The PagN protein of Salmonella enterica serovar Typhimurium is an adhesin and invasin
© Lambert and Smith; licensee BioMed Central Ltd. 2008
Received: 09 May 2008
Accepted: 08 September 2008
Published: 08 September 2008
The pagN gene of Salmonella enterica serovar Typhimurium is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection. The PagN protein displays similarity to the Hek and Tia invasins/adhesins of Escherichia coli. Thus far no function has been ascribed to the PagN protein.
Here we show that the outer membrane located PagN protein mediates agglutination of red blood cells and that this can be masked by LPS. When expressed in Escherichia coli the PagN protein supports adhesion to and invasion of mammalian cells in a manner that is dependent on cytoskeletal rearrangements. S. enterica sv Typhimurium pagN mutants display a reduction in adhesion to and invasion of epithelial cells. Finally, we demonstrate that over-expression of PagN in a SPI-1 mutant can partially compensate for the lack of a functional invasasome.
PagN is an outer membrane protein that may contribute to the virulence of S. Typhimurium. This protein is a haemagglutinin and contributes to the adherence to mammalian cells. In addition, PagN can mediate high-level invasion of CHO-K1 cells. Previously,pagN mutants have been shown to be less competitive in vivo and thus this may be due to their lessened ability to interact with mammalian cells. Finally PagN can be added to an ever-growing repertoire of factors that contribute to the pathogenesis of Salmonella.
Salmonella enterica serovar Typhimurium infects a wide range of animal hosts and typically causes a self-limiting gastroenteritis . Initial symptoms include nausea and vomiting, which are followed by abdominal pain and diarrhoea. S. Typhimurium adheres to intestinal epithelial cells using a myriad of fimbriae including Type 1 fimbriae . Outer membrane proteins such as the plasmid-encoded Rck [3–5] and the OmpD  protein also mediate attachment to epithelial cells. Deletion of either rck or ompD results in decreased invasion of intestinal epithelial cells [5, 6]. Upon adhesion the subsequent uptake of Salmonella into mammalian cells is a complex process that is coordinated by a series of proteins that are encoded within the SPI-1 and SPI-5 pathogenicity islands . The SPI-1 locus encodes a type three secretion system (T3SS), also known as an invasasome, that delivers effector protein including the S almonella outer proteins (Sop) SopE, SopE2, SopB, the secreted protein tyrosine phosphatase SptP, and the S almonella invasion proteins (Sip), SipA and SipC into cells . These effectors have profound effects on the regulation of actin cytoskeletal structure and on mammalian cell membrane plasticity . The net result of injecting these effectors into the host cell is bacterial-promoted endocytosis .
The PhoPQ system regulates the transcription of a multitude of virulence genes of Salmonella . This regulatory system is composed of the PhoQ membrane-bound sensor kinase and the PhoP response regulator. In response to specific stimuli, PhoQ modifies the phosphorylation state of the cytoplasmic DNA-binding protein PhoP, increasing its affinity for specific DNA targets . Genes that are PhoP-activated are known as pags (PhoP-activated genes) whilst those that are repressed are known as prgs (PhoP-repressed genes) . Three different cues have been proposed to activate the PhoPQ system inside macrophages: a mild acidic pH, antimicrobial peptides, and low Mg2+ concentration . The pagN gene is a PhoP-activated gene . The pagN ORF was originally identified in a TnphoA random-mutagenesis screen of the S. Typhimurium strain 14082s . In that study bacteria with active phoA gene-fusions that displayed decreased fusion-protein activity on acquisition of the phoP12 allele (which results in a PhoP null phenotype) were identified. Through the use of in vivo expression technology (IVET), Heithoff et al. identified over 100 genes that were specifically expressed during infection of BALB/c mice . These authors showed that pagN (also termed iviVI-A in that study), although not expressed in S. Typhimurium strains grown on typical laboratory medium, was required for survival in BALB/c mice under the conditions of the IVET selection. Subsequently, Conner et al. noted a decrease in competitiveness for a mutant with deletions in the region of pagN/iviVI-A when mixed with its parental strain . Expression of the pagN gene has been shown to respond to pH and Mg2+ concentration in vitro . As with other pags, and in a PhoP-dependent manner, pagN expression was induced in conditions of acidic pH and low Mg2+ concentration. Furthermore, expression of pagN has been demonstrated to be highly up-regulated within cultured RAW264.7 macrophages and Henle-407 enterocytes .
Whilst the function of PagN is unknown it does display similarity to known invasins of Escherichia coli. PagN is ~54% similar to the Hek and Tia adhesins/invasins of pathogenic E. coli [13–15]. Based on this homology it is possible that PagN may be an adhesin and/or invasin. Here we show that PagN, when expressed in E. coli, can agglutinate erythrocytes as well as mediate adhesion to and invasion of mammalian cells. Furthermore S. Typhimurium mutants defective in pagN display reduced adhesion to and invasion of mammalian cells. Finally, multi-copy expression of PagN can compensate for the loss of the SPI-1 encoded T3SS.
PagN is a haemagglutinin but not an autoagglutinin
In addition to promoting haemagglutination Hek is also an autoagglutinin . Autoagglutination can be conveniently measured in a kinetic manner by a spectrophotometric assay. Expression of the Hek protein from pHek6 in E. coli XL-1 Blue caused bacteria to settle out of solution at a rate of 0.024 OD600 nm units/min whereas bacteria expressing PagN settled at rate of 0.0035 OD600 nm units/min almost identical to that of the vector control strain. Thus, E. coli expressing PagN did not support autoagglutination.
Next we sought to establish if PagN could support haemagglutination when expressed in multicopy in S. Typhimurium. Plasmid pPagN2.3 was introduced into S. Typhimurium LT-2 and cultured in minimal MOPS medium. Growth in this medium leads to maximal induction of the PhoP-dependent pagN promoter. Expression of PagN was confirmed by SDS-PAGE analysis of outer membrane extracts (data not shown). Surprisingly S. Typhimurium/pPagN2.3 did not mediate agglutination of red blood cells (Fig. 1C). Laboratory strains of E. coli such as K-12 or B completely lack the O antigen component of lipopolysaccharide (LPS) . It has been reported that porins in E. coli are partially obscured by the LPS core and completely blocked by O antigen sugars  and indeed in S. Typhimurium the O antigen prevents the outer membrane PgtE protease from interacting with its substrate . S. Typhimurium has a large O antigen, which may serve to mask PagN thus limiting access to its receptor. To investigate this hypothesis, the plasmid pPagN2.3 was transformed into S. Typhimurium strain CH133, a galE mutant. The galE gene encodes the enzyme UDP-galactose 4-epimerase which is the first enzyme catalyzing O antigen biosynthesis. Therefore S. Typhimurium strain CH133 lacks the O antigen component of LPS. Haemagglutination assays using this strain showed that PagN, when over-expressed in a rough strain, was capable of promoting agglutination (Fig. 1C). These data suggest that LPS is capable of 'masking' PagN and preventing functional receptor binding during haemagglutination.
PagN is an adhesin and invasion
Invasion of epithelial cells by bacteria is often accomplished by inducing cytoskeletal rearrangements by manipulating polymerisation of actin. Actin filaments have a fast and slow growing end; cytochalasins inhibit elongation at the fast growing end . Specifically, cytochalasin D affects the polymerisation of actin in two ways: it increases the initial rate but markedly reduces the final extent of the Mg2+-induced polymerization process . To assess the role of actin filament polymerization in PagN-mediated invasion, confluent CHO-K1 monolayers were pre-incubated with cytochalasin D (1 μg/ml) for 30 min at 37°C before infection with bacteria. Cytochalasin D was present throughout the invasion assay. Treatment with this inhibitor lead to a 30-fold reduction in invasion, which was comparable to the level seen with the vector control in the absence of cytochalasin D (Fig. 2).
PagN contributes to Salmonellaadhesion to and invasion of epithelial cells
The contribution of PagN to the invasion of CHO-K1 cells by S. Typhimurium strain SL1344 was determined. Invasion promoted by S. Typhimurium strain SL1344 and its pagN mutant derivative, ML6, were tested with non-polarized cells in a standard invasion assay. These data established that wild-type S. Typhimurium strain SL1344 were internalized in significantly greater numbers than the pagN mutant strain ML6. Data from triplicate wells revealed that wild-type levels were reduced from 18.26 ± 2.35% to 9.99 ± 1.37% for a pagN mutant (P < 0.05). The PagN-defective bacteria displayed a consistent ~2-fold reduction in CHO-K1 cell invasion as compared to the wild-type parent.
PagN failed to promote haemagglutination when overexpressed in S. Typhimurium LT2. This inability to promote haemagglutination may be explained by the differences between the LPS of E. coli laboratory strains and S. Typhimurium strain LT2. Laboratory strains of E. coli such as K-12 and B completely lack the O antigen component of LPS  whereas S. Typhimurium has a large O antigen containing OAc, Abe, Man, Gal, and Glc. When over-expressed in the rough S. Typhimurium strain CH133, PagN promoted haemagglutination. PagN contains fifteen positively charged amino acids that we predict to be surface-exposed. It is likely that these amino acids might interact with negatively charged LPS thus masking PagN and preventing interactions between the protein and its mammalian target.
Expression of PagN in E. coli resulted in adhesion to and invasion of mammalian cell lines. High levels of invasion promoted by PagN were observed for CHO-K1 cells. Invasion of HT-29 cells by E. coli expressing PagN was much lower. It may be that the receptor for PagN is less abundant in HT-29 cells, expressed by fewer cells or possibly that the receptor is not as accessible in this cell line. Another possibility may be that the bacterial cells do not survive within the environs of HT-29. HT-29 is known to produce antibacterial peptides and these are deleterious for E. coli. HT-29 cells express human beta defensin 1  and also up-regulate expression of LL-37 in response to E. coli . A similar difference in the efficiency of invasion of CHO-K1 cells versus the T84 intestinal epithelial cell line was previously noted for the Hek protein .
Mutation of pagN in S. Typhimurium resulted in a decrease in invasion of and adhesion to CHO-K1 and HT-29 cells. Indeed, this defect was more pronounced for rough strains. Though mutation of pagN did not completely abrogate adhesion and invasion the magnitude of the effect is of the same order as that seen for OmpD  and Rck proteins . Thus outer membrane proteins such as PagN may play an incremental role in Salmonella interactions with host cells. The pagN gene is activated by PhoP and is maximally expressed intracellularly as observed by gene fusion [11, 12] or microarray analysis . Thus bacteria that exit epithelial cells or macrophages might be expected to have an optimal level of expression and this might facilitate subsequent interactions with mammalian cells that the pathogen encounters. The fact that PagN can functionally substitute for the lack of a functional T3SS may have biological relevance. Within macrophages the SPI-1 T3SS is strongly downregulated  and Salmonella that exit such cells may have to rely on PagN to facilitate subsequent interactions with cells until such time as the T3SS is maximally expressed.
The primary sequence of PagN has similarity to the Tia and Hek adhesions/invasins. Both Tia and Hek bind heparin sulphate proteoglycan (HSPG) [13, 29]. Studies are currently in progress to establish if PagN utilizes HSPG for adhesion/invasion.
We have shown that PagN can mediate interactions with mammalian cells when expressed in E. coli and S. Typhimurium. pagN is upregulated in vivo and contributes to the competitiveness of S. Typhimurium. This may be due to the adhesive and invasive characteristics of PagN. PagN in multicopy can complement an invA mutation. PagN may thus prove to be a useful tool for studying Salmonella in the intracellular environment in the absence of the SPI-1 T3SS.
Bacterial strains, plasmids and culture conditions
Bacterial strains used in this work
LT-2 galE503 pagN::spc
LT-2 galE503 invA::cat pagN::spc
LT-2 galE503 invA::cat
F' endA1 hsdR17 (rk-mk+) glnV44 thi-1 recA1 gyrA (Nalr) relA1 Δ(lacIZYA-argF)U169 deoR (Φ80dlacΔ (lacZ)M15)
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F' proAB lac q ZΔM15 Tn10 (Tetr)]
Bacterial plasmids used in this work
Cmr Tetr General cloning vector
Apr ColE1 replicon
Apr Cloning vector
Apr hek gene in Pbskii+
Apr Spcr pMB1 replicon
Apr invA gene with 500 bp flanking DNA cloned into pBSKII+
Apr invA::cat gene with 500 bp flanking DNA cloned into pBSKII+
Apr Temperature-sensitive plasmid carrying the red and gam genes of λ-phage under control P araBAD promoter
Apr MBP fusion vector
New England Biolabs
Apr pagN ORF inserted into pTrc99a
Apr pagN ORF flanked by NcoI and BamHI sites inserted into pBSKII+
Apr pagN ORF, excluding the DNA corresponding to the signal sequence of the PagN protein, inserted into pMAL-c2
Apr pagN gene inserted into pBR322
Apr pagN gene with 500 bp flanking DNA cloned into pBSKII+
Apr Spcr pagN::spc
Apr Cloning vector with an IPTG-inducible P trc promoter
Eukaryotic cell lines and growth conditions
All cell lines used were obtained from ATCC (Manassas, VA, U.S.A.). The mammalian cells lines used were HT-29 (ATCC HTB-38) and CHO-K1 (ATCC CCL-61). CHO-K1 cells were grown in a 1:1 mixture of DMEM and Ham's F12 medium supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS) (Life Technologies) at 37°C in 5% CO2. HT-29 cells were grown in McCoy's medium supplemented with 10% (v/v) FBS.
Recombinant DNA techniques
Plasmid DNA was isolated using the Genelute Plasmid Miniprep kit (Sigma-Aldrich) or the Qiagen Plasmid Midi kit. Restriction endonucleases were purchased from New England Biolabs and used according to the manufacturer's instructions. Standard methods were used for the ligation of DNA fragments and transformation of plasmid DNA [30, 31]. The synthesis of oligonucleotide primers and DNA sequencing was performed by MWG Biotech, Ebersberg.
Cloning of the pagNgene
The pagN ORF from S. Typhimurium strain LT2 and 500 bp of DNA flanking the ORF were amplified by PCR using primers PagNF (5'-AGA TAA TTG CTC GCC ATT CG-3') and PagNR (5'-ATG GAG GGT TCC AGA TCT CC-3'). The purified pagN PCR product was cloned into pBSKII + cloning vector (pBSKII+) cut with EcoRV creating the multi-copy plasmid pPagN2.3. The pPagN2.3 plasmid was sequenced to confirm the DNA sequence of the insert (Table 1). The pagN gene and putative promoter region was excised from pPagN2.3 using PshAI and SacI and the resulting DNA fragment was ligated into vector pBR322. The structure of the resulting plasmid was confirmed by restriction endonuclease digestion and the recombinant was designated pML10 (Table 1).
The pagN ORF was amplified with primers pagNF2 (5'-GCT AGG ATC CCG ATA GTG TTT AAA AGG CG-3') and pagNR2 (5'-GCC TCC ATG GAA AAC TTT GCA GTC TGC-3'). The resultant PCR product was digested with BamHI and ligated into plasmid pBSKII+ digested with BamHI and EcoRV. The resulting plasmid was sequenced to confirm the sequence of the insert and the plasmid was designated pML4. The PagN expression vector pML1 was constructed by excising the pagN ORF from pML4 using NcoI and BamHI. This DNA fragment was cloned into pTrc99a, placing it under the control of the IPTG-inducible Ptrc promoter.
The pagN ORF was amplified by PCR using the primer set pagNF2 and PagNR3 (5'-GCT ATC TAG ACG ATA GTG TTT AAA AGG CG-3') and S. Typhimurium strain LT2 genomic DNA as a template. The PagNR3 primer had an XbaI site incorporated into it, resulting in a unique XbaI site at the 3' end of the pagN ORF. The PCR product was digested with XbaI and BsaBI. The BsaBI site is at nucleotide position 75 of the pagN ORF, corresponding to amino acid residue number 26. Plasmid pMALc-2 (New England Biolabs) was digested with EcoRI and the 5' overhangs were blunted by digestion with Mung Bean Nuclease and was then digested with XbaI. The digested PCR product was cloned downstream, and in frame with the malE gene. The recombinant plasmid was sequenced to confirm the DNA sequence of the insert and named pML7. SDS-PAGE analysis of E. coli XL-1 Blue containing the pML7 plasmid revealed one major inducible protein with an apparent molecular weight of ~65 kDa, corresponding to the predicted size of the MBP-PagN fusion protein. An anti-PagN serum was produced by inoculation of rabbits with the MBP-PagN protein fusion.
Cloning of the invAgene
A 1499-bp fragment of the 2058-bp invA gene of S. Typhimurium strain LT2 was amplified by PCR using the primers invA-allele-F (5'-CAA ACG CTG CAA AAC TTC AG-3') and invA-allele-R (5'-TTG ATT TCC TGA TCG CAC TG-3'). The PCR product was ligated into the pBSKII+ plasmid that had been digested with EcoRV creating the plasmid pInvA
Disruption of the pagN and invAgenes of S. Typhimurium
An interrupted pagN gene was constructed in vitro and transferred to the bacterial chromosomes using an allele-replacement system based on the gene products of the λ phage red operon. The λ-phage redγβα genes were supplied on the temperature sensitive pKOBEGA plasmid under the control of the arabinose-inducible PBAD promoter . A 2.2 kb spectinomycin resistance encoding fragment from the pHP45Ω plasmid was excised using HindIII, blunted using the DNA polymerase I large (Klenow) fragment and ligated into a BsaBI site 233 bp from the start of the pagN ORF in pPagN2.3 yielding plasmid pPagNKO. The disrupted pagN gene from pPagNKO was amplified by PCR and transferred to S. Typhimurium chromosome as previously described . The invA gene of S. Typhimurium strain LT-2 was interrupted in a similar manner. A 2.0 kbp chloramphenicol resistance cassette, excised from pACYC184, was used to interrupt the invA gene of plasmid pInvA, resulting in the plasmid pInvAKO. The disrupted invA gene from pInvAKO was amplified by PCR and transferred to the S. Typhimurium chromosome as described above.
The ability of bacterial strains to agglutinate erythrocytes was determined using a 3% (v/v) suspension of human blood group A containing 100 mM mannose. Mannose was added to block adhesion by type 1 fimbriae. Bacterial cultures were harvested by centrifugation at 15,800 × g for 1 min and resuspended in PBS to an optical density of 1.0 at 600 nm. The bacteria were then serially 2-fold diluted with PBS in a final volume of 100 μl in a 96-well microtiter plate. An equal volume of the 3% blood suspension was added to each well and the plate was incubated at room temperature for 2 hours or at 4°C overnight to allow un-agglutinated erythrocytes to settle out of suspension. Agglutination was seen as a diffuse carpet of erythrocytes spread over the whole well surface. Non-agglutinated cells were seen as a tight button in the centre of the wells.
Overnight cultures were harvested by centrifugation, resuspended in PBS and normalized to an optical density of 8 OD600 nm. Samples were taken from the surface of the cultures at regular intervals to determine the OD600 nm. Assays were performed in duplicate and the rate of autoaggregation was determined by the mean decrease in optical density over time. Rates of autoaggregation were determined using Kaleidagraph software.
Preparation and analysis of whole cell lysates and membrane proteins
Samples enriched for outer-membrane proteins were prepared as previously described . Proteins samples were separated by SDS-PAGE using the method of Laemmli and visualized following staining with coomassie brilliant blue R-250 or were transferred to Immobile-P PVDF membrane (Millipore), processed for western blotting, probed with an anti-MBP-PagN antibody and the blot was developed using the SuperSignal West Pico chemiluminescent HRP substrate (Pierce).
Preparation and analysis of LPS
Overnight cultures (2 ml) of the bacteria to be examined were grown in the presence of appropriate antibiotics. 1.5 ml of each culture was collected by centrifugation at 19,000 × g for 2 min. Bacteria were resuspended in 100 μl of 2× Laemmli buffer . Lysis of resuspended bacteria was achieved by boiling the samples for 10 min at 100°C. Lysed bacteria were cooled on ice for 5 min. All protein present in the sample was digested with 20 μl of Proteinase K (10 mg/ml), leaving the LPS component of the outer membrane intact. Digestion of protein was carried out at 60°C for 1 h. A 1:6 dilution of each sample was prepared in Laemmli buffer. As before samples were stored at -20°C and boiled for 5 min prior to use. LPS samples separated by SDS-PAGE were fixed overnight in fixing solution (25% (v/v) isopropanol, 7% (v/v) acetic acid). Gels were then oxidized for 5 min in oxidation buffer (0.7% (v/v) periodic acid in 2.67% (v/v) fixing solution) and subsequently washed four times for 15 min in dH2O. Gels were then stained for 10 min in freshly prepared staining solution 0.8 M NaOH (0.1 M), 2% NH4OH (v/v), 3% (w/v) AgNO3). Excess stain was washed away with 3 × 10 min washes with dH2O. Gels were subsequently placed in developing solution (0.04% (v/v) formalin, 2% (w/v) Na2CO3). Image development was stopped with 10% (v/v) acetic acid.
Cell association and invasion assays
Cell association and invasion assays were performed as previously described . CHO-K1 cells were seeded into 12 well trays at densities of 3.0 × 104, while HT-9 cells were seeded at a cell density of 2.5 × 105 cells/well. Cells were grown to confluence.
Salmonella strains used in these assays were grown overnight in a MOPS-based minimal media adjusted to pH 5.8. E. coli strain DH5α was grown overnight, and subsequently sub-cultured at a 1:50 dilution in 10 ml. At an OD600 nm of 0.4–0.6, PagN expression was induced with IPTG (1 mM). Bacteria were grown for a further 3 h. Prior to their addition to confluent mammalian cells, bacteria were washed with PBS and diluted 1:500 in warm tissue culture medium containing D (+)-mannose (100 mM). Mammalian cell monolayers were washed once with warm PBS and bacteria-containing medium (1 ml) was added to each well. The infected cells were centrifuged at 600 × g for 5 min to initiate contact between bacteria and the mammalian cells and to synchronize the time of infection. Cells were incubated at 37°C in 5% CO2 for 1 h to allow adhesion to and invasion of the cultured cells. Infected monolayers were washed thrice with warm PBS to remove any non-adherent bacteria. To determine the total number of cell-associated bacteria the monolayer was disrupted by treatment with 0.1% Triton X-100 and the released bacteria were enumerated by spreading serial dilutions on agar plates. To determine the number of intracellular bacteria, a standard gentamicin protection assay was performed. Following the 1 h infection, cells were incubated with medium containing gentamicin (100 μg/ml) for 90 min at 37°C in 5% CO2, washed with PBS, disrupted with 0.1% Triton X-100 and the released bacteria enumerated as before. The total viable counts of the bacterial inocula were also determined and the cell-association or invasion efficiencies were expressed as the percentage of bacteria recovered from triplicate wells. The student t-test was performed where appropriate. Experiments were performed at least three times and data from a typical experiment is presented.
This work was supported with grants from the Irish Higher Educational Authority (IITAC project) and Health Research Board Project grant RP85/2001. The authors acknowledge Dr S Yasuda (Shigen) for donation of plasmids.
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