Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo

Microbes and Infection, 3, 2001, 1345 1352 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457901014964/REV Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo Carmen R. Beuzón, David W. Holden* Department of Infectious Diseases, Imperial College School of Medicine, Flowers Building, Armstrong Road, London SW7 2AZ, UK ABSTRACT In the Salmonella mouse model of systemic infection, high dose inoculation results in the multiplication of many of the cells present in the inoculum, rather than the clonal amplification of a small number. This characteristic has allowed the development of methods to screen multiple strains for either virulence attenuation or gene expression within the same animal. Mixed infections with mutant and wild-type strains are used to provide a sensitive measure of virulence attenuation referred to as the competitive index. We have recently used a variation of this method, involving mixed infections of single and double mutant strains, to study virulence gene interaction in vivo. 2001 Éditions scientifiques et médicales Elsevier SAS competitive index / gene interactions / COI / STM / IVET 1. Introduction Salmonella typhimurium encounters a diversity of environments in the mouse throughout the course of systemic infection. After oral inoculation and survival in the stomach and intestine, it penetrates the gut epithelium by invading the M cells in the Peyer s patches of the ileum [1], and reaches the spleen and liver where it multiplies intracellularly [2, 3], eventually resulting in fatal bacteraemia. S. typhimurium pathogenesis is a complex, multifactorial process that results from the activity of many bacterial gene products: it has been estimated that at least 4% of its genome is involved in virulence [4]. Many of these genes are clustered together on pathogenicity islands (PAIs) which appear to have been acquired by horizontal transfer from unknown sources. SPI-1 and SPI-2 are two PAIs that encode structurally similar but functionally distinct type III secretion systems (TTSS) which translocate virulence proteins from bacterial to host cells during the infectious cycle [5]. SPI-1 plays an important role in invasion of epithelial cells [6, 7], whereas SPI-2 is required for bacterial replication within macrophages [8] and systemic growth in the mouse [9, 10]. In addition to these, numerous other genes have been shown to be necessary for S. typhimurium virulence [11]. Many of them are involved in intracellular survival and replication; these include the spv locus, present on a large plasmid [12], the ompr/envz genes, which encode a two-component regulatory system that *Correspondence and reprints. E-mail address: d.holden@ic.ac.uk (D.W. Holden). Microbes and Infection responds to changes in ph, osmolarity and temperature [13, 14], and the phop/phoq genes which encode another two-component regulatory system that controls the expression of more than 40 virulence genes [15]. Although the biochemical functions of some of these genes have been elucidated, little is known about their regulation in vivo and how they interact during the infectious process. The study of interactions between bacterial strains and cultured host cells has led to the identification of genes involved in specific aspects of virulence (e.g., invasion of epithelial cells [16]). However, the complexity of Salmonella virulence obviously limits the use of this type of analysis. In recent years there has been a shift towards more sophisticated in vivo virulence studies, and some of these rely on the use of mixed infections. The application of mixed infections in the analysis of the Salmonella mouse interaction will be the subject of this review, with particular emphasis on our recent application of the competitive index to the analysis of gene interactions in vivo. 2. Use of mixed infections in the analysis of Salmonella virulence Intraperitoneal (i.p.) inoculations of mice with a mixture of different Salmonella strains were first used more than 40 years ago to study fundamental aspects of the infection process. Meynell and Stocker [17] inoculated mice with a mixture of S. paratyphi-b strains carrying one of three different flagellar antigens, or a mixture of Xyl + Gal + and Xyl Gal S. typhimurium strains. All these strains have a similar growth rate in vivo. They demonstrated that 1345

Forum in Immunology Beuzón and Holden at high dose inocula, the ratio between these strains in the blood after infection was the same as their ratio in the inoculum. They concluded from these results that if the inoculum dose is sufficiently high, systemic S. typhimurium infection of mice results from the multiplication of many of the cells present in the inoculum, rather than the clonal expansion of a small number. Therefore, mice can support the growth of multiple Salmonella strains simultaneously, and at high dose inoculum the probability of any given bacterium to multiply in a mixed infection will be equivalent to its ability to multiply when infecting by itself. These findings have allowed the recent development of several techniques for the identification and characterisation of virulence functions of Salmonella and other pathogens in vivo. 2.1. High-throughput screening methods to identify Salmonella virulence factors in vivo based on the use of mixed infections Characterisation of virulence genes frequently involves analysis of levels of attenuation by infection studies with individual mutant strains [18]. Individual analysis of bacterial strains carrying reporter gene fusions has also been used to study gene expression in vivo [19]. The fact that the Salmonella mouse model can support mixed systemic infections where any given strain has the same probability to multiply as in single infection has made possible the simultaneous high-throughput analysis of either virulence attenuation or gene expression of multiple strains within the same animal. These new methods use the mouse as a selective environment: in vivo expression technology (IVET) is based on positive selection of in vivo-induced genes [20], whereas signature-tagged mutagenesis (STM) is based on in vivo negative selection of attenuated strains [9]. 2.1.1. IVET When a pathogen enters the host, altered patterns of gene expression enable it to respond to the challenges of the new environment. Therefore, a method to select promoters activated in vivo provides a means to identify genetic loci that could be important for bacterial virulence. The first IVET method consisted of the inoculation of mice with a pool of strains carrying chromosomal fusions to a promoterless reporter gene essential for growth within the host, whose expression could therefore be positively selected for in vivo [20]. Several versions of the IVET method have been used in different pathogen host systems, and the results and implications from these have been discussed in detail in other reviews [21 24]. 2.1.2. STM STM is a technique that labels mutants with different identifying DNA signature-tags [9]. Strains carrying tagged mutations can be identified on the basis of their tags, allowing them to be individually detected in mixed infection. PCR amplification of the tags from a pool of STM mutants is carried out using primers common to all of them, and the resulting products are used as hybridisation probes. Comparative hybridisation with probes obtained prior to inoculation and after bacterial recovery from the host enables attenuated mutants to be identified [9]. STM therefore takes advantage of the capability of the animal pathogen interaction model to support the simultaneous growth of multiple bacterial strains. To date, over 50 attenuated mutants have been identified by STM in S. typhimurium and hundreds of them in several other pathogens [25]. The technical aspects, results and limitations of STM in Salmonella and other pathogens have been discussed in detail previously [22, 25 29]. 3. Functional characterisation of virulence determinants The use of techniques such as IVET and STM has identified a large number of genes that are important for Salmonella virulence. In some cases, DNA sequence analysis has provided information on their specific functions. However, in other cases the information obtained by sequence analysis is not sufficient to suggest a function for the genes. In these cases, phenotypic characterisation of mutant strains is a useful way to shed light on gene function. In the case of Salmonella, a series of assays can be done to relate these genes to one or more aspects of Salmonella virulence, for example: resistance to serum complement and antimicrobial peptides, invasion of epithelial cells, and survival and replication within macrophages. In addition to these, more sophisticated in vivo studies can be done to establish the time and the body site where the virulence attenuation first becomes apparent. 3.1. Competitive index Historically, in vivo determination of the contribution of a given gene to virulence has relied on the use of the LD 50 test. The LD 50 is defined as the number of bacteria necessary to kill 50% of the infected animals. The LD 50 is a useful measure of virulence, but it has limitations: it is relatively crude and reflects only the cumulative effect of all the steps involved in causing lethality. A failure of a mutation to raise the LD 50 does not necessarily mean that the mutation did not affect an important virulence determinant, but could reflect a situation in which the effect was not sufficient to be detected by the assay. The competitive index (CI) is an alternative measure that uses mixed infections to determine the degree of virulence attenuation caused by a given mutation. Equivalent numbers of two strains (the wild-type and a mutant strain) are combined (input) and used to inoculate an animal host. Bacteria are recovered after an appropriate period of time from a target organ (output) and the numbers of each strain are enumerated by colony-forming units (CFU); strains are usually distinguished by different antibiotic resistance markers. The CI is defined as the ratio between the mutant strain and the wild type in the output divided by the ratio of the two strains in the input [30, 31]. The CI is a more sensitive measure of virulence attenuation than the LD 50 because it reflects bacterial numbers directly rather than survival of the host animals, and it can therefore distinguish between mutant strains whose attenuation is too subtle to be detected by LD 50. By testing the virulence levels of mutant versus wild-type strains within the same animal, animal to animal variation is decreased. 1346 Microbes and Infection

Salmonella virulence gene interactions in vivo Forum in Immunology Furthermore, the LD 50 is calculated from the number of animals surviving the infection whereas the CI is obtained from the numbers of bacteria before and after the infection, providing a figure that is based on a much larger sample size. Therefore, the CI constitutes a more accurate indication of virulence attenuation using fewer animals. An obvious theoretical disadvantage of CI is that the defect of some attenuated strains might be complemented in trans by the wild-type strain present in the same inoculum. The CI is also a versatile method where route of infection, dose, strain combination and ratio, time and site of recovery can be modified to provide a more detailed description of the contribution to virulence of a given gene. All these factors have made CI analysis an increasingly popular method to determine virulence attenuation [32 35]. 4. In vivo analysis of gene interactions in Salmonella by mixed infections An in vivo assay to establish links between a new virulence determinant and known virulence genes or regulatory circuits operating in the Salmonella infection process could be a useful source of information about its potential function. Two approaches have been described to study gene interactions in vivo, both relying on the additivity of phenotypic effects of mutations. Because Salmonella virulence is multifactorial, combinations of mutations in genes with different virulence functions should result in strains with increased attenuation. 4.1. Early uses of competitive index to study gene interactions Bäumler and collaborators [36] were the first to apply mixed infections to study Salmonella gene interactions in vivo. They wished to determine if the functions of S. typhimurium lpfc (encoding long polar fimbriae) and inva (encoding a structural component of the SPI-1 TTSS) in colonisation and penetration of the mouse gut epithelium were independent. They constructed an inva lpfc double mutant strain and compared it after oral mixed inoculation to the wild-type strain. If these gene functions are independent, the attenuation of an inva lpfc mutant strain is predicted to be similar to the combined attenuation of an inva mutant strain plus a lpfc mutant. They infected mice by the oral route, with mixed inocula of the wild-type strain and either the double or the single mutant strains. Bacteria were recovered from the spleen 5 days later and the bacterial CFU of each strain determined, distinguished by antibiotic resistance markers. The attenuation of the double mutant strain was greater than the predicted combined effect of the single mutations, suggesting that mutations resulting in the loss of function of these systems have a synergistic effect on the attenuation of S. typhimurium virulence. The simplest explanation for these results is therefore that S. typhimurium possesses two independent mechanisms to colonise and penetrate the gut epithelium and that the absence of one of them can be partially compensated by the action of the other. Microbes and Infection 4.2. In vivo cancelling out to analyse Salmonella gene interactions by mixed infections Bäumler and collaborators [36] showed that mixed infection with double mutant and wild-type strains could be a useful tool to study Salmonella gene interactions in vivo. However, determination of phenotypic additivity by comparison to the wild-type strain is difficult in cases where the level of attenuation caused by a single mutation is very high (C.R. Beuzón and D.W. Holden, unpublished results). To detect differences in attenuation between double and single mutant strains affected in systemic infection, we have developed a modification of the competitive index which involves co-infecting the same animal with single and double mutant strains [3, 37]. In a mixed infection with a strain carrying mutations in two genes and a strain carrying only one of these mutations, the attenuation caused by the shared mutation ought to be equivalent and therefore cancel out. The CI obtained after cancelling out the effect of one mutation represents the contribution that the other mutation has to the attenuation of the double mutant strain. For the sake of clarity we will refer to this modified competitive index as COI ( cancelled out competitive index). COI is defined as the ratio of the double mutant to single mutant strain in the output, divided by the ratio of the two strains in the input. Direct comparison of virulence of single and double mutant strains within the same animal gives a more reliable account of the functional relationship of the genes than comparisons carried out using results from different animals, because it eliminates the effects of animal to animal variation. Furthermore, the use of COI to analyse gene interactions in vivo provides a more relevant environment in which to assess the significance of gene function and interaction than in vitro models. 4.2.1. COI predictions If we consider that two virulence genes, a and b, contribute independently to Salmonella virulence, the presence or absence of a functional copy of gene a would not affect the way in which gene b would contribute to virulence. Therefore the COI(a versus a b ) would be similar to the corresponding CI(wt versus b ) (this situation is represented in figure 1). On the other hand, null mutations in two genes that contribute equally to the same specific function (e.g., by encoding different steps of the same biosynthetic pathway or components of a macromolecular structure) would cause a similar attenuation of virulence singly or combined. Therefore, the double mutant strain would be no more attenuated than the single mutants. In this case, the CIs of the single mutant strains would be lower than 1.0 and similar to each other, whereas the corresponding COIs would be similar to 1.0 (figure 2). The analysis of two gene products not involved in the same specific function, but linked because the action of one is completely dependent on the action of the other would potentially give similar results. Hence, any analysis resulting in COIs of approximately 1.0 requires further study before it can be concluded that the genes are involved in the same function. 1347

Forum in Immunology Beuzón and Holden Figure 1. COI determination and theoretical predictions in the case of two genes contributing independently to virulence. This figure has been adapted from Beuzon et al., Infect. Immun. 69 (2001) 7254 7261 with permission from the American Society of Microbiology. Figure 2. COI determination and theoretical predictions in the case of two genes involved in the same specific function.this figure has been adapted from Beuzon et al., Infect. Immun. 69 (2001) 7254 7261 with permission from the American Society of Microbiology. There are clearly other possibilities apart from those discussed above. For example, gene products that are components of regulatory circuits can partially overlap in some of the functions they regulate, or contribute to different degrees to the same function. In these cases, the predicted COIs would not correspond to those shown in figures 1 and 2. In the case of genes involved in alternative but independent processes, where the loss of one of the processes is compensated by the action of the other (such as lpfc and inva), COIs would be greater than the corresponding CI. 4.3. Analysis of gene interaction in systemic infection by COI To determine if COI could be used to indicate phenotypic additivity of mutations affecting independent functions involved in systemic infection, i.p. mixed infections of mice were carried out using single and double mutant strains affected in purd (a gene encoding a component of the purine biosynthetic pathway [38]) and ssav (a gene encoding a structural component of the SPI-2 secretion [39], necessary for secretion [40]). The COI(ssaV versus purd ssav ) in the spleen, 48 h after inoculation was not statistically different from the CI(purD versus wild-type) (table I). Similarly, the reciprocal COI(purD versus purd ssav) was not statistically different from CI(ssaV versus wild-type). These results indicate that a mutation in purd causes the same attenuation to a strain independently of whether that strain has a functional SPI-2 TTSS or not, and vice versa; in other words, the functions carried out by their gene products contribute independently to Salmonella virulence [3, 37]. Strains carrying mutations in either ssav or sseb (a gene predicted to encode a component of the SPI-2 translocon) [32, 40], were similarly inoculated i.p. in mixed infections with a strain carrying mutations in both genes. The COIs obtained were similar to 1.0 (table I), as predicted from the knowledge that their gene products contribute to the same Salmonella virulence function [32, 39, 41]. Similar results were obtained from the analysis of ssav and ssec, another gene from SPI-2 (table I) [3]. 4.3.1. Independent functions In the original work by Shea et al. [3], the COI was used to analyse the possibility of a functional relationship between the spv operon and SPI-2 TTSS. The spv and SPI-2 genes have been shown to be important for systemic infection, and mutations in both systems cause a similar 1348 Microbes and Infection

Salmonella virulence gene interactions in vivo Forum in Immunology Table I. COI analysis of S. typhimurium mutants. Mixed infection CI or COI Controls wt v ssav 0.006 wt v purd 0.0005 ssav v ssav, purd 0.0038 b purd v ssav, purd 0.019 b wt v sseb 0.016 ssav v ssav, sseb 1.2 a sseb v ssav, sseb 0.82 a wt v ssec 0.013 ssec v ssav, ssec 1.28 a spv wt v spva 0.068 ssav v ssav, spva 0.052 b ompr/envz wt v ompr 0.008 ssav v ssav,ompr 0.823 a ompr v ssav,ompr 0.21 a sifa wt v sifa 0.012 ssav v ssav, sifa 1.34 a sifa v ssav, sifa 1.17 a a Not significantly different to 1.0 (P < 0.05). b Not significantly different to the CI of the corresponding single mutant strain versus wild type strain. This table has been adapted from Beuzón et al.[37] by permission of Oxford University Press. level of attenuation. The spv genes are located on the virulence plasmid [12] and are necessary for intracellular replication in mice [42]. Although it has been recently shown that SpvB has ADP-ribosyltransferase activity [43], its target, and the overall virulence function of the spv locus remains to be determined. The CI of an ssav mutant strain versus the wild-type strain was not significantly different from its corresponding COI(spvA versus spva ssav ) indicating that these genes contribute independently to Salmonella virulence. Microbes and Infection 4.3.2. Related functions COI analysis has also been carried out to determine a functional link between the SPI-2 TTSS, OmpR/EnvZ and SifA [37]. Strains carrying mutations in ompr/envz are highly attenuated in systemic infection of mice, but mutations in genes originally known to be regulated by OmpR could not account entirely for its virulence defect [44, 45]. It has been shown recently that transcription of ssra, a gene encoding a component of SsrA/SsrB, the SPI-2 twocomponent regulatory system [46 48], is modulated by OmpR in infected tissue cultured cells [49]. It was therefore possible than the attenuation of ompr mutants was attributable to an in vivo role in regulation of SPI-2 expression. To analyse this possibility, strains carrying mutations in either ssav or ompr were combined in mixed infections with a strain carrying both mutations. The COIs for the double mutant strain were not significantly different from 1.0, suggesting that these genes are involved in the same virulence function, which provides an explanation for the attenuation of an ompr mutant strain. SifA is encoded on a PAI located within the potabcd operon on the Salmonella chromosome [41]. Strains carrying mutations in sifa, ompr or envz are all defective for the formation of tubular membranous structures known as Sifs, which are normally induced in epithelial cells by wild-type bacteria [41, 50, 51]. sifa mutant strains are attenuated in virulence following oral or i.p. inoculation of mice [37, 41]. The COIs for a strain carrying mutations in sifa and ssav were similar to 1.0 (table I). These results suggested that these genes are involved in the same function in S. typhimurium virulence [37]. Further analysis was carried out to determine the nature of this potential interaction. The expression of sifa was found to be strictly regulated by SsrA/SsrB. A sifa mutant strain has an intracellular replication defect in macrophages similar to that of some SPI-2 mutants [37]. Analysis of the N-terminal region of the predicted sequence of SifA showed similarities to proteins that are secreted by Salmonella TTSSs. This sequence has been proposed to be a secretion and translocation signal [52]. All these results suggest that SifA could be secreted by the SPI-2 TTSS. The failure of strains carrying mutations in SPI-2 TTSS genes to induce Sif formation in epithelial cells supports this possibility [37]. Furthermore, the failure of a sifa mutant strain to induce Sif formation can be complemented in epithelial cells transfected with a plasmid expressing SifA, suggesting that the site of action of SifA is in the host cell cytosol or on the cytosolic face of the vacuolar membrane [37]. SifA was also found to be necessary to maintain the membrane of the Salmonella-containing vacuole, since sifa mutant bacteria lose their vacuolar membrane and are released into the cytosol of the host cell by 10 h after infection [37]. These results confirmed that, as indicated by COI analysis, sifa and SPI-2 are involved in the same virulence function. 4.3.3. Compensatory functions COI has been recently used in our laboratory to study the functional relationship between two different iron uptake systems (ABC transporters) of Streptococcus pneumoniae in vivo (Brown et al., Mol. Microbiol. 40 (2001) 572 585). Mixed infections of mice with strains carrying null mutations in genes encoding either one or both transporters were carried out by two different routes of inoculation and recovery: intranasal inoculation recovering bacteria from lungs and spleen, and i.p. inoculation recovering bacteria from spleen. The results are consistent in showing that these two transport systems act independently to provide the iron necessary for bacterial growth in vivo. The COIs for these systems were significantly lower than the corresponding CIs. Therefore, the loss of both systems reduces dramatically the ability of the organism to survive and replicate in vivo, whereas the loss of function of one system can be partially compensated for by the other. 4.4. Conclusions From its original use to study the aetiology of Salmonella systemic infection [17], to its more recent applications in high-throughput screening methods [9, 20] or in vivo analysis of gene interactions [3, 36, 37], mixed infections have proved a valuable tool in advancing our understanding of Salmonella virulence. The length, dose and route of infection are important factors to consider when using mixed infections to obtain 1349

Forum in Immunology representative recovery of inoculated strains. For example, with respect to the route of inoculation, M cells of the Peyer s patches are considered a bottleneck that restricts the number of bacteria that can translocate across the gut epithelium (discussed in [22]). Nevertheless, it has been shown that up to 30 different S. typhimurium strains are capable of reaching the spleen after oral inoculation [53]. To obtain representative numbers of virulent strains in the output from a mixed infection, the mice have to be inoculated with a relatively high dose of bacteria [17]. High dose inoculation raises the potential problems that some attenuated strains might be complemented in trans by the wild-type strain in the same animal, or might benefit from immune breakdown caused by such a high inoculum. In either case, this could lead to attenuated strains being mistaken as virulent. However, it is interesting to note that the frequency with which avirulent mutants of S. typhimurium were detected by STM [9] is very similar to the frequency obtained by Bowe et al. [4] when screening individual mutant strains at a lower dose. When standard CI analysis is used to address the attenuation of mutant strains, the CI value must be determined with accuracy to be used as a reference for the virulence attenuation caused by that mutation. The number of animals used needs to be sufficiently high to provide a low standard deviation for the CI value. On the other hand, COI values only need to be accurate enough to be assigned to one of three categories: similar to 1.0, similar to the corresponding CI, or lower than the corresponding CI. Once the COI has been deemed to fall into one of these categories, the actual numerical value of the index is irrelevant. This approach therefore reduces the number of animals needed in each analysis. For the COI studies described here, in most cases three mice were sufficient to obtain a meaningful interpretation. The recent application of COI analysis to study virulence gene interaction in S. pneumoniae (Brown et al., Mol. Microbiol. 40 (2001) 572 585) shows that this method can be applied to other host pathogen interactions. In the case of Salmonella, the diversity of serovars and animal models of infection provides a fertile ground for the application of this method, and could potentially allow a rapid assessment of gene interactions which might differ between the specific host serovar combinations, and thereby help in studies of host specificity. Acknowledgments We would like to express our gratitude to Herb Arst for help in making COI predictions, and for critical review. We thank members of our laboratory for helpful discussion. Work from our laboratory described in this review was supported by the Medical Research Council and Wellcome Trust (UK). 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