1 Department of Molecular Genetics and Microbiology, PO Box 100266, University of Florida, Gainesville, FL 32610, USA
2 Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangzhou, China
Correspondence
Shouguang Jin
sjin{at}mgm.ufl.edu
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ABSTRACT |
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These authors contributed equally to this work.
Present address: Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, Republic of Korea.
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INTRODUCTION |
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TTSS of P. aeruginosa responds to various environmental signals, such as low calcium and direct contact with tissue-culture cells (Yahr et al., 1995; Frank, 1997
). Upon activation, the type III secretion apparatus translocates effector molecules into the cytoplasm of host cells, resulting in cell rounding and lifting and cell death by necrosis or apoptosis (Finck-Barbancon et al., 1997
; Pederson et al., 1999
; Kaufman et al., 2000
). There are four known effector molecules, including ExoS and ExoT, two homologous toxins with both ADP-ribosyltransferase and GTPase-activating protein activities, an acute cytotoxin, ExoU, with lipase activity, and an adenylate cyclase, ExoY (Yahr et al., 1996
, 1998
; Finck-Barbancon et al., 1997
; Hauser et al., 1998
; Sato et al., 2005
). It is well known that ExoS preferentially ADP-ribosylates several Ras family members (GTP-binding proteins) required for the regulation of intracellular vesicle transport, cell proliferation and differentiation (Coburn & Gill, 1991
; Ganesan et al., 1998
). The ADP-ribosyltransferase activity of ExoS has also been shown to be essential in triggering programmed cell death in various types of tissue-culture cells (Kaufman et al., 2000
; Jia et al., 2003
).
The type III secretion complex of P. aeruginosa is highly similar to that of members of the genus Yersinia and, when expressed in cells of this genus, the ExoS protein can be translocated into mammalian cells via the yersinia TTSS (Frithz-Lindsten et al., 1997, 1998
). Both organisms harbour multiple regulators to tightly control the expression of the large type III secretion gene clusters in response to low-calcium environmental stimuli. The mechanism by which the extracellular calcium concentration is translated into a transcriptional signal remains a mystery. In P. aeruginosa, the expression of type III-related genes is regulated coordinately by a transcriptional activator, ExsA (Yahr & Frank, 1994
; Hovey & Frank, 1995
). ExsA is a DNA-binding protein that recognizes a consensus sequence (TNAAAANA) located approximately 5152 bp upstream of the transcriptional start site to stimulate the expression of type III genes, including the exsA gene itself. More recently, a number of genes have been shown to affect the expression of type III genes, including adenylcyclase (cyaA), pseudouridinase (truA) and pyruvate dehydrogenase (aceAB) (Dacheux et al., 2002
; Wolfgang et al., 2003
; Ahn et al., 2004
). Meanwhile, the type III genes are also regulated negatively by ExsD, the RhlI/RhlR quorum-sensing system and stationary-phase sigma factor RpoS (McCaw et al., 2002
; Hogardt et al., 2004
).
In this report, we describe the finding that optimal secretion of P. aeruginosa type III effector molecules requires a protein factor, designated type III secretion factor (TSF), in addition to the calcium chelator. The TSFs in L broth and serum were identified as caseins and albumin, respectively. Further analysis revealed that low-affinity calcium-binding proteins have TSF activity, whilst high-affinity calcium-binding proteins do not. Significance of the current finding in understanding the pathogenhost interaction is discussed.
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METHODS |
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Purification and quantification of secreted ExoSFLAG.
Strain PAKexoST/pHW0029 was grown overnight in 100 ml L broth supplemented with 5 mM EGTA. Bacterial cells were collected by centrifugation at 15 000 g for 15 min and the culture supernatant was decanted into a new container with an equal volume of saturated ammonium sulfate and kept at 4 °C overnight. Protein precipitates were recovered by centrifugation and dissolved in 10 ml PBS. The protein sample was mixed with an anti-FLAG antibody and incubated at 4 °C for 2 h, followed by an additional 2 h incubation with 0·1 vol. protein Gagarose. The beads were washed twice with PBS-T buffer (100 mM phosphate buffer, 150 mM NaCl, 0·2 % BSA and 0·05 % Tween 20) and the bound ExoSFLAG was eluted by adding 100 µg FLAG peptide ml1 (Sigma) and reacting for 20 min at room temperature with gentle agitation. The quantity of eluted ExoSFLAG protein was measured by the bicinchoninic acid (BCA) protein-assay method. As a standard marker, serially diluted BSA solutions were reacted for 30 min at 37 °C with the BCA reagent mixture (Pierce). A560 values of the reactions in the plates were measured to calculate the amount of ExoSFLAG in the samples. The quantified ExoSFLAG was also evaluated by SDS-PAGE and Western blot analysis.
Sandwich ELISA for detection of ExoSFLAG in bacterial-culture supernatant.
As capturing antibody, rabbit anti-ExoS antibody was diluted in 0·1 M carbonate buffer (pH 9·6) and used to coat 96-well microtitre plates (Nunc). After overnight incubation at 4 °C, the plates were washed three times with PBS-T and each well was blocked with 50 µl 2 % BSA (Sigma) in PBS for 2 h at 37 °C. The plates were then washed and 50 µl culture supernatant or positive control (serial dilutions of a known amount of purified ExoSFLAG) was applied to each well. The plates were incubated at 37 °C for 1 h, washed and then 50 µl mouse anti-FLAG M2 mAb (diluted 2000-fold in PBS-T) was added to each well. After incubation at 37 °C for 1 h and washing, 50 µl HRP-conjugated goat anti-mouse IgG diluted in PBS-T was added to each well. The plates were incubated at 37 °C for 1 h, washed and then 50 µl citrate/phosphate buffer (pH 5·0) containing o-phenylenediamine (OPD; Sigma) as peroxidase substrate was added together with H2O2. A490 was measured with an automated ELISA reader (Dynatech Laboratories) after 30 min incubation at room temperature. Each sample was run in triplicate and the mean value of the A490 readings for the triplicate wells was taken. The amount of ExoSFLAG in each sample was determined by using a standard curve generated with a known amount of serially diluted ExoSFLAG protein. ExoS in culture supernatants is shown as ng ml1 in all figures.
Isolation of type III activating factor.
To identify the active fraction of the TSF from serum and L broth, the samples were separated into <10, 1050, 50100 and >100 kDa fractions by using corresponding molecular mass cut-off membrane filters (Amicon). A gel-filtration column (Bio-Rad Bio-Silect SEC 250-5, 300x7·8 mm) was further used for fractionation of serum and L broth on an HPLC system (Bio-Rad BioLogic DuoFlow system), using 0·1 M sodium phosphate, 0·15 M NaCl, pH 6·8, as the mobile phase. Each fraction was tested for the ability to activate ExoSFLAG secretion by sandwich ELISA. A 40 µl aliquot of each fraction was added to 360 µl DMEM containing 5 mM EGTA and 108 cells of PAKexoST/pHW0029 in 24-well plates. After incubating for 3 h at 37 °C in a CO2 incubator, 50 µl aliquots of samples were subjected to sandwich ELISA as described above.
Other methods.
A standard -galactosidase assay (Miller, 1972
) was conducted to determine the expression of exoS : : lacZ, exsA : : lacZ and exoT : : lacZ fusion genes. Standard methods were used for plasmid DNA preparation, restriction-enzyme digestion and cloning (Sambrook et al., 1989
).
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RESULTS |
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Caseins in L broth function as TSFs
Size fractionation of L broth by HPLC showed multiple fractions with TSF activity (Fig. 6d). As the protein components in L broth come from yeast extract and tryptone, we traced the TSF activities of these two components. Interestingly, both yeast extract and tryptone showed TSF activities, with tryptone being more active (data not shown). As tryptone is a pancreatic-digest product of caseins, the result indicated that TSF activity is associated with peptide fragments derived from caseins. Confirming this prediction, casein protein obtained from a commercial source (Sigma) had not only high TSF activity, but also heat-stable and protease-sensitive characters. Dose-dependent TSF activities of L broth and caseins were also verified. As seen from Western blots in Fig. 7
, ExoS secretion was only seen in the presence of L broth or casein in a concentration-dependent manner, further confirming the TSF function of the caseins.
The casein complexes of all species contain two types of proteins: the - and
-caseins, which bind calcium and aggregate, and
-casein, which does not bind calcium, but stabilizes the
- and
-caseins to yield the micelle or colloid (Swaisgood, 1993
). Pure individual casein protein components,
-,
- and
-caseins, were obtained from a commercial source (Sigma) and tested for their TSF activities. As shown in Fig. 8(a)
, both
- and
-caseins showed high TSF activity, but
-casein lacked TSF activity, suggesting the possible involvement of Ca2+-binding ability in TSF activity. Depletion of the
-casein from whole casein by using an anti-
-casein antibody linked to agarose beads did not eliminate the TSF activity, presumably due to the presence of
-casein. However, adsorption of the
-casein through anti-
-casein antibodyagarose abolished the TSF activity (Fig. 8c
), demonstrating that individual casein proteins function as TSFs.
Low-affinity, high-capacity Ca2+-binding proteins display TSF activity
There is no apparent amino acid sequence similarity or common structural motif between albumin and casein proteins; thus, the TSF function may involve certain physical properties. One common feature of albumin and casein is their low-affinity, high-capacity binding of calcium (Aguanno & Ladenson, 1982; Swaisgood, 1993
; Vorum et al., 1995
; Farrell et al., 2002
). To test whether the Ca2+-binding property is associated with the TSF activity, we surveyed a number of well-studied Ca2+-binding proteins, including high- and low-affinity Ca2+-binding proteins. Calcium-binding proteins localized within the cytoplasm (CaBP, CaM, troponin and S100), membrane-anchored (calreticulin and annexin V) and secreted (albumin, casein and
-lactalbumin) have been tested. As shown in Fig. 9
, albumin, casein,
-lactalbumin and calreticulin, representing low-affinity Ca2+-binding proteins, showed high TSF activities, whereas annexin V, CaBP, CaM and S100, representing high-affinity Ca2+-binding proteins, exhibited no TSF activity, with the exception of troponin. Troponin is composed of three subunits, C, I and T, of which the C subunit is known to have a high-affinity Ca2+-binding ability (Farah & Reinach, 1995
; Borovikov, 1999
). Tests of individual subunits of the troponin demonstrated that the TSF activity is not associated with the C subunit; rather, it is associated with the T subunit (Fig. 9
). Recently, the troponin T from avian flight muscle has been shown to have a Ca2+-binding activity (Zhang et al., 2004
). These data together suggested that low-affinity Ca2+-binding proteins possess TSF activity, although the reason for requirement of this calcium-binding property for TSF activity is not clear.
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DISCUSSION |
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To identify factors affecting the TTSS, we have established a sensitive sandwich ELISA system for the detection of secreted ExoSFLAG and optimized in vitro type III-inducing conditions. To test multiple samples at once, bacterial cells were grown in multiwell plates and the culture supernatants were transferred into 96-well microtitre assay plates for direct quantification of secreted ExoSFLAG. This method of ExoS detection has several advantages over conventional Western blot analysis; first, the assay does not require concentration of the bacterial-culture supernatant; second, it can accurately quantify the amount of secreted ExoSFLAG protein; and third, hundreds of samples can be assayed at once. With this sensitive and high-throughput assay system, we were able to pursue the mysterious TSF in serum and L broth. This assay system has also proven to be a powerful tool in screening transposon insertional mutant banks for altered type III secretion patterns, which otherwise were impossible to screen for a large number of mutants by using Western blot analysis (J. Kim & S. Jin, unpublished results).
Serum albumin is an abundant, multifunctional protein. It is the major protein component of blood plasma, present at a concentration of around 0·6 mM, but can also be found in bodily tissues and secretions. The protein binds calcium, but its primary role is to transport fatty acids. BSA is a heart-shaped, monomeric protein of 65 kDa and, upon denaturation, it assumes an L-shaped form (Curry et al., 1999). Calcium binding by albumin is a complex process characterized by multiple binding sites whose affinity and binding capacity are variable, depending on parameters such as temperature, pH and ion strength (Fogh-Andersen, 1977
; Besarab et al., 1981
; Kragh-Hansen & Vorum, 1993
). In previous reports, serum has been shown to be essential for the activation of TTSSs of members of the genera Shigella, Salmonella and Yersinia (Ménard et al., 1994
; Zierler & Galán, 1995
; Lee et al., 2001
) and BSA was shown to trigger type III secretion in yersiniae (Lee et al., 2001
). Based on the similarities among the TTSSs of these organisms and P. aeruginosa, it is likely that the albumin in serum is required for the activation of TTSSs in these bacteria. Also, interestingly, two previous reports have demonstrated a role of BSA and caseins in facilitating the secretion of ATP-utilizing enzymes by mucoid P. aeruginosa, as well as Mycobacterium bovis, to the surrounding medium (Zaborina et al., 1999a
, b
). Relevance of this observation to the activation of type III secretion is not clear.
In eukaryotic cells, cytoplasmic calcium concentrations in a resting state are low (108107 M), whilst extracellular concentrations are high (103 M). Accordingly, intracellular Ca2+-binding proteins have high binding strengths (logK=7), whilst extracellular Ca2+-binding proteins have low binding strengths (logK=34) (Vogel, 2002). Structurally, a major family of calcium-binding proteins is the EF-hand superfamily, so called because they all contain the EF-hand helixloophelix Ca2+-binding motif. Classical EF-hand proteins include calmodulin, parvalbumin and troponin C, whilst non-classical EF-hand proteins include the S100 protein family and calbindins (Persechini et al., 1989
; Berchtold, 1993
). However, no common structural motifs have been identified among low-affinity Ca2+-binding proteins. As low-affinity Ca2+-binding proteins displayed TSF activity, regardless of their amino acid sequences, this implies the possible involvement of Ca2+-binding activity in regulation of type III secretion. This also explains the observation that yeast extract also contains TSF molecules, as all living organisms encode low-affinity Ca2+-binding proteins. Efforts are under way to identify the minimal functional peptide fragments with TSF activity, with the hope to understand the features essential for TSF function, especially the requirement for a low-affinity Ca2+-binding ability. The observation that high-affinity Ca2+-binding proteins do not have TSF activity suggests that intracellular P. aeruginosa is unlikely to secrete type III effectors, which might increase the intracellular life span of the bacteria once inside the host cells.
The process of translocation is contact dependent and occurs without secretion to the surrounding medium during infection of eukaryotic cells (Rosqvist et al., 1994). Translocation has therefore been described as polarized, i.e. secretion takes place only at the zone of contact between the pathogen and the host cell. Based on the fact that many low-affinity Ca2+-binding proteins function as TSFs, the TSF-like molecules are probably abundant in host tissues, even on the host-cell surface; however, a low-Ca2+ environment is only found in the microenvironment surrounding host-cell surfaces or host cytosol (Heizmann & Berchtold, 1987
; Vogel, 2002
). Therefore, through co-evolution with its hosts, P. aeruginosa may have chosen abundant host proteins (low-affinity Ca2+-binding proteins) as TSF molecules to perceive the host environment while sensing high- or low-Ca2+ environments to distinguish distal vs proximal to the host-cell surface, ensuring type III injection into host cells rather than wasting into the surrounding tissues.
The requirement of TSF and EGTA for TTSS effector secretion indicates the existence of bacterial cell-surface component(s) interacting with the TSF, possibly in a Ca2+ concentration-dependent manner. In members of the genus Yersinia, three proteins, YopN, LcrG and TyeA, are involved in preventing secretion in vitro into Ca2+-containing media and the surrounding culture media during infection of eukaryotic cells. Strains mutated for any of these genes display de-repressed Yop expression and secretion in vitro, i.e. high levels of expression and secretion are seen, irrespective of the Ca2+ concentration (Forsberg et al., 1991; Nilles et al., 1997
; Day & Plano, 1998
; Cheng & Schneewind, 2000
; Matson & Nilles, 2001
). Differing from the genus Yersinia, mutation in the pcrV gene of P. aeruginosa has been shown to result in constitutive secretion of type III effector molecules (Sawa et al., 1999
; McCaw et al., 2002
). However, no interactions between PcrV and the identified TSF molecules were observed (J. Kim & S. Jin, unpublished results), suggesting alternative target molecules for the TSF. Efforts are under way to identify the TSF-binding bacterial cell-surface component(s) involved in the control of type III secretion.
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ACKNOWLEDGEMENTS |
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Received 17 June 2005;
accepted 22 June 2005.
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