami and co-workers observed attenuation of plasmid-cured S . dublin by the oral and intraperitoneal routes of inoculation of mice . A subsequent study from the same laboratory 27 delineated the pathogenesis mediated by the S . dub/in plasmid with results very similar to those outlined above for S . typhimurium, 1 " 2 i .e ., the plasmid was necessary for spreading infection beyond the intestines of mice . Further work associating the S . dub/in plasmid with virulence was reported by Manning et al." using transposon mutagenesis and plasmid curing . Nakamura et al. associated loss of a 54 kb plasmid from S . enteritidis with attenuation in terms of the subcutaneous LD50 in mice29 and determined that virulence plasmid-cured strains could immunize mice against salmonella infection ." Hovi et al." subsequently reintroduced the S . enteritidis virulence plasmid into a cured derivative and restored virulence . The S. enteritidis virulence plasmid did not affect lipopolysaccharide or serum resistance . 31 Barrow and co-workers have identified virulence plasmids for S . ga/linarum 32 and S . pullorum. 33 Plasmid-cured S . gallinarum and S . pullorum retained serum resistance but were attenuated for multiplication within the reticuloendothelial system of chicks . A 50 kb plasmid is necessary for virulence of S , cho/eraesuis in mice inoculated intraperitoneally ." Several groups have demonstrated the functional interchangeability of plasmids from different species and serotypes of Salmonella.' 1,31,35-37 In summary, the virulence plasmid appears to be necessary for S . typhimurium to invade beyond the intestines after oral inoculation of mice, but the mechanism for this phenomenon is not known at either the cellular or molecular levels . The virulence plasmid does not directly affect virulence traits such as cellular adherence-invasion, serum-resistance and phagocytosis and killing by macrophages . Virulence plasmids of other species and serotypes of Salmonella appear to be similarly involved in spreading infection beyond the intestines . Perhaps the virulence plasmid affects the hostpathogen interaction at a more complicated level such as regulation of the host response to infection with S . typhimurium . In dissecting the pathobiology of why wild-type salmonellae are isolated in higher numbers than are plasmid-cured salmonellae from either mesenteric lymph nodes or spleens of orally inoculated mice, three general explanations are put forward . The virulence plasmid may affect the rates of (1) growth, (2) death, and/or (3) movement through tissues for salmonellae . These rates do not indicate specific mechanisms in themselves, but must be able to account for differences in numbers of bacteria recovered . For example, if the virulence plasmid encoded factors mediating immunosuppression in mice, the rates of growth and death might be affected . Genetics of the virulence plasmid Soon after the discovery that a virulence plasmid existed in S . typhimurium, 8 plasmids with DNA homology were found in other species and serotypes of Salmonella . Popoff et a/. 38 determined that large plasmids of S . typhimurium, S. enteritidis, S . dublin, S . paratyphi C, S. newport, and S . abortusovis were related at the level of DNA homology and restriction fragment mapping . Figure 1 summarizes the physical and genetic maps of the S. typhimurium virulence plasmid with detail of the virulence region . Baird et al." initially identified a locus of the S . typhimurium and S. dublin plasmids associated with virulence through the use of Tn 10 and TnA insertion mutations which attenuated virulence (Fig . 1, map B) . Through restriction mapping of this region they estimated that between 13 and 23 kb of homologous sequence could be involved in virulence . The homology between S . typhimurium and S . dublin extends to the thick vertical bar at the right hand ends of maps B and D . Therefore, mutations which had been
7
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identified to the right in the non-homologous region of the S . dub/in virulence plasmid are not shown . By using large scale deletion mutagenesis of the S . typhimurium virulence plasmid, Michiels et a/. 40 constructed a restriction map of the entire plasmid and identified a region of no more than 38 kb involved with virulence . The right portion of the 38 kb region is contained in Fig . 1, map C . The virulence region of Michiels et a/. 40 was very similar to that of Baird et a/. 39 Studies of Beninger et a/. 35 on the S. dub/in virulence plasmid further localized a virulence region to a 4 kb EcoRl fragment within the aforementioned virulence regions [Fig . 1, map D (=)] . Poppe et a/. 41 have recently used a subclone consisting of a 3 .75 kb Hindlll fragment from cloned S . typhimurium virulence genes of Gulig and Curtiss 42 (see below) and found homology with plasmids of five other serotypes . Rhen et al." examined Tn5 insertions within the consensus virulence region and identified two loci containing virulence genes (Fig . 1, map E) . In contrast to the studies discussed above which relied on either transposon insertion or deletion mutagenesis to identify virulence genes of the salmonella plasmids, Gulig and Curtiss 42 identified a region necessary for virulence through cloning . Libraries of cloned virulence plasmid sequences were placed into plasmid-cured S . typhimurium, and virulence-conferring clones were selected in mice by allowing the cured salmonellae containing cloned virulence genes to infect the spleens of orally inoculated mice . Three cosmid clones with insert sequences of approximately 23 kb, each of which conferred a wild-type level of virulence, were isolated . Restriction analysis indicated that the insert sequences shared a common 14 kb region . The 14 kb region [Fig . 1, map A (=)], which coincided with the aforementioned virulence regions identified by others 31,31,4' encoded at least three proteins with molecular weights of 28 k, 29 k, and 32 k . The cloned sequence of plasmid pYA403, which conferred complete virulence on plasmid-cured S . typhimurium in terms of LD 50 and infection of spleens after oral inoculation of mice, 42 is shown as the detailed region around virulence loci in Fig . 1 (map A) . A 3 .2 kb BamHI fragment alone conferred substantial virulence to plasmid-cured S . typhimurium and encoded the 28 k protein [Fig . 1, map A ( L I ] . A particular Tn5 insertion, vir-22 : :Tn5, essentially eliminated plasmid mediated virulence of S . typhimurium and inhibited production of the 28 k protein (Fig . 1, map A) . Gulig and Chiodo have since confirmed that the 28 k protein is a major virulence factor encoded by the S . typhimurium plasmid and have determined the DNA sequence of the gene encoding the 28 k protein, virA (Abstracts Annual Meeting Amer . Soc . Microbiol ., 1989, p . 66) . Deletion of the right half of the 14 kb virulence region 42 (Fig . 1, lane A) beyond the Xhol site from the S . typhimurium virulence plasmid did not affect virulence as determined by splenic infection after oral inoculation of mice (Gulig, unpublished results), thus narrowing the virulence region to approximately 8 kb . Williamson et a1. identified 11 and recently cloned 36 an 8 kb region of the S. dub/in virulence plasmid [Fig . 1 (G)], which appeared to confer virulence to S. typhimurium and S . dub/in . However, the virulence assay consisted of noting the sick appearance of mice . Since quantitative analysis of virulence by using either splenic infection or LD 50 was not carried out, the extent of the virulence conferred by the cloned sequence is not known . This region is similar to the shortened virulence region of Gulig (unpublished results) mentioned above . An interesting finding of Williamson et a/. 36 was that the copy number of the vector used in cloning affected the ability of cloned sequences to confer virulence, with low copy vectors functioning better than high copy vectors . Most recently, Norel et a/. 44 cloned a 22 kb fragment of the S . typhimurium virulence plasmid which conferred full virulence to plasmid-cured S . typhimurium and encoded
Salmonella virulence plasmids
9
at least 10 proteins . By using extensive Tn5 mutagenesis of cloned sequences, they identified two virulence loci termed virA and virB. virA corresponds to the 8 kb Sallregion [Fig . 1, map F (=)] of Williamson et al. 1136 and encodes the 28 k protein identified by Gulig and Curtiss 42 in addition to several other proteins . virB maps to the repA region identified by Michiels et al.,40 which also encodes incompatibility (inc) and partitioning (par) functions (Tinge and Curtiss, Abstracts Annual Meeting Amer . Soc . Microbiol ., 1989, p . 216) . virB : :Tn5 insertions 14, 65
Xhol
and 10 of Norel et al." mapped in a locus encoding a 43 k protein, and virB : :Tn5 insertions 125 and 178 inhibited production of a 38 k protein . Tinge and Curtiss identified products of the parA and pars genes as 44 k and 38 k proteins, respectively (Abstracts Annual Meeting Amer . Soc . Microbiol ., 1989, p . 216) . virB mutations may therefore indirectly diminish virulence by affecting plasmid stability or copy number . This possibility is supported by the fact that pYA403 of Gulig and Curtiss, 42 which conferred full virulence on plasmid-cured S. typhimurium, does not encode the repA ('virB') locus (Fig . 1, map A) . Most, if not all, virulence plasmid-encoded proteins are not detectably expressed by S . typhimurium grown in vitro (Gulig and Curtiss, unpublished results) . Therefore, either minute quantities of the plasmid-encoded virulence factors are sufficient for virulence, or virulence genes are expressed only with the appropriate stimuli in vivo in the infected host . The virulence plasmids of other pathogenic bacteria such as Shigella spp ." and Yersinia spp . 46 each possess unique mechanisms of regulation of plasmidencoded virulence genes . The results discussed above of VandenBosch and coworkers studying rsk 1521 indicate that regulatory systems may be present on the salmonella virulence plasmids .
Summary Related high molecular weight plasmids of several serotypes and species of Salmonella have been associated with virulence in a variety of animal models of infection . The primary virulence plasmid phenotype is in the ability of salmonellae to spread beyond the initial site of infection, the intestines . The mechanism of this plasmid-mediated invasive infection has not been identified, but may be a complex interaction in the host-pathogen relationship . A common region of the salmonella plasmids has been associated with virulence, and specific virulence genes and their products are now being identified ; however, much is yet to be accomplished in this field . The combined analysis of pathogenesis and genetics associated with the salmonella virulence plasmids may identify new systems of bacterial virulence and the genetic basis for this virulence . I thank Dr Roy Curtiss III, Dr Richard Lottenberg, Hank Lockman and Steve Tinge for their critical review of this manuscript .
References 1 . 2. 3. 4. 5.
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6 . Spratt BG, Rowbury RJ, Meynell GG . The plasmid of Salmonella typhimurium LT2 . Mol Gen Genet 1973 ; 121 : 347-53 . 7 . Bagdasarian M, Hryniewicz M, Zdzienicka M . Integrative suppression of a dnaA mutation in Salmonella typhimurium . Mol Gen Genet 1975; 139 : 213-31 . 8 . Jones GW, Rabert DK, Svinarich DM, Whitfield HJ . Association of adhesive, invasive, and virulent phenotypes of Salmonella typhimurium with autonomous 60-megadalton plasmids . Infect Immun 1982 ;38 :476-86 . 9 . Hackett J, Kotlarski I, Mathan V, Francki K, Rowley D . The colonization of Peyer's patches by a strain of Salmonella typhimurium cured of the cryptic plasmid . J Infect Dis 1986 ; 153 : 1119-25 . 10 . Gulig PA, Curtiss R III . Plasmid-associated virulence of Salmonella typhimurium . Infect Immun 1987 ; 55 :2891-901 . 11 . Williamson CM, Baird GD, Manning EJ . A common virulence region on plasmids from eleven serotypes of Salmonella . J Gen Microbial 1988 ; 134 : 975-82 . 12 . 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Evidence of coordinate regulation of virulence in Salmonella typhimurium involving the rsk element of the 95-kilobase plasmid . Infect Immun 1989; 57 :2566-8 . 16 . Rhen M, Sukupolvi S . The role of the traT gene of the Salmonella typhimurium virulence plasmid for serum resistance and growth within liver macrophages. Microbial Pathogenesis 1988 ; 5 : 275-85 . 17 . Rhen M, O'Connor D, Sukupolvi S . The outer membrane permeability mutation of the virulenceassociated plasmid of Salmonella typhimurium is located in a traT-like gene . FEMS Microbiol Lett 1988;52 :145-54 . 18 . Sukupolvi S, O'Connor D, Makela PH . The effects of traT insertion mutations on detergent sensitivity and serum resistance of Escherichia coli and Salmonella typhimurium . FEMS Microbiol Lett 1987 ; 43 : 81--7 . 19 . Sukupolvi S, O'Connor D, Edwards MF . The TraT protein is able to normalize the phenotype of a plasmid-carried permeability mutation of Salmonella typhimurium . J Gen Microbiol 1986 ; 132 : 207985 . 20 . VandenBosch JL, Rabert DK, Jones GW . Plasmid-associated resistance of Salmonella typhimurium to complement activated by the classical pathway . Infect Immun 1987 ; 55 : 2645-52 . 21 . VandenBosch JL, Rabert DK, Kurlandsky DR, Jones GW . Sequence analysis of rsk, a portion of the 95-kilobase plasmid of Salmonella typhimurium associated with resistance to the bactericidal activity of serum . Infect Immun 1989; 57 : 850--7 . 22 . Carter PB, Collins FM . The route of enteric infection in normal mice . J Exp Med 1974; 139 : 1189203 . 23 . Fields PI, Swanson RV, Haidaris CG, Heffron F . Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent . Proc Natl Acad Sci USA 1986; 83 : 5189-93 . 24 . O'Brien AD, Scher I, Formal SB . Effect of silica on the innate resistance of inbred mice to Salmonella typhimurium infection . Infect Immun 1979 ; 25 : 513-20 . 25 . Terakado N, Sekizaki T, Hashimoto K, Naitoh S . Correlation between the presence of a fifty-megadalton plasmid in Salmonella dublin and virulence for mice . Infect Immun 1983 ; 41 : 443-4 . 26 . Chikami GK, Fierer J, Guiney DG . Plasmid-mediated virulence in Salmonella dublin demonstrated by use of a Tn5-oriT construct . Infect Immun 1985 ; 50 : 420-4 . 27 . Heffernan EJ, Fierer J, Chikami G, Guiney D . Natural history of oral Salmonella dublin infection in BALB/c mice : effect of an 80-kilobase-pair plasmid on virulence . J Infect Dis 1987 ; 155 : 1254-9 . 28 . Manning EJ, Baird GD, Jones PW . The role of plasmid genes in the pathogenicity of Salmonella dublin . J Med Microbiol 1986 ; 21 : 239-43 . 29 . Nakamura M, Sato S, Ohya T, Suzuki S, Ikeda S . Possible relationship of a 36-megadalton Salmonella enteritidis plasmid to virulence in mice . Infect Immun 1985 ; 47 : 831-3 . 30 . Nakamura M, Sato S, Ohya T, Suzuki S, Ikeda S, Koeda T . Plasmid-cured Salmonella enteritidis AL1 192 as a candidate for a live vaccine . Infect Immun 1985 ; 50 : 586-7 . 31 . Hovi M, Sukupolvi S, Edwards MF, Rhen M . Plasmid-associated virulence of Salmonella enteritidis. Microbial Pathogenesis 1988 ; 4 : 385-91 . 32 . Barrow PA, Simpson JM, Lovell MA, Binns MM . Contribution of Salmonella galinarum large plasmid toward virulence in fowl typhoid . Infect Immun 1987 ; 55: 388-92 . 33 . Barrow PA, Lovell MA . The association between a large molecular mass plasmid and virulence in a strain of Salmonella pullorum . J Gen Microbiol 1988 ; 134 : 2307-16 . 34 . Kawahara K, Haraguchi Y, Tsuchimoto M, Terakado N, Danbara H . Evidence of correlation between
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50-kilobase plasmid of Salmonella choleraesuis and its virulence . Microbial Pathogenesis 1988 ; 4 : 155--63 . 35 . Beninger PR, Chikami G, Tanabe K, Roudier C, Fierer J, Guiney DG . Physical and genetic mapping of the Salmonella dublin virulence plasmid pSDL2 . Relationship to plasmids from other Salmonella strains . J Clin Invest 1988 ; 81 : 1341-7 . 36 . Williamson CM, Pullinger GD, Lax AJ . Identification of an essential virulence region on Salmonella plasmids . Microbial Pathogenesis 1988 ; 5 : 469-73 . 37 . Barrow PA, Lovell MA. Functional homology of virulence plasmids in Salmonella gallinarum, S . pullorum and S. typhimurium . Infect Immun 1989 ; 57 : 3136-41, 38 . Popoff MY, Miras I, Coynault C, Lasselin C, Pardon P . Molecular relationships between virulence plasmids of Salmonella serotypes typhimurium and dublin and large plasmids of other Salmonella serotypes . Ann Microbiol (Paris) 1984 ; 135 : 389-98 . 39 . Baird GD, Manning EJ, Jones PW. Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium . J Gen Microbiol 1985; 131 : 1815-23 . 40 . Michiels T, Popoff MY, Durviaux S, Coynault C, Cornelis G . A new method for the physical and genetic mapping of large plasmids : application to the localisation of the virulence determinants on the 90 kb plasmid of Salmonella typhimurium . Microbial Pathogenesis 1987 ; 3 : 109--116 . 41, Poppe C, Curtiss R III, Gulig PA, Gyles CL . Hybridization studies with a DNA probe derived from the virulence region of the 60 Mdal plasmid of Salmonella typhimurium . Can J Microbiol (in press) . 42 . Gulig PA, Curtiss R Ill . Cloning and transposon insertion mutagenesis of virulence genes of the 100kilobase plasmid of Salmonella typhimurium . Infect Immun 1988 ; 56 : 3262-71 . 43 . Rhen M, Virtanen M, Makela PH . Localization by insertion mutagenesis of a virulence-associated region on the Salmonella typhimurium 96 kilobase pair plasmid . Microbial Pathogenesis 1989 ; 6 : 1538. 44 . Norel F, Coynault C, Miras I, Hermant D, Popoff MY . Cloning and expression of plasmid DNA sequences involved in Salmonella serotype typhimurium virulence . Mol Microbiol 1989; 3 : 733-43 . 45 . Maurelli AT, Blackmon B, Curtiss R Ill . Temperature-dependent expression of virulence genes in Shigella species . Infect Immun 1984 ; 43 : 195-201 . 46 . Goguen JD, Yother J, Straley SC . Genetic analysis of the low calcium response in Yersinia pestis mu dl (Ap lac) insertion mutants . J Bacteriol 1984 ; 160 : 842 8 .
Mini-review Virulence plasmids of Salmonella typhimurium and other salmonellae Paul A . Gulig Department of Immunology and Medical Microbiology, University of Florida College of Medicine, Box J-266, Gainesville, FL 32610, U .S.A .
Introduction In the 1970s several groups identified a large plasmid in strains of Salmonella typhimurium ;' -7 however, with few exceptions, no phenotype could be ascribed to the plasmid . Hence, the plasmid was called a 'cryptic plasmid' . The cryptic plasmid was able to suppress the fertility of the F plasmid,` and the plasmid could integrate into the chromosome of dnaA mutants of S. typhimurium to drive chromosomal replication .' In 1982 Jones et al.' described the large 'cryptic' plasmid of S . typhimurium as being associated with virulence . Since then, considerable work has been published concerning the pathogenesis associated with and genetics of the high molecular weight virulence plasmids of S. typhimurium and other serotypes and species of Salmonella . Although a consensus exists as to the genetic loci involved with plasmidmediated virulence of salmonellae, much is yet to be learned about how the virulence plasmids contribute to pathogenicity . This review examines these two subjects concerning the S. typhimurium virulence plasmid in addition to correlating results with those obtained with other salmonellae including S . dublin, S. enteritidis, S . choleraesuis, S . gallinarum and S . pullorum .
Pathogenesis associated with the virulence plasmid Jones et al.' initially described the virulence phenotype conferred on S . typhimurium by the cryptic plasmid . In studies including infection of mice and cultured animal cells, they concluded that the virulence plasmid was involved in the mannose-resistant adherence to and invasion of mammalian cells by salmonellae . Jones and co-workers also reported that the virulence plasmid could integrate into the salmonella chromosome resulting in lack of expression of the virulence phenotype . The plasmid could later excise and again confer virulence . Subsequent investigations by other laboratories confirmed the role of the plasmid in virulence, but failed to identify a relationship between the virulence plasmid and interactions of S . typhimurium with cultured animal cells ."' As discussed below, the adherence-invasion deficient phenotype is the result of the integration of the plasmid into the chromosome in the strain of Jones et al.' and not loss of the plasmid by curing . This integration is an artifact of the use of Tn 10 to label the plasmid with tetracycline resistance . Others have since observed an integration event mediated by Tn 10 but not by other transposons ." The S . typhimurium virulence plasmid is primarily responsible for spreading infection 0882-4010/90/010003+09 $03 .00/0
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1990 Academic Press Limited
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P . A . Gulig
beyond the intestines . Hackett et a/. 9 determined that virulence plasmid-cured S . typhimurium was proficient at infecting the Peyer's patches of orally inoculated mice . Pardon et al." and Gulig and Curtiss 10 subsequently identified the principal virulence attribute of the plasmid as the spreading of infection beyond the small intestine to deeper tissues such as the mesenteric lymph nodes or spleen since plasmid-cured derivatives were attenuated in infection of these sites . Gulig and Curtiss 10 reported that parenterally administered cured S . typhimurium, although detectably attenuated, retained considerable virulence as measured by growth within spleens and LD 50 . These results suggested that the plasmid primarily affects early infection between the intestines and mesenteric lymph nodes, as opposed to later events such as growth within the reticuloendothelial system . In contrast, Pardon et a/. 12 observed greater attenuation of plasmid-cured S . typhimurium after parenteral inoculation . The exact mechanism by which the virulence plasmid mediates the spreading of infection beyond the intestines to deeper tissues is not known . Interestingly, Hoertt et al."' reported evidence suggesting that the S . typhimurium virulence plasmid may encode factors mediating splenomegaly and non-specific immunosuppression . Until recently, a controversial issue has been the role of the virulence plasmid in resistance of S . typhimurium to complement-mediated bacteriolysis of normal serum . Helmuth et al." originally reported a correlation of the presence of high molecular weight plasmids among isolates of S . typhimurium and serum resistance in unrelated strains . Gulig and Curtiss 10 found no role for the virulence plasmid in serum resistance by constructing plasmid-cured derivatives of three different strains of S . typhimurium, all of which retained serum resistance . Hackett et a/. 9 ' 14 constructed a plasmid-cured S. typhimurium derivative that was serum sensitive, suggesting that the virulence plasmid was necessary for serum resistance . Hackett et al. also cloned from the S . typhimurium virulence plasmid a gene encoding an 11 000 molecular weight (11 k) protein which conferred increased, but not complete, serum resistance to E. co/i K-12 and their plasmid-cured S . typhimurium, J42 . 14 However, during construction, the plasmid-cured S . typhimurium strain also acquired a rough lipopolysaccharide phenotype . This result was not reproduced by others in the -construction of isogenic sets of wild-type and plasmid-cured strains ."" Because the virulence plasmid was not reintroduced into cured S . typhimurium J42 to demonstrate restoration of wildtype virulence and lipopolysaccharide phenotypes, 14 it is possible that the rough lipopolysaccharide and serum sensitivity of strain J42 were caused by a secondary mutation selected during curing . In this regard, Gulig and Curtiss (unpublished results) isolated at least one plasmid-cured S . typhimurium derivative that did not regain virulence upon re-introduction of the virulence plasmid, indicating that unidentified secondary mutations had occurred . Since complete, smooth lipopolysaccharide is essential for serum resistance of S. typhimurium, the serum sensitivity of J42 of Hackett et a/. 14 could have been caused by rough Iipopolysaccharide . The exact role of the 11 k protein in serum resistance has not been defined in wild-type S . typhimurium . A traT-like gene is present on the S . typhimurium virulence plasmid ."" This gene is functionally homologous to the traT gene of the F and other plasmids in terms of conferring limited serum resistance under specific conditions .""' For example, Rhen and Sukupolvi 16 observed increased serum resistance contributed by TraT in plasmidcured S . typhimurium with a rough lipopolysaccharide chemotype ; the relevance of these observations to smooth salmonellae is unclear since others have found that smooth, virulence plasmid-cured S . typhimurium is serum resistant .' 0,15 It is important to note that the TraT protein from S. typhimurium has a molecular weight of 27 k, 16 and that the restriction map of the loci encoding the 11 k protein 14 and TraT are different . Therefore, the 11 k protein is probably not related to TraT . It is possible that
Salmonella virulence plasmids
5
the 1 1 k and TraT proteins play secondary roles in serum resistance of S . typhimurium, but the serum resistance of plasmid-cured S . typhimurium precludes an essential role . VandenBosch et al. determined that S . typhimurium containing a TOO-mediated, chromosomally integrated virulence plasmid was serum sensitive 20 and hypothesized that the plasmid was therefore involved with serum resistance . VandenBosch et al. 21 subsequently cloned from the virulence plasmid a locus termed rsk (regulation of serum killing) which restored serum resistance to plasmid-integrated salmonellae . However, VandenBosch et al. 15 observed that S. typhimurium completely lacking the virulence plasmid, as examined by Gulig and Curtiss, 70 is serum resistant and proficient in infection of cultured animal cells . Furthermore, cloned rsk exerts no effects on virulence of plasmid-cured S . typhimurium ." The cloned rsk gene therefore appears to be involved in regulation of several chromosomally encoded virulence functions that, in an undetermined manner, are affected by the integration of the virulence plasmid into the salmonella chromosome . VandenBosch et al. suggest that rsk could bind and titrate a regulatory factor ." In summary, the virulence plasmid is not necessary for S. typhimurium to be resistant to killing by complement of normal serum . However, the plasmid does encode factors such as the 11 k and TraT proteins and rsk, which may have minor or indirect roles in serum resistance . Several groups have hypothesized that the virulence plasmid affects the resistance to killing by macrophages or bacterial growth within these host cells . However, direct proof of these hypotheses is lacking . S. typhimurium probably first encounters macrophages at the Peyer's patches and subsequently at the mesenteric lymph nodes and spleen ." S . typhimurium is considered to be a facultative intracellular pathogen and is able to at least survive within murine macrophages . 23 Contrary to the macrophage resistance hypotheses, Gulig and Curtiss 10 examined salmonella-macrophage interactions under a variety of conditions in vitro and in vivo and did not detect differences in phagocytosis and killing of wild-type and plasmid-cured S. typhimurium . For example, after intraperitoneal injection of mice with wild-type and cured salmonellae, equal numbers of each strain were associated with macrophages at 1 h post-injection (i .e . the plasmid did not affect phagocytosis) . Furthermore, equal numbers of each strain survived within peritoneal macrophages, as determined by culturing infected host cells from lavages in the presence of gentamicin for 2 h . To determine whether macrophages were responsible for preventing spreading infection by plasmid-cured S. typhimurium (i .e . the plasmid encoded macrophage-resistance), mice were treated with intravenous silica to deplete functional macrophages . 24 In silica-treated mice, wild-type S . typhimurium remained 100-fold more virulent than a plasmid-cured derivative in terms of spreading to the spleen after oral inoculation (Gulig and Curtiss, unpublished results) . Therefore, a host factor(s) other than macrophages was responsible for preventing splenic infection by cured salmonellae . These data must be considered with the contrasting results of Pardon et al." who observed decreased splenic infection by plasmid-cured S. typhimurium in intravenously inoculated mice . In summary, the virulence plasmid does not seem to directly affect a salmonellaemacrophage interaction at the level of phagocytosis or killing by macrophages . Many different groups have determined that related high molecular weight plasmids are involved with virulence of other serotypes of Salmonella . In an epidemiological investigation of diverse Salmonella isolates, Helmuth et al." identified high molecular weight plasmids in strains of S . typhimurium, S . enteritidis, S. dublin and S. choleraesuis . Terakado et al. 25 initially correlated the loss of a 75 kb plasmid of S . with a decrease in virulence in terms of intraperitoneal LD 50 . The requisite
dub/in
experiment to confirm this plasmid-pathogenesis relationship was performed by Chikami et a/. 26 by reintroducing a transposon-labeled virulence plasmid back into a
6
P . A. Gulig
plasmid-cured S. dub/in derivative and demonstrating that virulence was regained . Chikami and co-workers observed attenuation of plasmid-cured S . dublin by the oral and intraperitoneal routes of inoculation of mice . A subsequent study from the same laboratory 27 delineated the pathogenesis mediated by the S . dub/in plasmid with results very similar to those outlined above for S . typhimurium, 1 " 2 i .e ., the plasmid was necessary for spreading infection beyond the intestines of mice . Further work associating the S . dub/in plasmid with virulence was reported by Manning et al." using transposon mutagenesis and plasmid curing . Nakamura et al. associated loss of a 54 kb plasmid from S . enteritidis with attenuation in terms of the subcutaneous LD50 in mice29 and determined that virulence plasmid-cured strains could immunize mice against salmonella infection ." Hovi et al." subsequently reintroduced the S . enteritidis virulence plasmid into a cured derivative and restored virulence . The S. enteritidis virulence plasmid did not affect lipopolysaccharide or serum resistance . 31 Barrow and co-workers have identified virulence plasmids for S . ga/linarum 32 and S . pullorum. 33 Plasmid-cured S . gallinarum and S . pullorum retained serum resistance but were attenuated for multiplication within the reticuloendothelial system of chicks . A 50 kb plasmid is necessary for virulence of S , cho/eraesuis in mice inoculated intraperitoneally ." Several groups have demonstrated the functional interchangeability of plasmids from different species and serotypes of Salmonella.' 1,31,35-37 In summary, the virulence plasmid appears to be necessary for S . typhimurium to invade beyond the intestines after oral inoculation of mice, but the mechanism for this phenomenon is not known at either the cellular or molecular levels . The virulence plasmid does not directly affect virulence traits such as cellular adherence-invasion, serum-resistance and phagocytosis and killing by macrophages . Virulence plasmids of other species and serotypes of Salmonella appear to be similarly involved in spreading infection beyond the intestines . Perhaps the virulence plasmid affects the hostpathogen interaction at a more complicated level such as regulation of the host response to infection with S . typhimurium . In dissecting the pathobiology of why wild-type salmonellae are isolated in higher numbers than are plasmid-cured salmonellae from either mesenteric lymph nodes or spleens of orally inoculated mice, three general explanations are put forward . The virulence plasmid may affect the rates of (1) growth, (2) death, and/or (3) movement through tissues for salmonellae . These rates do not indicate specific mechanisms in themselves, but must be able to account for differences in numbers of bacteria recovered . For example, if the virulence plasmid encoded factors mediating immunosuppression in mice, the rates of growth and death might be affected . Genetics of the virulence plasmid Soon after the discovery that a virulence plasmid existed in S . typhimurium, 8 plasmids with DNA homology were found in other species and serotypes of Salmonella . Popoff et a/. 38 determined that large plasmids of S . typhimurium, S. enteritidis, S . dublin, S . paratyphi C, S. newport, and S . abortusovis were related at the level of DNA homology and restriction fragment mapping . Figure 1 summarizes the physical and genetic maps of the S. typhimurium virulence plasmid with detail of the virulence region . Baird et al." initially identified a locus of the S . typhimurium and S. dublin plasmids associated with virulence through the use of Tn 10 and TnA insertion mutations which attenuated virulence (Fig . 1, map B) . Through restriction mapping of this region they estimated that between 13 and 23 kb of homologous sequence could be involved in virulence . The homology between S . typhimurium and S . dublin extends to the thick vertical bar at the right hand ends of maps B and D . Therefore, mutations which had been
7
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identified to the right in the non-homologous region of the S . dub/in virulence plasmid are not shown . By using large scale deletion mutagenesis of the S . typhimurium virulence plasmid, Michiels et a/. 40 constructed a restriction map of the entire plasmid and identified a region of no more than 38 kb involved with virulence . The right portion of the 38 kb region is contained in Fig . 1, map C . The virulence region of Michiels et a/. 40 was very similar to that of Baird et a/. 39 Studies of Beninger et a/. 35 on the S. dub/in virulence plasmid further localized a virulence region to a 4 kb EcoRl fragment within the aforementioned virulence regions [Fig . 1, map D (=)] . Poppe et a/. 41 have recently used a subclone consisting of a 3 .75 kb Hindlll fragment from cloned S . typhimurium virulence genes of Gulig and Curtiss 42 (see below) and found homology with plasmids of five other serotypes . Rhen et al." examined Tn5 insertions within the consensus virulence region and identified two loci containing virulence genes (Fig . 1, map E) . In contrast to the studies discussed above which relied on either transposon insertion or deletion mutagenesis to identify virulence genes of the salmonella plasmids, Gulig and Curtiss 42 identified a region necessary for virulence through cloning . Libraries of cloned virulence plasmid sequences were placed into plasmid-cured S . typhimurium, and virulence-conferring clones were selected in mice by allowing the cured salmonellae containing cloned virulence genes to infect the spleens of orally inoculated mice . Three cosmid clones with insert sequences of approximately 23 kb, each of which conferred a wild-type level of virulence, were isolated . Restriction analysis indicated that the insert sequences shared a common 14 kb region . The 14 kb region [Fig . 1, map A (=)], which coincided with the aforementioned virulence regions identified by others 31,31,4' encoded at least three proteins with molecular weights of 28 k, 29 k, and 32 k . The cloned sequence of plasmid pYA403, which conferred complete virulence on plasmid-cured S . typhimurium in terms of LD 50 and infection of spleens after oral inoculation of mice, 42 is shown as the detailed region around virulence loci in Fig . 1 (map A) . A 3 .2 kb BamHI fragment alone conferred substantial virulence to plasmid-cured S . typhimurium and encoded the 28 k protein [Fig . 1, map A ( L I ] . A particular Tn5 insertion, vir-22 : :Tn5, essentially eliminated plasmid mediated virulence of S . typhimurium and inhibited production of the 28 k protein (Fig . 1, map A) . Gulig and Chiodo have since confirmed that the 28 k protein is a major virulence factor encoded by the S . typhimurium plasmid and have determined the DNA sequence of the gene encoding the 28 k protein, virA (Abstracts Annual Meeting Amer . Soc . Microbiol ., 1989, p . 66) . Deletion of the right half of the 14 kb virulence region 42 (Fig . 1, lane A) beyond the Xhol site from the S . typhimurium virulence plasmid did not affect virulence as determined by splenic infection after oral inoculation of mice (Gulig, unpublished results), thus narrowing the virulence region to approximately 8 kb . Williamson et a1. identified 11 and recently cloned 36 an 8 kb region of the S. dub/in virulence plasmid [Fig . 1 (G)], which appeared to confer virulence to S. typhimurium and S . dub/in . However, the virulence assay consisted of noting the sick appearance of mice . Since quantitative analysis of virulence by using either splenic infection or LD 50 was not carried out, the extent of the virulence conferred by the cloned sequence is not known . This region is similar to the shortened virulence region of Gulig (unpublished results) mentioned above . An interesting finding of Williamson et a/. 36 was that the copy number of the vector used in cloning affected the ability of cloned sequences to confer virulence, with low copy vectors functioning better than high copy vectors . Most recently, Norel et a/. 44 cloned a 22 kb fragment of the S . typhimurium virulence plasmid which conferred full virulence to plasmid-cured S . typhimurium and encoded
Salmonella virulence plasmids
9
at least 10 proteins . By using extensive Tn5 mutagenesis of cloned sequences, they identified two virulence loci termed virA and virB. virA corresponds to the 8 kb Sallregion [Fig . 1, map F (=)] of Williamson et al. 1136 and encodes the 28 k protein identified by Gulig and Curtiss 42 in addition to several other proteins . virB maps to the repA region identified by Michiels et al.,40 which also encodes incompatibility (inc) and partitioning (par) functions (Tinge and Curtiss, Abstracts Annual Meeting Amer . Soc . Microbiol ., 1989, p . 216) . virB : :Tn5 insertions 14, 65
Xhol
and 10 of Norel et al." mapped in a locus encoding a 43 k protein, and virB : :Tn5 insertions 125 and 178 inhibited production of a 38 k protein . Tinge and Curtiss identified products of the parA and pars genes as 44 k and 38 k proteins, respectively (Abstracts Annual Meeting Amer . Soc . Microbiol ., 1989, p . 216) . virB mutations may therefore indirectly diminish virulence by affecting plasmid stability or copy number . This possibility is supported by the fact that pYA403 of Gulig and Curtiss, 42 which conferred full virulence on plasmid-cured S. typhimurium, does not encode the repA ('virB') locus (Fig . 1, map A) . Most, if not all, virulence plasmid-encoded proteins are not detectably expressed by S . typhimurium grown in vitro (Gulig and Curtiss, unpublished results) . Therefore, either minute quantities of the plasmid-encoded virulence factors are sufficient for virulence, or virulence genes are expressed only with the appropriate stimuli in vivo in the infected host . The virulence plasmids of other pathogenic bacteria such as Shigella spp ." and Yersinia spp . 46 each possess unique mechanisms of regulation of plasmidencoded virulence genes . The results discussed above of VandenBosch and coworkers studying rsk 1521 indicate that regulatory systems may be present on the salmonella virulence plasmids .
Summary Related high molecular weight plasmids of several serotypes and species of Salmonella have been associated with virulence in a variety of animal models of infection . The primary virulence plasmid phenotype is in the ability of salmonellae to spread beyond the initial site of infection, the intestines . The mechanism of this plasmid-mediated invasive infection has not been identified, but may be a complex interaction in the host-pathogen relationship . A common region of the salmonella plasmids has been associated with virulence, and specific virulence genes and their products are now being identified ; however, much is yet to be accomplished in this field . The combined analysis of pathogenesis and genetics associated with the salmonella virulence plasmids may identify new systems of bacterial virulence and the genetic basis for this virulence . I thank Dr Roy Curtiss III, Dr Richard Lottenberg, Hank Lockman and Steve Tinge for their critical review of this manuscript .
References 1 . 2. 3. 4. 5.
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6 . Spratt BG, Rowbury RJ, Meynell GG . The plasmid of Salmonella typhimurium LT2 . Mol Gen Genet 1973 ; 121 : 347-53 . 7 . Bagdasarian M, Hryniewicz M, Zdzienicka M . Integrative suppression of a dnaA mutation in Salmonella typhimurium . Mol Gen Genet 1975; 139 : 213-31 . 8 . Jones GW, Rabert DK, Svinarich DM, Whitfield HJ . Association of adhesive, invasive, and virulent phenotypes of Salmonella typhimurium with autonomous 60-megadalton plasmids . Infect Immun 1982 ;38 :476-86 . 9 . Hackett J, Kotlarski I, Mathan V, Francki K, Rowley D . The colonization of Peyer's patches by a strain of Salmonella typhimurium cured of the cryptic plasmid . J Infect Dis 1986 ; 153 : 1119-25 . 10 . Gulig PA, Curtiss R III . Plasmid-associated virulence of Salmonella typhimurium . Infect Immun 1987 ; 55 :2891-901 . 11 . Williamson CM, Baird GD, Manning EJ . A common virulence region on plasmids from eleven serotypes of Salmonella . J Gen Microbial 1988 ; 134 : 975-82 . 12 . Pardon P, Popoff MY, Coynault C, Marly J, Miras I . Virulence-associated plasmids of Salmonella serotype Typhimurium in experimental murine infection . Ann Inst Pasteur Microbiol 1986 ; 137 : 4760 . 12a . Hoertt BE, Ou J, Kopecko DJ, Baron LS, Warren RL . Novel virulence properties of the Salmonella typhimurium virulence-associated plasmid : immune suppression and stimulation of splenomegaly . Plasmid 1989 ; 21 : 48-58 . 13 . Helmuth R, Stephan R, Bunge C, Hoog B, Steinbeck A, Bulling E . Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes . Infect Immun 1985 ; 48 :175-82 . 14 . Hackett J, Wyk P, Reeves P, Mathan V . Mediation of serum resistance in Salmonella typhimurium by an 11 -kilodalton polypeptide encoded by the cryptic plasmid . J Infect Dis 1987 ; 155 : 540-9 . 15 . VandenBosch JL, Kurlandsky DR, Urdangaray R, Jones GW . Evidence of coordinate regulation of virulence in Salmonella typhimurium involving the rsk element of the 95-kilobase plasmid . Infect Immun 1989; 57 :2566-8 . 16 . Rhen M, Sukupolvi S . The role of the traT gene of the Salmonella typhimurium virulence plasmid for serum resistance and growth within liver macrophages. Microbial Pathogenesis 1988 ; 5 : 275-85 . 17 . Rhen M, O'Connor D, Sukupolvi S . The outer membrane permeability mutation of the virulenceassociated plasmid of Salmonella typhimurium is located in a traT-like gene . FEMS Microbiol Lett 1988;52 :145-54 . 18 . Sukupolvi S, O'Connor D, Makela PH . The effects of traT insertion mutations on detergent sensitivity and serum resistance of Escherichia coli and Salmonella typhimurium . FEMS Microbiol Lett 1987 ; 43 : 81--7 . 19 . Sukupolvi S, O'Connor D, Edwards MF . The TraT protein is able to normalize the phenotype of a plasmid-carried permeability mutation of Salmonella typhimurium . J Gen Microbiol 1986 ; 132 : 207985 . 20 . 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