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1 Centre for Infectious Disease Research and Biosafety Laboratories, Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
2 Institute of Clinical Microbiology, Immunology and Hygiene, FAU Erlangen-Nürnberg, 91054 Erlangen, Germany
Correspondence
Dipshikha Chakravortty
dipa{at}mcbl.iisc.ernet.in
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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yejF strain showed increased sensitivity to protamine, melittin, polymyxin B, human defensin (HBD)-1 and HBD-2, and was compromised in its capacity to proliferate inside activated macrophages and epithelial cells. Inside Intestine 407 cells, Salmonella was found to co-localize with human defensins HD-5 and HBD-1; this suggests that the ability to counteract AMPs in the intracellular milieu is important for Salmonella. In a murine typhoid model, the yejF strain displayed decreased virulence when infected intragastrically. These findings suggest that the putative transporter encoded by the yejABEF operon is involved in counteracting AMPs, and that it contributes to the virulence of Salmonella.
Three supplementary tables and four supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Multicellular organisms use various defence strategies to protect themselves from microbial infections. Production of antimicrobial peptides (AMPs) is one of these strategies. AMPs are widely distributed in phylogenetically diverse animal, plant and bacterial species (Hancock & Chapple, 1999
; Lehrer & Ganz, 1999). Most of these peptides are cationic in nature, and they interact with the bacterial cytoplasmic membrane, which usually comprises negatively charged phospholipids. AMPs exhibit broad antimicrobial activity against Gram-negative and Gram-positive bacteria, fungi and enveloped viruses (Lehrer, 2007). They also function as chemotactic agents, cytotoxins and opsonins (Ganz et al., 1990).
A continuous monolayer of gastrointestinal epithelial cells functions as the primary physical barrier against microbes, such as Salmonella, that enter the host through the gastrointestinal tract. Intestinal epithelial cells, especially Paneth cells, produce several AMPs that are lethal to invading bacteria (Ayabe et al., 2000; Ouellette et al., 1994). Murine macrophages produce cathelin-related antimicrobial peptides (CRAMPs), which serve as antimicrobial agents against intracellular Salmonella (Rosenberger et al., 2004). To cause disease, pathogenic bacteria, such as Salmonella, must endure the battery of these AMPs. Mutants of Salmonella that are sensitive to AMPs are compromised in their virulence (Groisman et al., 1992). Bacteria have developed many strategies to protect themselves from AMPs. For example, they reduce the net negative charge on the cell envelope by modifying lipid A; this helps to reduce the attraction of cationic AMPs towards bacteria. They also pump AMPs out of the bacteria using energy-dependent transporters, and cleave AMPs using surface-associated proteases. A putative ATP-binding cassette (ABC) transporter encoded by the sapABCDF operon is required to counteract AMPs, and it contributes to the virulence of Salmonella (Groisman et al., 1992). The presence of such a variety of strategies to counteract AMPs suggests the significant role of AMPs in host defence against microbes (Guina et al., 2000; Guo et al., 1998; Peschel, 2002; Shafer et al., 1998; Stumpe et al., 1998).
The S. Typhimurium genome contains a gene cluster located at minute 49 that encodes the components of a putative ABC-type dipeptide/oligopeptide/nickel transport system. This putative operon consists of four genes: yejA (STM 2216), which encodes a putative periplasmic binding protein; yejB (STM 2217) and yejE (STM 2218), which encode putative permease components; and yejF (STM 2219), which encodes the ATPase component of this transporter. Qimron et al. have screened for genes that interfere with major histocompatibility complex (MHC) class I presentation in S. Typhimurium. In their screen, they identified that the yej operon interferes with MHC I presentation. They showed that specific CD8 T cells were elicited at a higher level in mice in response to immunization with yej mutants, compared with immunization with the parental strain (Qimron et al., 2004). Antal et al. have observed that a small non-coding bacterial RNA of 62–64 nt, RydC, regulates the expression of the yejABEF gene cluster, and overexpression of RydC abolishes the expression of these genes (Antal et al., 2005). It has also been reported that the yejE and yejF genes of S enterica serovars Typhimurium and Typhi are upregulated inside host macrophages (Eriksson et al., 2003; Faucher et al., 2006), indicating the importance of these genes inside host cells. Apart from these reports, not much is known about the function of the yejA, yejB, yejE and yejF genes.
The objective of the present study was to probe into the other possible functions of yejA, yejB, yejE and yejF genes, and to investigate their role in the virulence of Salmonella. In this study, we show that mutants of the yejABEF operon are more sensitive to AMPs, are compromised to proliferate inside activated macrophages and epithelial cells, and have decreased virulence in a murine typhoid model.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Bacteria were grown at 37 °C in Luria broth (LB), unless mentioned otherwise. Carbenicillin, kanamycin, nalidixic acid (all at 50 µg ml–1) and chloramphenicol (10 µg ml–1) were added when required for selection of strains. F medium composition was as follows: 1x N-salts (10x N-salts: 50 mM potassium chloride, 75 mM ammonium sulphate, 5 mM potassium sulphate, 10 mM potassium dihydrogen phosphate and 1 M Bistris), 8 µM magnesium chloride, 38 mM glycerol and 0.1 % Casamino acids, pH 5.2. The growth study in a lower K+ concentration was done as described (Parra-Lopez et al., 1994). The composition of the medium was: 50 mM MES, 5 mM Na2HPO4, 10 mM (NH4)2SO4, 10 mM KCl, 1 mM MgSO4, 4 mM Tricine and 10 µM FeSO4. Glucose was added to a final concentration of 5 mM, and glycerol and DL-lactate to a final concentration of 10 mM each.
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Bacterial RNA extraction from infected cells.
Bacterial RNA from infected cells was isolated as described previously (Eriksson et al., 2003). Twelve hours after infection, cells were lysed on ice by incubating for 30 min in 0.1 % SDS, 1 % acidic phenol and 19 % ethanol in water. Salmonella were isolated from the lysate by centrifugation, and RNA was isolated using TRI reagent (Sigma), according to the manufacturer's protocol. In each case, bacteria were recovered from a six-well plate of infected cells and pooled to isolate RNA. RNA from in vitro-grown bacteria was obtained by growing bacteria statically at 37 °C to mid-exponential phase in DMEM medium, under 5 % CO2. These conditions mimicked those used for the cell-infection experiments.
RT-PCR.
Bacterial RNA (extracted from infected cells, bacteria grown in LB medium or bacteria grown in DMEM medium) was reverse transcribed using a reverse transcription system (Promega) and a gene-specific primer (yejF or 16S rRNA). It was then amplified (35 cycles) using primers generated to amplify the intergenic regions of the yejABEF genes, to check co-transcription (Mason et al., 2005). To check the expression level inside eukaryotic cells, cDNA was amplified (30 cycles) using primers (Table S1, available with the online version of this paper) generated to amplify the intergenic region of the yejE and yejF genes. Control reactions were done without reverse transcription to verify the absence of DNA in RNA preparations.
To check the expression of genes encoding human defensin (HD)-5 and human β defensin (HBD)-1, total RNA was isolated from 500 000 Intestine 407 cells, seeded in a 35 mm Petri dish, using TRI reagent (Sigma). RNA was reverse transcribed (Promega), and the cDNA was used to amplify the cDNA of the genes encoding HD-5, HBD-1 and hypoxanthine phosphoribosyl transferase (HPRT) (see Table S1).
Construction of gene deletions in S. Typhimurium.
Knockout constructions were done as described (Datsenko & Wanner, 2000). This method has been used successfully to knock out single genes, as well as an operon. Salmonella transformants carrying a Red helper plasmid (pKD46) were grown in LB with carbenicillin and 10 mM L-arabinose (Sigma), at 30 °C, to an OD600 of 0.35–0.4, and then made electrocompetent by washing three times with ice-cold 10 % (v/v) glycerol and MilliQ water. The PCR product containing the chloramphenicol-resistance gene (from pKD3) or the kanamycin-resistance gene (from pKD4), flanked by sequences upstream and downstream of the target genes, was obtained using specific primers (Table S1). PCR products were digested with DpnI to digest the methylated template. Electroporation was done according to the manufacturer's instructions (Bio-Rad), using 500 ng PCR product. Transformants were selected on LB agar containing chloramphenicol or kanamycin. The knockouts were confirmed using specific primers (Table S1). The yejF knockout was also confirmed with a reverse primer (Table S1) specific for the CAT gene of pKD3.
Construction of the yejFsap strain.
pKD46 was transformed into the sap strain. To this, the PCR product containing the chloramphenicol-resistance gene (from pKD3), flanked by sequences upstream and downstream of the yejF gene, was electroporated. There was a possibility that the chloramphenicol cassette could replace the kanamycin cassette that was present in the sap strain, because of the presence of the flippase recombinase target (FRT) sequence. To avoid such recombinants, transformants were selected on LB agar containing kanamycin and chloramphenicol. The double-knockout strain was confirmed using specific primers designed for both yejF and the sap operon (see Table S1).
Complementation of yejF.
First, the yejF gene was cloned in pET-21a(+) using BamHI and XhoI. The yejF fragment was subcloned, after digesting with BamHI and XhoI, into the BamHI and SalI sites of pTrc99A. The plasmid was electroporated into the yejF strain to obtain the pTrc99AyejF strain. This strain showed a significant decrease in virulence in a murine typhoid model. This non-specific effect of some cloning vectors on virulence has been described recently (Knodler et al., 2005). Hence, the yejF fragment from pTrc99A was moved to pQE60, after digesting with BamHI and HindIII, to obtain the pQE60yejF strain.
AMP sensitivity assay.
This was done as described by Fields et al. (1989), with some modifications. Bacteria in exponential phase were seeded in a 96-well plate at a concentration of 2x104 to 5x104 bacteria per well, in 50 µl of a solution containing 0.5 % tryptone and 0.5 % sodium chloride. A 100 µl volume of specific AMP was added at a specified concentration (polymyxin B, 0.15 µg ml–1; protamine, 40 µg ml–1; melittin, 5 µg ml–1; HBD-1 and HBD-2, 2 µg ml–1 each) and incubated at 37 °C in shaking conditions for 1 h. The samples were then plated at different dilutions on LB plates. For the HBD-1 sensitivity assay, 10 mM potassium phosphate buffer was used, as the presence of sodium chloride is known to inhibit the action of HBD-1 (Singh et al., 1998), and the incubation period was increased to 4 h. Data are presented as percentage survival relative to the untreated sample. Protamine, melittin and polymyxin B were purchased from Sigma. HBD-1 and HBD-2 were purchased from CytoLab/PeproTech Asia.
Intracellular proliferation assay.
Cells at a concentration of 1x105 to 2x105 per well were plated in 24-well plates 24 h prior to infection. These cells were infected with a specific strain at an m.o.i. of 1. To check transcription level of the yej genes by RT-PCR, an m.o.i. of 1 : 100 was used. Macrophages were infected with bacteria from an overnight culture. Overnight cultures were diluted to a ratio of 1 : 33 and grown for 3 h to late-exponential phase prior to the infection of epithelial cells. The plate was centrifuged at 120 g for 5 min and incubated at 37 °C for 20 min. The cells were then washed with PBS to remove excess bacteria and fresh medium containing 100 µg gentamicin ml–1 was added. After 1 h, the medium was discarded and the cells were washed with PBS, and then added to medium containing 25 µg gentamicin ml–1 and incubated at 37 °C for further time periods. After specified incubation periods, cells were lysed with 0.1 % Triton X-100 (Sigma), the lysate was plated on LB agar containing the appropriate antibiotic at different dilutions and the colonies were counted to calculate the c.f.u. Fold intracellular replication was calculated by dividing the intracellular bacterial load at 16 h by the intracellular bacterial load at 2 h.
Confocal microscopy.
To study the morphology of bacteria after treating with HBD-2, 2x104 to 5x104 exponential-phase bacteria were seeded on coverslips in 24-well plates and treated with HBD-2 at a concentration of 2 µg ml–1 for 30 min at 37 °C. Bacteria were then fixed using 3.5 % paraformaldehyde for 20 min. They were stained for LPS using monoclonal anti-S. Typhimurium LPS antibody (HyTest), raised in mouse, as the primary antibody, and Cy3-conjugated anti-mouse IgG antibody (Dianova), raised in goat, as the secondary antibody. Both antibodies were diluted in PBS containing 2 % BSA and 2 % goat serum for staining. Images were taken using a confocal laser-scanning microscope (Zeiss LSM Meta). Bacteria showing blebbings were counted. The percentage of bacteria showing blebbings relative to total number of bacteria counted was calculated.
To study the localization of HD-5 and HBD-1, Intestine 407 cells were seeded on coverslips in a 24-well plate and infected with either the wild-type (WT) strain or the yejF strain, as described above. At specified time points, cells were fixed with 3.5 % paraformaldehyde and stained for HD-5, or HBD-1 and LPS. Affinity-purified anti-HD-5 antibody or anti-HBD-1 antibody (Alpha Diagnostic International), raised in rabbit, was used as the primary antibody, and Cy2-conjugated anti-rabbit antibody, raised in goat (Jackson ImmunoResearch Laboratories), was used as the secondary antibody. LPS staining was done using the antibodies described above. All antibodies were diluted in PBS containing 0.1 % saponin, 2 % BSA and 2 % goat serum, for staining. After staining, images were taken using a confocal laser-scanning microscope (Zeiss LSM Meta).
Scanning electron microscopy.
Bacteria (108) were treated with 1 mg polymyxin B ml–1 for 1 h and fixed using Karnovsky's fixative (2 % gluteraldehyde and 2 % paraformaldehyde in 0.1 M phosphate buffer). After fixing, samples were treated with 1 % osmium tetroxide for 1 h. Samples were subsequently washed and dehydrated in a series of ethanol washes (70 % for 10 min, 90 % for 10 min and 100 % for 30 min) and air-dried prior to sputter coating with gold. Samples were then analysed using a scanning electron microscope (FEI Quanta 200).
Mouse experiments.
Six- to eight-week-old BALB/c mice (Central Animal Facility, Indian Institute of Science, Bangalore, India) were maintained under specific-pathogen-free conditions. All procedures with animals were carried out in accordance with institutionally approved protocols. Mice were infected intragastrically or intraperitoneally under aseptic conditions with the indicated doses. For the survival assay, 10 mice were used for each strain of bacteria and observed twice daily for survival. In organ infiltration studies, liver, spleen and Peyer's patches were taken aseptically, 4 days after infection. The organs were weighed and homogenized in a tissue homogenizer, in 1 ml PBS. The homogenate was centrifuged and plated at different dilutions. For liver and spleen, LB agar was used, and for Peyer's patches, Salmonella Shigella agar (HiMedia) was used. The number of c.f.u. was calculated per gram weight of organ for liver and Peyer's patches, and per organ for spleen.
Determination of mouse competitive indices.
This was done as described (Beuzon & Holden, 2001). The WT and yejF strains were diluted and mixed at a ratio of 1:1 to a final bacterial concentration of 107 bacteria ml–1. A 1 ml volume of this bacterial suspension was inoculated intragastrically into 6- to 8-week-old BALB/c mice. Five mice were used for each pair of strains. Four days after infection, the mice were sacrificed, and spleens and Peyer's patches were removed and homogenized in sterile PBS. The numbers of WT and yejF strains in each infected organ were then quantified by serial dilution and plating on selective media. The yejF strain was marked with chloramphenicol resistance, whereas the WT strain harboured carbenicillin resistance. The competitive index was calculated by dividing the number of mutant bacteria recovered from infected animals by the number of WT bacteria recovered. This value was then corrected by the initial ratio of mutant to WT bacteria used to infect the mice. This experiment was performed twice.
Statistical methods.
For AMP sensitivity assays and the intracellular survival assay, Student's t test was employed. For mouse experiments, the Mann–Whitney U test was employed.
Bioinformatics analysis.
Amino acid sequence identity and similarity were estimated with the help of the ‘Align two sequences’ option of BLAST.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), and the Dpp transporter, which is involved in dipeptide transport (Abouhamad et al., 1991) (Table S2, available with the online version of this paper). The sap operon is known to contribute to virulence in Salmonella spp., Haemophilus spp. and Erwinia spp. (Groisman et al., 1992; Lopez-Solanilla et al., 1998; Mason et al., 2005) by counteracting AMPs. The Opp transporter has also been implicated in the virulence of Staphylococcus aureus (Mei et al., 1997). Based on these facts, we hypothesized that the transporter system encoded by the yejABEF operon (henceforth referred to as the yej operon) might be involved in conferring virulence to Salmonella, and may be involved in counteracting AMPs, similar to the transporter system encoded by the sap operon.
The yejA, B, E and F genes are co-transcribed as a single message in S. Typhimurium
Before investigating the biological importance of the yejA, B, E and F genes, we demonstrated that these genes are co-transcribed as a single message using specific primers (Table S1) designed to amplify the nucleotide sequence spacing the junction region between each pair of adjacent genes (Fig. 1a). RNA was isolated from a stationary-phase culture, and reverse transcribed using a yejF-specific primer. The cDNA was used to amplify the intergenic regions. PCR products were detected for intergenic regions of the yejA, B, E and F genes. No product was detected for the rtn–yej region, or control samples lacking reverse transcriptase (Fig. 1a). This result demonstrated that yejA, B, E and F genes constitute an operon.
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). A similar finding was recently observed in Salmonella Typhi in THP-1 cells (Faucher et al., 2006). To follow up this observation, RT-PCR was done to check the expression of yej genes inside J774A.1 and Intestine 407 cells. Twelve hours after infection, bacteria were recovered, RNA was isolated, and reverse transcription was done as described in Methods. Intergenic primers between the yejE and yejF genes were used for PCR (Fig. 1b
). The results were analysed using Multi Gauge densitometry software and, after normalizing with an internal control (16S rRNA), it was observed that the yej genes are upregulated about sevenfold in J774A.1, which is a macrophage cell line, and in Intestine 407, which is an epithelial cell line. This result confirmed the previous result, and also indicated the importance of the yej operon in the epithelial cells that Salmonella encounters first during its course of infection.
Deletion of the yejF gene confers susceptibility to AMPs
yejF codes for a protein that has two putative ATP-binding cassettes, and could be the ATPase components for the transporter system encoded by the yej operon. We thus reasoned that YejF is energizing this transport system, and that deletion of the yejF gene would abolish the function of this transporter. We adopted the Lambda Red recombinase system to delete yejF, as described in Methods (Datsenko & Wanner, 2000). The yejF strain grew normally in LB medium when compared with the WT strain (data not shown).
Based on our hypothesis that the yej operon may confer resistance to AMPs, we investigated the sensitivity of the yejF strain to different AMPs. Salmonella spp. have been isolated from insects, amphibians, birds and a variety of mammals. Salmonella must have strategies to counteract AMPs found in all of these organisms. Protamine, melittin and polymyxin B represent AMPs from different organisms. Protamine is derived from salmon sperm. The role of the sap operon in counteracting AMPs was identified by Groisman et al. (1992) by screening for mutants that were sensitive to protamine. Melittin and polymyxin B are derived from honey bee and Paenibacillus polymyxa, respectively. It was observed that the yejF was more sensitive to protamine, melittin and polymyxin B (Fig. 2a and Fig. S1, available with the online version of this paper) when compared with the WT. Sensitivity to polymyxin B, a product of another bacterium, reflects the importance of the yej transporter in competing with other bacteria in the environment. S. Typhimurium sap mutant strains have been reported to be sensitive to protamine (Groisman et al., 1992). We compared the protamine sensitivity of the yejF and sap strains with that of the sapyejF strain, in which transporters encoded by both the yej and the sap operons were inactivated. It was observed that yejF, sap and sapyejF strains were equally sensitive to 40 µg protamine ml–1 (Fig. 2c). This may be due to other genes that have roles in counteracting AMPs in Salmonella taking over the function, at least to some extent, of counteracting AMPs when yej and sap systems are non-functional. The yejB and yejE strains were also sensitive to protamine and polymyxin B, confirming the role of the yej operon in conferring resistance to AMPs in Salmonella (Fig. 2b). Surprisingly the yejA strain did not show any increased sensitivity to polymyxin, protamine (Fig. 2b) or melittin (data not shown), suggesting that YejA is dispensable in this aspect.
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yejF strain to AMPs, we looked at the morphology of bacteria treated with polymyxin B using a scanning electron microscope. In the case of the yejF strain, we could see many damaged bacteria with membrane irregularities (inset, Fig. 3a), and many bacteria with their extruded cytoplasm formed an irregular mass of debris. When we scored for disrupted bacteria, we found 16 % (20 out of 122) of the WT strain, and 54 % (63 out of 117) of the yejF strain, showed either membrane irregularities and/or other features of bacterial lysis (Fig. 3a).
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, 2004). To further characterize the role of the yej operon in counteracting mammalian AMPs, we investigated the sensitivity of the yejF strain to HBD-2 and HBD-1. As expected the yejF strain was more sensitive to both HBD-2 and HBD-1 at 2 µg ml–1 concentration when compared with the WT strain (Fig. 2d and e). To further confirm this, we looked at the morphology of the bacteria after HBD-2 treatment (2 µg ml–1) using a confocal laser-scanning microscope. A total of 84 % of cells of the yejF strain showed blebbings on the membrane; this is a characteristic feature of bacteria exposed to AMPs, as observed in Escherichia coli, Pseudomonas aeruginosa, Staph. aureus and Mycobacterium tuberculosis (Lehrer et al., 1989; Sawyer et al., 1988; Shai, 2002; Shimoda et al., 1995). However, only 11 % of cells of the WT strain showed such membrane defects at this concentration of HBD-2 (Fig. 3b).
In E. coli, the SapD protein, which is a product of the sap operon, confers ATP dependence to the Trk potassium-uptake system that mediates potassium homeostasis, contributing to extracytoplasmic protease activity by OmpT, and the subsequent degradation of toxic peptides (Harms et al., 2001; Stumpe & Bakker, 1997; Stumpe et al., 1998). However, the yejF strain, which lacks the sapD homologue yejF, did not show any growth defect at a lower potassium concentration, unlike sap (Fig. 2f). This showed that the mechanism of counteraction of AMPs by the transporter encoded by the yej operon is not connected to potassium transport, unlike that of the sap operon.
yejF strain is attenuated for replication inside activated macrophages and epithelial cells
Since yejF is upregulated inside the macrophage-like cell line J774A.1, we looked for the intracellular replication capacity of the yejF strain in RAW 264.7, J774A.1 and elicited mouse peritoneal macrophages (data not shown). We did not find any defect in the capacity of the yejF strain to replicate inside any of these macrophage cell lines (Fig. 4a). This result was in accordance with the finding of Qimron et al. (2004). Brodsky et al. showed that mig-14 confers resistance to AMPs in Salmonella (Brodsky et al., 2002). It has also been reported that a mig-14 mutant was compromised in its replication inside activated macrophages, but not inside naïve macrophages (Brodsky et al., 2005). Increased expression of CRAMP in activated macrophages has been implicated for this phenotype of mig-14 mutant of Salmonella (Brodsky et al., 2005). We looked at the replication of the yejF strain in RAW 264.7 cells, which were activated by pre-treating with interferon-
and LPS. In agreement with the previous observation that activated macrophages limit intracellular replication of Salmonella (Rosenberger & Finlay, 2002), we did not observe any significant replication of the WT strain in activated RAW 264.7 cells, and the yejF strain showed decreased survival (Fig. 4b). The yejF strain has also been shown to be compromised in its replication within phorbol-12-myristate-13-acetate-activated U937 cells, which are derived from human monocytes, and are known to produce LL-37 (Agerberth et al., 2000) (Fig. S2, available with the online version of this paper).
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upregulate a variety of effector functions, including production of reactive oxygen and reactive nitrogen species (Ding et al., 1988), increased expression of AMPs (Brodsky et al., 2005; Hiemstra et al., 1993), and increased trafficking of phagocytosed material to the lysosome (Ishibashi & Arai, 1990). Though the yejF strain can be sensitive to any of the above antimicrobial mechanisms of activated macrophages, the compromised phenotype of the yejF strain inside activated macrophages is most likely to be due to its sensitivity to AMPs such as CRAMP, as suggested by the results of our in vitro studies (Figs 2 and 3). Moreover, CRAMP is known to colocalize with Salmonella inside murine macrophages and impair its replication (Rosenberger et al., 2004).
Salmonella encounters epithelial cells in the intestine before entering macrophages. Epithelial cells are known to express genes that encode for AMPs like defensins (Fahlgren et al., 2004; Mineshiba et al., 2005; O'Neil et al., 1999; Ogushi et al., 2001). We checked for the intracellular replication capacity of the yejF strain in Intestine 407 cells, COLO 205 cells, HeLa cells and Caco-2 cells; these are epithelial cell lines derived from different tissues. It has been reported that Intestine 407 cells express genes coding for HBD-3 and HBD-4 (Fahlgren et al., 2004), HeLa cells and Caco-2 cells express HBD-2, to which yejF was more sensitive (Mineshiba et al., 2005; Ogushi et al., 2001, 2004), and Caco-2 cells also express HBD-1 constitutively (O'Neil et al., 1999). The yejF strain was attenuated in intracellular replication in these epithelial cells (Fig. 4c, d and e). However, the invasion was not defective, as the number of colonies of the yejF strain after 30 min of infection was comparable to that of the WT strain (Fig. 4d). The yejB and yejE strains were also defective in intracellular replication within Intestine 407 cells (Fig. 4f). However, the yejA strain did not show any such defect, in agreement with the results of AMP-sensitivity experiments (Fig. 4f). These findings suggest that the yej operon confers the ability to proliferate inside activated macrophages and epithelial cells.
The minimal medium F, which is low in magnesium and phosphorous, has a low pH of 5.0, and is poor in aromatic amino acids. This medium mimics the environment of a Salmonella-containing vacuole inside host cells (Chakravortty et al., 2005). The yejF strain grew normally in F medium (data not shown), indicating that this strain is not compromised in its capacity to utilize the limited nutrition available inside the phagosomes of host cells, but that it may be compromised in its resistance to host intracellular defences, such as AMPs.
Surprisingly, unlike yejF, yejE and yejB strains, the yejA strain did not show any difference when compared with the WT strain in its survival upon treatment with protamine, polymyxin B (Fig. 2b) and melittin (data not shown), and the yejA strain proliferated normally in Intestine 407 cells (Fig. 4f). yejA encodes a putative periplasmic-binding protein of the Yej transporter. Periplasmic-binding proteins bind to the substrates and deliver them to the import complex in the inner membrane of Gram-negative bacteria. The different phenotype of the yejA strain, when compared with the yejB, yejE and yejF strains, was unexpected, as periplasmic-binding proteins are essential for the function of bacterial importers. However, it has been reported that some of the mutants of histidine and maltose transporters can function independent of their periplasmic-binding protein (Petronilli & Ames, 1991; Treptow & Shuman, 1988). So it is possible that YejA is dispensable for the function of Yej transporter, at least in counteracting AMPs. The transporter activity of the Yej transporter has to be investigated to validate this.
HD-5 and HBD-1 co-localize with intracellular Salmonella in Intestine 407 cells
It has been reported that HD-5 is secreted by Paneth cells into the crypts of the small intestine, and that it can clear Salmonella both in vitro and in vivo (Ghosh et al., 2002; Salzman et al., 2003). Transgenic mice expressing HD-5 are markedly resistant to oral challenge with S. Typhimurium (Salzman et al., 2003). However, the intracellular role of HD-5 in clearing intracellular pathogens has not been established. We observed uniform distribution of HD-5 inside the cytoplasm of Intestine 407 cells, and the WT and the yejF strains were co-localized with HD-5 at 2 and 12 h (Fig. 5 and Fig. S3, available with the online version of this paper) post-infection. Similarly, HBD-1 was also found to co-localize with the WT and the yejF strain (Fig. 6). We confirmed the expression of HD-5 and HBD-1 in Intestine 407 cells by RT-PCR (Fig. S4, available with the online version of this paper). These findings indicate that S. Typhimurium is exposed to lethal AMPs, such as HD-5, in the intracellular environment. Because of the non-functional Yej transporter, the yejF strain is more sensitive to intracellular AMPs such as HD-5, and is compromised in its proliferation within Intestine 407 cells. However, the mechanism of entry of AMPs into the Salmonella-containing vacuole remains to be investigated. These results provide a strong indication of the intracellular role of defensins in clearing intracellular pathogens, such as Salmonella, from epithelial cells; this role remains largely unexplored.
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yejF strain is attenuated for survival in vivoyejF strain in a murine model of typhoid fever. The intragastric route was chosen for infection, as bacteria are exposed to the intestinal epithelium and its secreted AMPs, such as cryptidins. Mice were intragastrically inoculated with the WT or the yejF strain, with a dose of 2x108 bacteria per mouse, and survival was monitored. Mice infected with the yejF strain had significantly higher survival rates (Fig. 7a). Then we analysed the bacterial load in Peyer's patches, liver and spleen after infecting mice intragastrically at a dose of 107 bacteria per mouse. After 4 days of infection, there was a significant decrease in the bacterial burden in all these organs of mice infected with the yejF strain when compared with mice infected with the WT strain (Fig. 7b–d). However, this difference was not observed after intraperitoneal infection (Fig. 7e). This observation clearly indicates that the transporter encoded by the yej operon is important in intragastric infections where Salmonella might encounter AMPs in both extracellular (intestinal epithelium) and intracellular environments (neutrophils, macrophages and epithelial cells).
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yejF strain, we tested for the ability of the yejF strain to compete with the WT strain after intragastric infection. Table 2 shows the competitive indices resulting from co-infection using the strains indicated. The competitive index represents the ratio (normalized to the ratio of inocula) of the number of mutant to WT bacteria recovered from infected spleens and Peyer's patches 4 days after the intragastric inoculation of 107 bacteria. The yejF strain showed a reduced competitive index in both Peyer's patches and spleen, indicating that attenuation of yejF diminishes its ability to compete for survival in these organs.
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). The decrease in bacterial load in the liver and spleen of the yejF-infected mice was most likely to be due to the decrease in the entry of bacteria to the systemic circulation, as most of the bacteria are cleared at the Peyer's patches, and also due to attenuated replication of the yejF strain inside activated macrophages. A similar observation was made in mutants of the PmrA–PmrB regulated locus pmrHFIJKLM, which is involved in resistance to polymyxin B. It has been shown that this locus is essential for the virulence of Salmonella when infected orally, but not intraperitoneally, in a murine model of typhoid fever (Gunn et al., 2000). In accordance with this report, our findings show the importance for Salmonella to counteract AMPs at the intestinal level, in order to cause systemic disease.
To best of our knowledge, this is the first report to show that the yej operon is important for virulence in S. Typhimurium. Our results, along with the findings of Qimron et al. (2004), show that the yej operon is important for both innate and adaptive immunity. The exact mechanisms of action, and the link between the two entirely different kinds of function, need to be elucidated. One interesting possibility, based on the model proposed by Parra-Lopez et al. (1993) for the Sap transporter, is as follows: Salmonella, using the Yej transporter, can transport immunogenic peptides, which are otherwise targeted for loading on to MHC I molecules, by the same mechanism by which it imports AMPs for degradation. This makes these peptides unavailable for MHC I presentation. In fact, it has been showed that phagosomes are competent organelles for antigen cross-presentation (Houde et al., 2003). Moreover, the MHC I molecule is known to co-localize with the Salmonella-containing vacuole, and peptides expressed in Salmonella have been reported to undergo MHC I presentation (Smith et al., 2005; Yrlid & Wick, 2000). This hypothesis could explain the superior CD8 response observed in mice infected with yej mutants (Qimron et al., 2004), and also their sensitivity to AMPs (Figs 2 and 3). These peptides, which are imported, can be a source of amino acids after proteolysis. Thus, the yej operon seems to have three functions: (i) interfering with MHC I presentation, which is a part of adaptive immunity; (ii) counteracting AMPs that are part of innate immunity; and (iii) providing a nutritive source in the form of peptides. All these functions are very crucial for Salmonella to survive and proliferate inside the hostile environment of the host.
The fact that importers are not found in mammalian hosts make them ideal targets for novel antimicrobial compounds. Drugs linked to the natural substrate of these transporters can easily cross the bacterial cell wall, and act more effectively. Further studies to understand the functional and structural details, and substrates, of transporters such as the one encoded by the yej operon, may help in the design of novel antimicrobial compounds. This is relevant in the present scenario, where pathogenic bacteria, such as Salmonella, are developing resistance to existing antibiotics (Glynn et al., 1998; Molbak et al., 1999).
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ACKNOWLEDGEMENTS |
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Edited by: P. H. Everest
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REFERENCES |
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Received 2 July 2007;
revised 17 September 2007;
accepted 27 September 2007.
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) and 
) strains in minimal medium with 10 mM K+. 
, WT.
