Characterization of the muts-proximal region of the Salmonella typhimurium SPI-1 identi es a group of pathogenicity island-associated genes

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1 FEMS Microbiology Letters 197 (2001) 203^208 Characterization of the muts-proximal region of the Salmonella typhimurium SPI-1 identi es a group of pathogenicity island-associated genes Alessandra Pancetti, Jorge E. Galän * Section of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale School of Medicine, New Haven, CT , USA Received 29 January 2001; accepted 16 February 2001 Abstract The virulence properties of Salmonella enterica are largely encoded within a set of horizontally acquired gene blocks termed pathogenicity islands. One such pathogenicity island, SPI-1, located at centisome 63 of the Salmonella chromosome between the muts and fhla genes, encodes a type III protein secretion system and an iron uptake system. We have characterized the muts-proximal border of this pathogenicity island and have identified two sets of genes, pigab and pigcd. All four genes have homologs of unknown function in several bacteria that share the ability to establish an intimate association with higher eukaryotic hosts. The expression of at least two of these genes, piga and pigb, is controlled by SprA, a transcription factor encoded within SPI-1 that controls the expression of genes associated with the type III secretion system of this island. In addition, we found that homologs of the pig genes are also found at different locations of the S. enterica chromosome in association with segments of DNA that exhibit features of pathogenicity islands. The presence of several apparently functional copies of these genes argues for an important role in the biology of this bacterial pathogen. Furthermore, they constitute a valuable tool to identify potential pathogenicity islands. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Type III secretion; Bacterial pathogenesis; Gene expression 1. Introduction * Corresponding author. Tel.: +1 (203) ; Fax: +1 (203) ; jorge.galan@yale.edu The virulence properties of Salmonella enterica are largely dictated by sets of genes encoded within discrete segments of its chromosome known as pathogenicity islands [1,2]. These genes are thought to have been acquired horizontally during the evolutionary process that eventually led to the close association of these bacteria with vertebrate hosts. One of these pathogenicity islands (SPI- 1), located at centisome 63 and anked by the fhla and muts genes [3,4], is thought to have been acquired very early in the evolution of Salmonella in an event that probably resulted in a signi cant niche expansion for these bacteria [5]. SPI-1 encodes a type III secretion system that is required for the stimulation of cellular responses that lead to bacterial internalization into non-phagocytic cells, the initiation of apoptosis in macrophages and the production of pro-in ammatory cytokines by intestinal epithelial cells [5]. This type III secretion system exerts its function by delivering a battery of e ector proteins that stimulate and/or interfere with cellular responses. Although all the components of the type III secretion system are encoded within SPI-1, the e ector proteins delivered by this system are encoded both within and outside of this pathogenicity island [5]. In addition to the invasion-associated type III secretion system, SPI-1 encodes an iron uptake system within its fhla-proximal region [6,7]. However, unlike the genes encoding the type III secretion system, the G+C content of the locus encoding the iron transport system is not signi cantly deviated from that of the rest of the Salmonella chromosome. This nding suggests that the acquisition of these two systems may have been the result of independent events. In this paper we report the characterization of the mutsproximal end of Salmonella typhimurium SPI-1. Within this region we have identi ed a group of genes with homologs in other Salmonella pathogenicity islands. These ndings suggest that this group of genes have been acquired horizontally by Salmonella in several independent / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (01)

2 204 A. Pancetti, J.E. Galän / FEMS Microbiology Letters 197 (2001) 203^208 events and therefore suggest an important role for these genes in the biology of this bacterial pathogen. 2. Characterization of the muts-proximal region of SPI-1 The muts-proximal region of the pathogenicity island-1 of S. typhimurium SL1344 was ampli ed by polymerase chain reaction with primers complementary to the neighboring genes invh [8] and muts [9] and its entire nucleotide sequence determined. Five putative open reading frames (ORFs) were identi ed within this region, oriented in the same transcriptional direction as invh (Fig. 1). These genes were called piga through pige (for pathogenicity island genes). The piga and pigb genes are organized in an operon located immediately downstream of invh. The start of the predicted pigb ORF overlaps with the end of piga by 1 nucleotide, indicating that these two genes may be translationally coupled. The pigc and pigd genes are organized in an operon which is separated from the pigab operon by a stretch of non-coding sequence containing remnants of a transposable element with a high degree of sequence similarity (95% sequence identity) to the Salmonella enteritidis IS1230c (accession number AJ002209). The start of the predicted pigd ORF also overlaps the coding sequence of the upstream gene pigc suggesting translational coupling. A remnant of a transposase similar to one previously described in Vibrio anguillarum (65% identity, accession number L ), and to the transposase of IS903 of Escherichia coli (61% identity; accession number F ), is located in the non-coding region downstream of the pigcd operon. The presence of remnants of transposable elements supports the notion that these genes must have been acquired horizontally. Consistent with this notion, the G+C content of the DNA of this region is 42%, signi cantly lower than that of the rest of the Salmonella chromosome (V52%) but very similar to the G+C content of SPI-1 (45%). Amino acid sequence comparison of the predicted Pig proteins with sequences in the available databases revealed intriguing similarities with proteins from other bacteria which, like Salmonella, are known to interact intimately with their eukaryotic hosts. PigA and PigB share signi cant sequence similarity with two putative proteins identi- ed in the chromosome of Yersinia pestis through genome sequencing (The Sanger Centre, UK). The putative Yersinia proteins share 39% identity and 56% similarity with PigA and 33% identity and 47% similarity with PigB. The organization of the genes encoding these proteins in Y. pestis is similar to that in S. typhimurium. PigC and PigD share amino acid sequence similarity to proteins identi ed through sequencing in Haemophilus in- uenzae [10], Mycobacterium tuberculosis [11], the plantassociated bacteria Rhizobium spp. [12], and a virulenceassociated plasmid of enteropathogenic E. coli [13]. In all cases the genes that encode these proteins are organized in a similar manner as to S. typhimurium and are also anked by insertion sequence (IS)- and/or transposase-like genes suggesting the possibility of their horizontal acquisition in these bacteria as well. The pige gene, located immediately downstream of a transposase-like sequence, encodes a predicted polypeptide that shares extensive amino acid sequence similarity to PrpB, an E. coli protein phosphatase encoded immediately Fig. 1. Genetic organization of the muts-proximal region of SPI-1. Horizontal arrows indicate the direction of transcription. Vertical arrows indicate the boundaries of SPI-1. The location of remnants of an IS and a transposase (tn) are indicated.

3 A. Pancetti, J.E. Galän / FEMS Microbiology Letters 197 (2001) 203^ Table 1 Bacterial strains and plasmids Strain or plasmid Relevant genotype Description, source and/or reference Strains: S. typhimurium SL1344 wild-type [24] SB160 invf::apht [15] SB161 vinvg [15] SB592 spra::apht [19] SB598 hila: :apht [20] SB1121 vpigcd: :apht this study SB1122 vpigab: :apht this study SB1133 pigab: :lacz SL1344 derivative with psb1509 integrated in the chromosome SB1135 spra pigab: :lacz P22HTint[SB592]CSB1133 SB1134 hila pigab: :lacz P22HTint[SB598]CSB1133 SB1136 vinvf pigab: :lacz SB160 derivative with psb1509 integrated in the chromosome SB1137 pigcd: :lacz SL1344 derivative with psb1510 integrated in the chromosome SB1139 spra pigcd: :lacz P22HTint[SB598]CSB1137 SB1138 hila pigcd: :lacz P22HTint[SB598]CSB1137 E. coli M2991 v(ara-leu)7697 arad139 vlacx74 gale galk vphoa20 thi rpse rpob arge(am) reca1 SM10Vpir thi thr leu tona lacy supe reca: :RP4-2-Tc: :Mu(Knr)Vpir [25] Plasmids: psb616 [26] psb375 vinvf [15] psb831 invh pigabcde this study psb1501 pigcd: :apht this study psb1502 pigab this study psb1504 pigab: :apht this study psb1507 piga: :M45 this study psb1508 pigb: :M45 this study psb1509 pigab: :lacz this study psb1510 pigcd: :lacz this study downstream of muts [14] and to the related E. coli protein PrpA. Although in S. typhimurium the PrpB homolog PigE is encoded immediately upstream of muts, it is likely that this gene is not part of SPI-1 but rather marks the boundary of SPI-1. Therefore, we would like to propose that the border of SPI-1 is not muts as originally proposed [4], but rather prpb. 3. E ect of loss-of-function mutations in the pigab and pigcd loci on Salmonella virulence The observation that the piga^d genes are organized into two distinctive operons and that this organization is maintained in their homologs in other bacteria and in other locations of the Salmonella chromosome suggests a Table 2 E ect of loss-of-function mutations on SPI-1-associated phenotypes Strain Relevant genotype Henle-407 invasion (%) a Macrophage cytotoxicity b Mouse virulence c SL1344 wild-type 100 þ /3 SB161 vinvg 2.7 þ ND SB1121 vpigcd::apht 71 þ /3 SB1122 vpigab::apht 100 þ /3 a Entry into Henle-407 cells was determined by the gentamicin protection assay ([27], p. 1987; [3], p. 686). Values are the result of three independent determinations and represent the percentage of the bacterial inoculum that survived 2 h of gentamicin treatment. Values were standardized to that of wild-type which was considered 100%. The actual internalization value for wild-type was 57 þ 9%. b Macrophage J774 cytotoxicity was determined by a dye exclusion assay [27]. Each plus sign represents cytotoxic e ects in approximately 25% of the cells. Results were recorded after 1 h of infection. A minimum of 500 cells were scored for each strain. c BALB/c mice were infected orally with 10 6 bacteria which represents V10 LD50 for the wild-type strain. Virulence is presented as the number of deaths over the number of inoculated mice. There were no signi cant di erences in the time to death among the di erent strains tested. ND, not determined.

4 206 A. Pancetti, J.E. Galän / FEMS Microbiology Letters 197 (2001) 203^208 Fig. 2. E ect of loss-of-function mutations in the SPI-1-encoded transcriptional regulatory genes hila, invf and spra on the expression of the pigab and pigcd loci. Levels of transcription of the di erent reporter gene fusions were measured by assaying L-galactosidase activity in bacterial cell lysates. The values represent the mean þ S.D. from one representative experiment performed with triplicate samples. Equivalent results were obtained in several repetitions of this experiment. functional relationship between the two genes within each operon. To investigate the potential contribution of the pig genes to phenotypes encoded within SPI-1, we constructed S. typhimurium mutant derivatives carrying lossof-function mutations in either the pigab (strain SB1122) or pigcd (SB1121) operons. The piga/pigb double mutant strain was constructed by generating an internal deletion immediately downstream of the piga transcription start site encompassing 200 bp of the pigb gene. The pigc/ pigd double mutant was constructed by generating a deletion that encompasses the entire pigc and pigd genes. The vpigab and vpigcd deletions were mobilized into the S. typhimurium chromosome by allelic exchange as previously described [15], generating the mutant strains SB1121 and SB1122, respectively (Table 1). These strains were tested for their ability to invade cultured Henle-407 cells or to induce apoptosis in cultured J774 macrophages, phenotypes known to be associated with genes encoded within SPI-1. As shown in Table 2, both mutant strains entered Henle-407 cells and induced apoptosis in macrophages in a manner that was indistinguishable from that of wild-type. We then examined the e ects of these mutations on S. typhimurium virulence in a mouse model of infection. The loss-of-function mutations in either pigab or pigcd had no apparent e ect on the virulence of S. typhimurium after oral inoculation into BALB/c mice (Table 2). These results indicate that the pig genes are not involved in any of these phenotypes or that their contribution to virulence is masked by the presence of functionally redundant genes elsewhere in the Salmonella chromosome. Table 3 Characteristics of the piga^d-associated chromosomal islands G+C content (%) Observations; examples of genes encoded within the island Boundaries of the island a Remnants of transposable elements in the island Size (kb) Gene Location (centisome) PigAB fhla/muts yes 42 present in S. typhimurium only; SPI-1 PigA2B amn/trna asn yes 49 present in S. typhimurium and S. typhi; no additional genes encoded in this islet PigA3B osmc/hya operon no 47 present in S. typhi only; genes encoding transporters; a Pho/ PhoQ-regulated locus encoding a multidrug e ux protein homolog PigCD fhla/pige yes 42 present in S. typhimurium and S. typhi; SPI-1 PigC2D glycil trna synthetase/cspa yes 52 present in S. typhi only; gene encoding a putative secreted protein with no apparent homolog PigC3D3/pigC4D4 94^96 V144 trna phe /dms operon yes 50 present in S. typhimurium and S. typhi; several genes encoding proteins with no apparent homologs; Vi antigen synthesis genes (S. typhi only); sope; phon PigC5D5 b???? 59 present in S. typhimurium only; homologs to silver nitrate resistance genes a The boundaries are de ned by regions that encode genes with homologs in E. coli. b These genes were detected within a V10-kb contig in the S. typhimurium database. Since the boundaries of this contig could not be determined, the location of these genes within the S. typhimurium chromosome as well as other characteristics could not be established.

5 A. Pancetti, J.E. Galän / FEMS Microbiology Letters 197 (2001) 203^ The expression of the pigab operon is in uenced by the SPI-1-associated regulatory protein SprA The location of the pig genes within SPI-1 suggested the possibility of a functional relationship with the type III secretion system encoded within this pathogenicity island. Although the absence of SPI-1-associated phenotypes in the pig mutants does not support this hypothesis, functional redundancy may be a plausible explanation for such a lack of phenotype. The expression of the genes that are associated with the type III secretion system encoded within SPI-1 is speci cally controlled by several transcriptional regulatory proteins that are encoded within this pathogenicity island [15^20]. To investigate further the possibility of a functional linkage between the SPI-1-encoded type III secretion system and the proteins encoded by the pig loci, we examine the e ect of mutations in genes encoding the SPI-1-associated transcriptional regulators HilA, InvF and SprA on pig gene expression. Operon fusions of the pigab and pigcd loci to lacz were constructed and introduced into the wild-type strain and the appropriate isogenic mutants following standard techniques as previously described [7]. The expression of the pig genes in the di erent genetic backgrounds was then monitored by measuring L-galactosidase activity [21]. As shown in Fig. 2, loss-of-function mutations in either invf or hila had little or no e ect on the expression of the pigab or pigcd genes. In contrast, introduction of a mutation in the SPI-1-associated transcriptional regulator SprA [8] resulted in a signi cant decrease in the transcription of the pigab operon although it had no e ect on the transcription of the pigcd operon. These results support the hypothesis that there is a functional link between PigA and PigB and the SPI-1-associated type III secretion system. However, mutations in pigab did not a ect the expression or secretion of proteins through the SPI-1-associated type III secretion system and PigA and PigB are not secreted via this pathway (data not shown). More experiments will be required to establish the nature of the potential association between PigA, PigB and the invasionassociated type III secretion system. 5. Homologs of the pig genes are present in other putative pathogenicity islands of S. typhimurium Comparison of the predicted sequence of the pig-encoded proteins with translated sequences in both the Salmonella typhi (The Sanger Center, ac.uk/) and S. typhimurium (Washington University Genome Center, genome sequence databases revealed the existence of several highly related proteins encoded outside SPI-1 (Table 3). At least two additional copies of the pigab operon and three of the pigcd operon were found in several di erent locations of both the S. typhi and S. typhimurium chromosomes. Interestingly, all these copies were found in association with putative pathogenicity islands. Although some of these regions do not encode known virulence genes, they all exhibit features of horizontally acquired genes such as the presence of remnants of transposable elements or bacteriophage genes, location in the vicinity of trna genes, and/or in some cases, nucleotide composition signi cantly deviated from that of the rest of the chromosome. For example, homologs of the pigab genes were found in S. typhi in the immediate vicinity of a recently reported PhoP/PhoQ-regulated locus that includes a 12-transmembrane domain protein homologous to multidrug transporters [22]. Also encoded in this V20 kb are several proteins with no homologs in E. coli. This observation in conjunction with a G+C content (47%) signi cantly deviated from that of the rest of the Salmonella chromosome suggests that this segment of the S. typhi chromosome may have been acquired horizontally and may represent a pathogenicity island. Two copies of the pigcd locus are found within a large insertion in the chromosomes of both S. typhi and S. typhimurium at centisome 94. This insertion, also located within a trna gene (trna phe ), does encode genes implicated in virulence, such as those involved in the synthesis of Vi antigen in S. typhi [23]. It is possible that the presence of the pig genes in association with horizontally acquired genes may be related to functions associated with the transfer or acquisition event and not with phenotypes potentially associated with virulence. However, unlike the commonly found remnants of transposases, integrases and bacteriophage genes in association with pathogenicity islands, the copies of the pig genes are complete and at least those encoded within SPI-1 are expressed. Furthermore, a copy of the pigab locus is inserted by itself within the trna asn locus and a copy of the pigcd locus is present in a small insertion next to the the glycyl trna synthetase gene. The latter involves only an additional gene that encodes a protein with no signi cant similarity to any other protein in the database. The maintenance of multiple copies of the pig genes argues for an important function in Salmonella biology. Our inability to identify a virulence phenotype associated with the SPI- 1-encoded pig genes may be therefore due to the presence of multiple copies of these genes with redundant function. The identi cation of all copies of pig-related genes and the subsequent construction of a Salmonella strain carrying null mutations in all these copies will be necessary to test this hypothesis. 6. Conclusions The characterization of the muts-proximal region of SPI-1 resulted in the identi cation of a group of genes that are exclusively associated with horizontally acquired genetic material and are found only in pathogenic or symbiotic bacteria. In addition, these ndings allowed the

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