Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain1
Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41080 Sevilla, Spain2
Author for correspondence: Francisco García-del Portillo. Tel: +34 91 5854923. Fax: +34 91 5854506. e-mail: fgportillo{at}cnb.uam.es
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ABSTRACT |
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Keywords: Dam methylation, membrane vesicle, protein secretion
Abbreviations: Dam, DNA adenine methylase; PAL, peptidoglycan-associated lipoprotein; PG, peptidoglycan (fraction); SP, secreted protein; SPI-1, Salmonella-pathogenicity island 1; TP, total protein; VES, vesicle; VSN, vesicle supernatant
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INTRODUCTION |
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Recent studies have shown that mutants of S. enterica serovar Typhimurium lacking Dam methylase are highly attenuated for virulence in the murine typhoid model (Heithoff et al., 1999 ; Garcia-del Portillo et al., 1999
; Low et al., 2001
). DNA methylation is thus essential for virulence. Current evidence suggests that the role of Dam methylation in Salmonella virulence is multifactorial: Dam- mutants show defects in several virulence-related traits such as invasion of the intestinal epithelium and cytotoxicity to M cells (Garcia-del Portillo et al., 1999
). In vitro infection assays have also shown that Dam- mutants survive within professional phagocytic cells while having reduced capacity to invade cultured non-phagocytic cells (Garcia-del Portillo et al., 1999
). This partial inability to invade epithelial cells was tentatively related to reduced secretion of InvJ and SipC, two invasion proteins encoded in the Salmonella-pathogenicity island 1 (SPI-1) (Garcia-del Portillo et al., 1999
). Besides the impaired secretion of invasion proteins, dam mutations also cause a marked increase in the amount and number of proteins that are present in the extracellular medium (Garcia-del Portillo et al., 1999
).
The spontaneous loss of membrane material by Gram-negative enteric bacteria is known to increase in the absence of a specific subset of inner-membrane, periplasmic and outer-membrane proteins that maintain envelope integrity. That is the case for the Brauns (murein) lipoprotein (Lpp); the peptidoglycan-associated lipoprotein (PAL) (Lazzaroni & Portalier, 1992 ); the periplasmic protein TolB; and the inner-membrane complex formed by TolQ, TolR and TolA proteins (Lazzaroni et al., 1999
). A series of studies have provided evidence for the existence of large complexes containing these proteins that establish a physical link between the inner and the outer membrane (Bouveret et al., 1995
, 1999
; Rigal et al., 1997
; Clavel et al., 1998
; Lazzaroni et al., 1999
). This function is essential for providing stability and integrity to the cell envelope. Thus, mutants defective in any of these proteins share distinct phenotypic traits such as shedding of membrane vesicles, release of periplasmic proteins and hypersensitivity to detergents (Lazzaroni et al., 1999
). Nevertheless, this so-termed membrane-leakage phenotype has been differentiated from the spontaneous shedding of outer-membrane vesicles that occurs, though to a much lesser extent, in actively growing bacteria (Kadurugamuwa & Beveridge, 1997
).
We describe below a correlation between the accumulation of proteins in the extracellular medium of S. enterica Dam- cultures and the disorganization of the cell envelope. Dam- mutants in exponential-growth phase are sensitive to deoxycholate and show reduced association to peptidoglycan of the envelope proteins PAL, TolB, OmpA and murein Lpp. As a result, lack of Dam methylation causes release of membrane vesicles to the extracellular medium of stationary cultures. Interestingly, these phenotypes occur concomitantly with the defect in secretion of SPI-1-encoded invasion proteins. Altogether, these data support the view that Dam methylation is necessary for both the proper activity of the SPI-1 type III secretion system and the maintenance of envelope integrity in Salmonella. We also describe that E. coli Dam- mutants do not display an equivalent phenotype of envelope instability.
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METHODS |
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Preparation of protein extracts.
Bacteria grown in LB medium to exponential (OD550 0·3) or stationary phase (OD550 1·0) were spun down by centrifugation at 10000 g, 15 min, 4 °C. The supernatant was filtered in a Millipore 0·22 µm pore size filter. Proteins were precipitated in 10% trichloroacetic acid (1 h, 4 °C), washed in acetone, suspended in PBS pH 7·4, and boiled in SDS-loading buffer as described by Kaniga et al. (1995) . These proteins constituted the secreted protein (SP) fraction. The bacterial pellet was washed twice in PBS pH 7·4 and boiled in SDS-loading buffer to obtain the cell-associated total protein (TP) fraction. To determine the presence of particulate material in the supernatant, the 0·22 µm filtrate was subjected to high-speed centrifugation (300000 g, 4 °C, 20 min) and the pellet was boiled in SDS-loading buffer. The proteins present in this pellet constituted the vesicle (VES) fraction. Proteins remaining in the supernatant after high-speed centrifugation were precipitated with the 10% trichloroacetic acid/acetone procedure as described above. This fraction was named vesicle-supernatant (VSN), which contained soluble extracellular proteins. All subcellular fractions were analysed for protein content by SDS-PAGE in Tris/Tricine buffer by using 8% or 10% acrylamide gels (Shägger & von Jagow, 1987
).
Analysis of membrane and peptidoglycan-associated proteins.
A subcellular fractionation method was followed to determine the relative amount of envelope proteins that were present in inner/outer membranes or firmly associated with peptidoglycan. Thus, 1010 bacteria were spun down (15000 g, 15 min, 4 °C), washed and suspended in PBS pH 7·4 buffer. The cells were disrupted by sonication. Unbroken cells were removed by low-speed centrifugation, 5000 g, 5 min, 4 °C. The supernatant was centrifuged at high speed (200000 g, 20 min, 4 °C) to obtain a pellet containing the envelope material, which was then treated with 0·4% Triton X-100 (3 h, 4 °C) and centrifuged at 15000 g, 30 min, 4 °C in a microfuge. The 0·4% Triton X-100 soluble material corresponded to the Sol fraction, enriched in inner-membrane proteins. The insoluble pellet was suspended in PBS pH 7·4 and corresponded to the fraction enriched in outer-membrane proteins (Ins fraction). Both the Sol and Ins fractions were finally suspended in SDS-sample buffer, boiled for 5 min and cleared by centrifugation before loading the samples in gels. To analyse proteins covalently bound or strongly associated with peptidoglycan,
2x1011 bacteria were used to purify peptidoglycan. Bacteria were spun down and washed as described above. The pellet was then suspended in 1·5 ml PBS pH 7·4 buffer, and slowly mixed with 1·5 ml boiling 8% SDS. Boiling at 100 °C was carried out for 3 h and then continued overnight at 80 °C. Macromolecular peptidoglycan was recovered by high-speed centrifugation (300000 g, 20 min, 30 °C). After four washing steps with warm (60 °C) distilled water, the pellet was suspended in 50 mM phosphate buffer pH 4·9 and digested overnight at 37 °C with N,O-diacetyl-muramidase (Cellosyl, Hoescht) (20 µg ml-1, 37 °C, 18 h). Samples were incubated at 100 °C for 20 min to inactivate the enzyme. Proteins released to the supernatant by peptidoglycan digestion were precipitated by simultaneous treatment with acid pH (pH 4·6 of muramidase buffer) and heat (100 °C, 10 min), a step included in the standard procedure to prepare muropeptides for HPLC analysis (Glauner, 1988
). After centrifugation at 15000 g, 15 min, 4 °C, the pellet containing the precipitated proteins and undigested peptidoglycan was suspended in 40 µl PBS pH 7·4. After addition of SDS-sample buffer, the sample was centrifuged (15000 g, 5 min, 4 °C) to remove undigested macromolecular peptidoglycan. The supernatant, containing proteins firmly associated with peptidoglycan (PG fraction) was then subjected to electrophoretic protein analysis. In all cases, the sample volume to load in the gels was adjusted by the optical density value of the culture at the time at which bacteria were collected.
Western analysis and antibodies.
Western analysis was performed using a Bio-Rad blotting system for protein transfer to nitrocellulose membranes. Specific proteins present in the different subcellular fractions were detected with the following primary antibodies: polyclonal rabbit anti-PAL and anti-TolB antibodies (a gift of Professor J.-C. Lazzaroni, CNRS-INSA-Université de Lyon, France), polyclonal rabbit anti-murein lipoprotein (Lpp) (a gift from M. Inouye, Robert Wood Johnson Medical School, Piscataway, NJ, USA), affinity-purified polyclonal rabbit anti-OmpA (a gift from Heinz Schwarz, Universität Tübingen, Germany), polyclonal rabbit-anti SipC from our sera collection raised against the SipC peptide ASDEARESSRKS, and polyclonal rabbit anti-ribosomal elongation factor Tu (a gift from M. Vicente, CNB-CSIC, Madrid, Spain). As secondary antibody, goat anti-rabbit IgG conjugated to peroxidase was used for the ECLassay as described by the manufacturer (Amersham-Pharmacia Biotech).
Electron microscopy.
The release of membrane vesicles was followed by transmission electron microscopy. Thus, the pellet obtained after high-speed centrifugation of culture supernatants (VES fraction) was fixed in 2% glutaraldehyde/2% paraformaldehyde in PBS buffer pH 7·4 (2 h, room temperature). After three washes (10 min each) in phosphate buffer, the pellets were post-fixed in 1% osmium tetroxide for 1 h at 4 °C. After three additional washes with PBS buffer pH 7·4, samples were dehydrated with increasing concentrations of ethanol (from 50% to 100%). Samples were then embedded in LR-White resin (London Resin Co., Reading, UK). Ultrathin sections were put onto nickel grids coated with a Formvar film, contrasted with lead citrate/uranyl acetate and visualized at 80 kV in a JEOL1200EX electron microscope. To visualize the integrity of bacterial envelope, the bacteria were fixed in the growth medium by adding a twofold concentrated glutaraldehyde/paraformaldehyde fixer solution. Fixation was maintained for 1 h at room temperature. After spinning down the bacteria, the pellet was washed twice with 0·1 M phosphate buffer pH 7·4 and processed for transmission electron microscopy as described for the pellet containing the VES fraction.
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RESULTS |
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It is known that specific invasion proteins, such as the Ipa proteins of Shigella flexneri, aggregate in the extracellular medium (Parsot et al., 1995 ). With this observation in mind, we compared the relative amount of the invasion protein SipC in the extracellular fractions SP, VES and VSN of Dam+ and Dam- strains. Western analysis showed that the amount of SipC secreted by the Dam- mutant was lower, in the range of 50%, than that of the wild-type (compare SP fractions, Fig. 1B
). However, most of the SipC protein detected in the SP fraction appeared as non-particulate material (VSN fraction) in both Dam+ and Dam- strains (Fig. 1B
). This result suggested that SipC neither forms extensive aggregates nor associates with the membranous particulate material released by Dam- mutants (Fig. 1B
). To further characterize the nature of the particulate material obtained upon high-speed centrifugation (VES fraction), this material was directly visualized by transmission electron microscopy. The analysis showed that intact flagella constituted the major extracellular particulate material of cultures containing the wild-type strain (Fig. 1C
). This observation was consistent with the protein pattern observed in the VES fraction prepared from the wild-type strain (Fig. 1A
). In contrast, the VES fraction of the Dam- mutant contained numerous membrane vesicles (Fig. 1C
). These results indicated that lack of Dam methylation causes exacerbated loss of envelope material in the form of membrane vesicles, a process that is accompanied by the presence of a large number of soluble proteins in the extracellular medium.
S. enterica mutants deficient in Dam methylase are sensitive to deoxycholate
The level of resistance to deoxycholate shows variations among strains of S. enterica serovar Typhimurium: for instance, strain 14028s is resistant to higher deoxycholate concentrations than SL1344 and LT2. However, under the conditions chosen for our experiments (LB plates containing 1% sodium deoxycholate), strains SL1344, 14028s and LT2 were all resistant to deoxycholate, while their Dam- derivatives were sensitive (Table 2). An additional observation was that, irrespective of their parental origin, Dam- mutants were more sensitive to deoxycholate during exponential growth than in the stationary phase (Table 2
). These data provide evidence that Dam- mutants of S. enterica have a defect in envelope stability, mostly during active growth. In contrast to S. enterica, a Dam- mutant of E. coli did not show deoxycholate sensitivity, even during exponential growth (Table 2
).
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DISCUSSION |
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In an attempt to characterize the basis for this phenotype, we separated the extracellular proteins by high-speed centrifugation into two fractions: (i) particulate material; and (ii) soluble material. In the Dam- mutant, over 15 proteins were visible in the particulate material. Some of these proteins were identified as flagellin (FliC), the outer-membrane proteins OmpA, OmpC, OmpX, and the murein lipoprotein Lpp by Western blotting and mass spectrometry analysis (data not shown). This result provided us with the first evidence for an involvement of Dam methylation in the maintenance of envelope integrity. In addition, electron microscopy analysis of fractions containing particulate material showed that stationary cultures of Dam- mutants contained large amount of membrane vesicles in the extracellular medium, whereas only flagella were visible in extracts from the wild-type strain. Interestingly, direct examination of whole bacteria in either exponential or stationary growth phases failed to show massive envelope defects in the Dam- mutant. Nevertheless, and in agreement with a recent study (Heithoff et al., 2001 ), our results show that Dam- mutants are highly sensitive to deoxycholate during active growth. The apparent contradiction between this sensitivity and the inability to observe major defects in actively growing Dam- mutants might indicate the existence of minor alterations in envelope stability sufficient to promote sensitivity to bile acids. These envelope alterations might be cumulative and, upon entrance into the stationary phase, culminate in envelope breakage in a subpopulation of bacteria.
The existence of an envelope defect in exponential cultures of Dam- mutants was confirmed by subcellular fractionation analysis: a set of proteins required for the maintenance of envelope stability were found to associate at lowered levels with the peptidoglycan of Dam- mutants. Among these proteins, a marked alteration of the association of TolB and PAL to peptidoglycan was observed in mutants lacking Dam methylase. Previous studies have shown that deficiency of any of these proteins in E. coli triggers release and leakage of membrane and periplasmic material into the growth medium (Bernadac et al., 1998 ; Lazzaroni et al., 1999
). Our study provides the first evidence for a similar envelope-stabilizing role of these proteins in S. enterica and, in addition, raises the hypothesis that such a role is controlled by Dam methylation. The fact that the alterations in proteinpeptidoglycan association were noticeable only in actively growing bacteria indicates that the envelope defect caused by lack of Dam methylation might reflect up-regulation of a natural process that occurs under conditions of high growth rate and cell wall turnover. This assumption agrees with previous studies that demonstrated the shedding of membrane vesicles as a process occurring predominantly in actively growing bacteria (Kadurugamuwa & Beveridge, 1995
, 1997
; Zhou et al., 1998
). Interestingly, and unlike Lpp and OmpA, lack of Dam methylation seems to affect the total amount of TolB and PAL proteins present in the envelope of actively growing bacteria. This observation contrasts with the similar levels of mRNA transcripts of the tolQRAB and pal operons detected in Dam+ and Dam- strains (unpublished observations), which suggests the existence of post-transcriptional regulatory mechanisms acting on TolB and PAL, and controlled by Dam methylation. These post-transcriptional regulatory mechanisms could be sustained by the stimulation of a normal physiological process (i.e. cell wall turnover). Alternatively, Dam methylation might control the synthesis of an as yet unknown cell function required for proper maintenance or positioning of the TolPAL protein complexes. The fact that the association to peptidoglycan of all the envelope proteins analysed was altered at some degree by the lack of Dam methylase suggests that a basic process of envelope physiology may be altered.
In addition to the set of proteins present in membranous material, about 2530 proteins were visible by Coomassie staining in the soluble, extracellular fraction (VSN) from Dam- mutants. Although we first considered that hypersecretion of VSN proteins could reflect some imbalance in protein secretion systems, two observations revealed that this was not the case. First, with the exception of the flagellin subunit FliC, the pattern of proteins present in the VSN fraction was unaltered when either the flagellar or invasion-specific type III systems were non-functional. Because most proteins secreted by S. enterica are either flagellar proteins or virulence-related proteins (Komoriya et al., 1999 ), we reasoned that many proteins present in the VSN fraction could derive from lysed bacteria. Furthermore, the detection of a ribosomal factor in the VSN fraction confirmed that the Dam- mutant undergoes lysis upon entering stationary phase. Some 25% of the Dam- bacterial population was estimated to lyse in stationary phase. Despite this small percentage, the concentration of extracellular proteins by acid precipitation hampered the visual analysis of secreted proteins. An example was the SipC protein, which is secreted at a 50% rate in a Dam- mutant (Fig. 1B
) (Garcia-del Portillo et al., 1999
), but was virtually masked by the bulk of proteins present in the extracellular fraction (SP) of the Dam- mutant.
Release of outer-membrane vesicles is a physiological process that occurs naturally in many bacterial pathogens. For instance, Shigella flexneri, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae, enterohaemorrhagic E. coli (EHEC) and Helicobacter pylori are all known to shed membrane vesicles into the culture medium (Kadurugamuwa & Beveridge, 1995 , 1997
, 1999
; Wai et al., 1995
; Keenan et al., 2000
). In some cases, these vesicles have been related to the transport of pathogenic factors. Examples include the VacA toxin of H. pylori (Keenan et al., 2000
), Shiga toxin and lipopolysaccharide by STEC (Shiga-toxin producing E. coli) and EHEC (Wai et al., 1995
; Meno et al., 2000
; Yokoyama et al., 2000
). In other cases outer-membrane vesicles have been shown to contain muramidases and other periplasmic enzymes (Kadurugamuwa & Beveridge, 1995
, 1999
). When these membrane vesicles fuse with the membrane of a different bacterial species, their content can trigger loss of viability of the recipient strain, or a physiological incorporation of this heterologous material into its envelope structure (Li et al., 1998
; Kadurugamuwa & Beveridge, 1999
). If outer-membrane shedding is a normal physiological process in S. enterica, it seems reasonable that it must be subjected to tight control. Our study suggests that Dam methylation may participate in such a control.
Unlike S. enterica, lack of Dam methylation did not cause any noticeable effect on envelope integrity or stability in E. coli. Biochemical analyses of protein content in extracellular fractions, deoxycholate sensitivity experiments, and studies of proteinpeptidoglycan interactions were all unable to show any difference between Dam+ and Dam- strains of E. coli. The basis of this unexpected difference between Salmonella and E. coli is at present unknown. One may, however, recall that the S. enterica genome contains genes with a putative role in peptidoglycan remodelling which are absent in E. coli (Hilbert et al., 1999 ).
Envelope instability might contribute to virulence attenuation of S. enterica Dam- mutants in the mouse typhoid model. Enhanced release of membrane vesicles through infected cells and tissues might cause overstimulation of the host immune response, as shown for Neisseria meningitidis (Mirlashari et al., 2001 ). Release of outer-membrane proteins such as OmpA, PAL and Lpp to the extracellular medium can occur in the presence of serum (Hellman et al., 2000a
, b
), and this process has been proposed as a major phenomenon mediating elicitation of immune host defence. Therefore, overstimulation of host defences during infection might render Dam- mutants unable to cause disease. These effects on the host would also sustain the vaccine properties of S. enterica Dam- mutants (Garcia-del Portillo et al., 1999
; Heithoff et al., 1999
, 2001
).
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ACKNOWLEDGEMENTS |
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Received 31 October 2001;
revised 12 December 2001;
accepted 19 December 2001.