1 Centre for Molecular Microbiology and Infection, Department of Biological Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
2 Molecular Infectious Diseases Group, Department of Paediatrics, Faculty of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK
Correspondence
Jun Yu
jun.yu{at}imperial.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Lipopolysaccaride (LPS) is a major component of the Gram-negative bacterial envelope. It consists of lipid A, as well as an inner and an outer core oligosaccaride to which a repeating O antigen region is attached. Loss of LPS has been shown in many organisms, including S. flexneri (Okamura et al., 1983; Rajakumar et al., 1994
), to affect virulence as well as other phenotypes such as colony morphology, biofilm production and outer membrane permeability (Genevaux et al., 1999
; Nesper et al., 2001
).
A galU mutant of S. flexneri, the LPS of which consisted of only the lipid A and inner core region, was found to invade epithelial cells well, but had lost its ability to disseminate through a epithelial cell monolayer owing to aberrant localization of IcsA (Sandlin et al., 1995). Instead of being directed to the old pole of the bacterium, IcsA appeared to be evenly distributed around the surface of the galU mutant. As would be expected, this mutant was unable to form plaques in L2 cell monolayers and failed to provoke keratoconjunctivitis in guinea pigs.
YihE is a 38·1 kDa cytoplasmic protein of unknown function that is present in many enteric bacteria. yihE lies upstream from dsbA and is co-transcribed with it. Although dsbA is expressed independently from a 70 promoter, the transcript that originates from the CpxRA-regulated promoter upstream of yihE continues through both genes. Mutation of yihE in Escherichia coli reduces the amount of DsbA present in the periplasm, but the folding of proteins that require DsbA, such as PhoA, is unaffected (Belin & Boquet, 1994
). The CpxRA two-component signal transduction system senses and responds to envelope stress caused by accumulation of misfolded proteins by up-regulating the expression of dsbA, ppiA and degP (Otto & Silhavy, 2002
). Monitoring the biogenesis of P-pili is one such example (Hung et al., 2001
). In addition, Cpx is responsible for surface sensing and adhesion in E. coli (Otto & Silhavy, 2002
).
On DNA array analysis, a yihE mutant (Sh54) of S. flexneri serotype 5a, which has a 398 bp internal deletion in yihE, shows a global change in gene expression during exponential growth (Li et al., 2001). Many genes involved in energy metabolism and carbon degradation are down-regulated while some stress-inducible genes are up-regulated, although dsbA is unaffected. Amongst the severely depressed genes are galETKM, the galactose utilization operon, the expression of which is reduced by as much as 11·2-fold. Depression of the gal operon to this degree may be expected to reduce the cell's ability not only to utilize galactose, but also to produce UDP-glucose and UDP-galactose. As both of these compounds are required for production of the outer core of LPS, we initiated the present study to investigate the consequences of the yihE mutation on LPS phenotype.
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METHODS |
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S. flexneri strains were grown at 37 °C overnight on tryptic soy agar (TSA) (Oxoid) containing 0·1 % Congo red. Red colonies were inoculated into tryptic soy broth and grown to early exponential phase for cell infection. Ampicillin was added to a final concentration of 200 µg ml1 to select for the presence of plasmid pAA101.
LPS preparation and electrophoresis.
LPS was prepared from Shigella strains as described by Hitchcock & Brown (1983). Bacteria grown overnight on TSA were resuspended in PBS to an OD600 of 0·8. The bacterial pellet from 1·5 ml of the suspension was then resuspended in 125 µl lysis buffer (0·1 M Tris/HCl pH 6·8, 2 % SDS, 4 %
-mercaptoethanol and 10 %, v/v, glycerol) and boiled for 10 min. Proteinase K (50 µg, Sigma) was added and the mixture incubated for 1 h at 60 °C. Samples were run on a Tris-Tricine gel (Schagger & von Jagow, 1987
) and visualized by silver staining (Tsai & Frasch, 1982
).
Antimicrobial sensitivity and growth supplement tests.
Antimicrobial sensitivity tests were performed in a microtitre plate in which the antibacterial agent had been serially (1 : 2) diluted from 100 mg l1 to 0·097 mg l1 (1 : 2 dilution from 20 % in the case of SDS). From a mid-exponential phase culture, 100 µl of bacterial suspension was added to an OD600 of approximately 0·05. The plates were incubated overnight at 37 °C, and bacterial growth scored by eye. Glucose and galactose requirements were tested by growth at 37 °C overnight on M9 minimal agar with either glucose or galactose (0·4 % in each case) as sole carbon source.
Infection of HeLa cells, cell stain and microscopy.
HeLa cells were cultured in minimal essential medium (MEM) (Gibco-BRL), supplemented with 10 % fetal bovine serum, at 37 °C under 5 % CO2. Gentamicin protection assays were carried out as described by Sansonetti et al. (1986). Infection was stopped at the appropriate time by extensive washing with PBS, and intracellular bacteria were recovered by lysing cells with 0·5 % sodium deoxycholate and plating out serial dilutions on TSA plates. For microscopy, cells were fixed with 3·7 % paraformaldehyde for 20 min and then stained with Giemsa, or Bodipy FL phallacidin and a mouse anti-IcsA antiserum followed by Texas red-conjugated goat anti-mouse antibody (Molecular Probes). Observations were made with a BH2-RFCA fluorescence microscope (Olympus Optical) and an LSM-510 confocal laser scanning microscope (Zeiss).
Plaque formation assay.
This was carried out according to Oaks et al. (1985) using confluent CaCo-2 cell monolayers.
Lactate dehydrogenase (LDH) activity assay.
The LDH assay was performed using the Cytotox 96 non-radioactive cytotoxicity assay system (Promega), according to the manufacturer's instructions. The assay measures the conversion of a tetrazolium salt to a red formazan product, detectable by absorbance measurement at 490 nm. For this, a TherMo Max Plate Reader (Molecular Devices) was used.
Sereny test.
The Sereny test for guinea pig keratoconjunctivitis was used to test the virulence of the S. flexneri strains in vivo.
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RESULTS |
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Intracellular motility of Sh54
galU and other LPS mutants all have defective intra- and intercellular motility due to aberrant localization of IscA (Sandlin et al., 1995). We therefore examined the motility of Sh54 by staining S. flexneri-infected HeLa cells with Bodipy FL phallacidin. As shown in Fig. 2
(a), M90TS was able to form characteristic actin comet tails in the host cells, indicating its ability to polymerize host actin to achieve intra- and intercellular spread. In contrast, the characteristic actin tails were not detected in Sh54-infected HeLa cells (Fig. 2b
). Furthermore, using an anti-IcsA antibody, IcsA was detected on the entire bacterial surface of Sh54 within the host (Fig. 2b
), indicating that Sh54 had similar aberrant localization of IcsA as the galU mutant reported previously (Sandlin et al., 1995
). The same fluorescent labelling procedure, however, was not sensitive enough to detect IcsA on the surface of M90TS within HeLa cells (Fig. 2a
).
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Sh54 is attenuated in virulence
Sh54 formed no plaques on CaCo-2 cell monolayers, but complementation with pAA101 restored the formation of wild-type plaques (Fig. 4). The aberrant intracellular motility of Sh54 (Fig. 2
) offered one explanation for the plaqueless phenotype, but not in a straightforward fashion, because Sh54 apparently possessed epithelial cytotoxic activity that led to self-destruction (Fig. 3
). The Sereny test (guinea pig keratoconjunctivitis) results indicated that Sh54 did not cause any manifestation of conjunctivitis during a period of 2 weeks' observation. M90TS, on the other hand, provoked a typical keratoconjunctivitis 72 h post-infection.
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DISCUSSION |
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With a truncated LPS, Sh54 is more sensitive than the wild-type to various antimicrobial agents (Table 1). It has been well documented that a deep rough LPS structure can increase permeability to hydrophobic agents such as SDS and vancomycin, possibly by increased levels of phospholipids in the outer leaflet of the outer membrane (Nesper et al., 2001
; Vaara, 1992
). Changes in LPS structure leading to increased sensitivity to gentamicin, possibly through an increase in self-promoted uptake, have also been documented (Genevaux et al., 1999
). This suggests that increased sensitivity to penicillin G may be due to subtle perturbations in the overall structure of the outer membrane. Transcriptome analysis revealed no significant difference between M90TS and Sh54 in the expression of the major porins (Li et al., 2001
), and the SDS-PAGE outer membrane protein profiles of M90TS, Sh54 and Sh54/pAA101 were identical (data not shown).
It is now clear that yihE and dsbA play different roles in modulating Shigella virulence, though their expression is subjected to the same transcriptional regulation by cpxRA (Belin & Boquet, 1994). yihE mainly dictates actin-based cellular motility via its role in LPS synthesis, through regulation of the galETK expression that determines surface expression of IcsA (Fig. 2
). DsbA, on the other hand, is vital for intracellular survival and growth (Yu, 1998
), though its key role in the secretion of Ipa proteins into the protrusions also affects cell-to-cell spread, and this, in part, contributes to the cellular growth defect of the mutants (Yu et al., 2000
).
Sh54 possesses an unusual epithelial cytotoxic phenotype, which is characteristic of neither wild-type nor dsbA mutants (Fig. 3). As the supernatant from Sh54 culture has no epithelial cytotoxicity, the undiscovered cytotoxic substance(s) is more likely to be produced after bacterial entry into the host cells. Despite having an epithelial cytotoxic phenotype in vitro, Sh54 is attenuated, as shown by the guinea pig keratoconjunctivitis model. In the light of the recent finding that LPS is required for epithelial adherence and activation of the ERK pathway leading to production of IL-8 (Köhler et al., 2002
), the attenuation of Sh54 in vivo is likely to be multi-factorial.
How inactivation of yihE leads to galEKT depression, and indeed a global change of gene expression, is an important question to answer for a better understanding not only of Shigella virulence but also of the bacterial stress response and bacterial physiology in general. It is known that the cpxRA two-component signal transduction system and E work together to govern the extracytoplasmic stress response caused by accumulation of misfolded proteins (Raivio & Silhavy, 1999
). Many genes in the cpxRA regulon encode either protein-folding catalysts (such as DsbA, which catalyses disulphide bond formation, and PpiA, which catalyses peptidyl-prolyl cis/trans isomerization), or proteases (such as DegP), which respond directly to the presence of misfolded proteins in the periplasm. The YihE protein, on the other hand, has been predicted to be cytoplasmic (Belin & Boquet, 1994
). So YihE probably plays a regulatory role in the bacterial envelope stress response, which is consistent with inactivation of yihE triggering a global change of gene expression (Li et al., 2001
).
To our knowledge, this is the first study that relates YihE, via regulation of the gal operon, with a phenotype showing an impaired pattern of LPS synthesis. Further functional studies on YihE are required for a full explanation of the phenotype observed.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 15 October 2003;
revised 19 December 2003;
accepted 19 December 2003.
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