HtrA chaperone activity contributes to host cell binding in Campylobacter jejuni
© Bæk et al; licensee BioMed Central Ltd. 2011
Received: 24 August 2011
Accepted: 22 September 2011
Published: 22 September 2011
Acute gastroenteritis caused by the food-borne pathogen Campylobacter jejuni is associated with attachment of bacteria to the intestinal epithelium and subsequent invasion of epithelial cells. In C. jejuni, the periplasmic protein HtrA is required for efficient binding to epithelial cells. HtrA has both protease and chaperone activity, and is important for virulence of several bacterial pathogens.
The aim of this study was to determine the role of the dual activities of HtrA in host cell interaction of C. jejuni by comparing an htrA mutant lacking protease activity, but retaining chaperone activity, with a ΔhtrA mutant and the wild type strain. Binding of C. jejuni to both epithelial cells and macrophages was facilitated mainly by HtrA chaperone activity that may be involved in folding of outer membrane adhesins. In contrast, HtrA protease activity played only a minor role in interaction with host cells.
We show that HtrA protease and chaperone activities contribute differently to C. jejuni's interaction with mammalian host cells, with the chaperone activity playing the major role in host cell binding.
KeywordsHtrA chaperone protease Campylobacter jejuni INT-407 phagocytosis virulence
The enteric pathogen Campylobacter jejuni is a frequent cause of bacterial food-borne infections worldwide . Acute gastroenteritis caused by C. jejuni is characterized by watery or bloody diarrhea, abdominal pain, fever, and malaise. While these symptoms typically last 3 - 7 days, serious complications may follow such as the acute autoimmune disease Guillan Barré Syndrome, affecting the peripheral nervous system. To cause disease in humans, C. jejuni must penetrate the mucus layer of the gastrointestinal epithelium and interact with the underlying epithelial cells . The importance of epithelial cell invasion in disease has been demonstrated in infected humans and animals [3, 4], and is emphasized by studies showing that C. jejuni mutants attenuated for virulence in animal models are less capable of invading intestinal epithelial cells in vitro[5, 6]. Upon invasion by C. jejuni, human epithelial cells respond by secreting cytokines, such as IL-8, which stimulate recruitment of inflammatory cells , including macrophages and dendritic cells that engulf and rapidly kill C. jejuni. Adherence to epithelial cells is a prerequisite for invasion, and capsular polysaccharides, motility, and a number of surface associated proteins including CadF, CapA, JlpA and FlpA are required for efficient adherence of C. jejuni to epithelial cells [8–14]. Furthermore, metabolic processes in C. jejuni are also important for invasion of epithelial cells [15, 16].
HtrA is a highly conserved periplasmic protein that possesses both protease and chaperone activity [17, 18], and it has been demonstrated that HtrA is important for virulence of a number of bacterial pathogens such as Salmonella enterica serovar Typhimurium , Listeria monocytogenes, Klebsiella pneumonia and Yersinia enterocolitica. It is well established that HtrA is important for stress tolerance and survival of most bacteria, because HtrA degrades and prevents aggregation of periplasmic proteins that misfold during stress [23–25], however, only a few studies have investigated the individual role of the protease and chaperone activity of HtrA in virulence. Recently, it was shown that Salmonella Typhimurium requires both the HtrA protease and chaperone activity to grow in the liver and spleen of infected mice . In contrast, only the chaperone activity of HtrA is important for spread of Shigella flexneri in cultured epithelial cells, possibly because HtrA is involved in the folding of the surface located virulence factor, IcsA [27, 28]. This is consistent with a model for outer membrane biogenesis in non-pathogenic Escherichia coli proposing that the chaperone activity of HtrA ensures a safe transit of proteins across the periplasm and their assembly into the outer membrane . Thus, even though HtrA is a conserved protein it is unpredictable whether the protease or chaperone activity is involved in virulence.
Several studies have suggested that HtrA is important for virulence of C. jejuni. Recently, an insect infection model was used to show that the outcome of a C. jejuni infection is affected by HtrA, as fewer Galleria mellonella larvae are killed by an htrA mutant than by the isogenic wild type strain . In addition, we previously showed that attachment of C. jejuni to epithelial host cells is highly dependent on HtrA . Furthermore, an htrA mutant was isolated from a C. jejuni transposon library screened for reduced invasion ability . While several studies have revealed a role of HtrA in virulence of pathogenic bacteria, the question whether HtrA employs the chaperone or protease activity to promote bacterial virulence has received only limited attention. In the present study, we explore the role of HtrA in the virulence of C. jejuni by assessing the requirement for each HtrA activity in the interaction with host cells.
Results and discussion
Interaction of C. jejuni htrA mutants with epithelial cells and macrophages
Interaction of C. jejuni htrA mutants with macrophages
30 ± 10
21 ± 9
KB1025 (htrA S197A)
96 ± 33
93 ± 44
Taken together, these results show that HtrA chaperone activity plays an important role in the attachment of C. jejuni to both epithelial cells and macrophages. We previously showed that the chaperone activity of HtrA is important for periplasmic protein homeostasis in C. jejuni under non-stress conditions , and we therefore speculate that HtrA of C. jejuni is involved in folding of periplasmic or outer membrane proteins. In E. coli and Shigella, HtrA acts as a chaperone that mediates proper folding and insertion of proteins into the outer membrane [27, 29]. In C. jejuni, HtrA may therefore be essential for the function of one or several adherence factors, which may explain why lack of HtrA has a 5-10 times larger effect on adherence than lack of any single surface adhesin, such as CadF, CapA, JlpA and FlpA [11–14]. Interestingly, C. jejuni lacking PEB4, a homolog of the periplasmic chaperone SurA, adheres to epithelial cells 50-100 fold less efficiently than wild-type , emphasizing the importance of periplasmic chaperones for the adherence of C. jejuni to host cells.
Surface properties and protein secretion of C. jejuni htrA mutants
It has long been known that HtrA is important for virulence of pathogenic bacteria, but not much attention has been given to the role of the individual activities of HtrA. This study demonstrates that particularly the chaperone activity of HtrA has a significant impact on the interaction between C. jejuni and host cells. Lack of HtrA reduced bacterial binding to epithelial cells 5-10 times more than lack of any known adhesin [11–14], suggesting a pleiotropic effect. Even though HtrA traditionally has been viewed as a stress response protein, our data indicate that HtrA has specific functions during infection that may be stress-independent. This suggestion correlates with growing evidence from other bacteria showing that the chaperone activity of HtrA is involved in folding of virulence factors.
Bacterial strains and growth conditions
C. jejuni NCTC11168 (National Collection of Type Cultures), C. jejuni NCTC11168 htrA::cat (LB1281, ), C. jejuni NCTC11168 htrA S197A (KB1025, ), and C. jejuni NCTC11168 flaAB (CV1178) were routinely grown on blood agar base II (Oxoid) supplemented with 5% bovine blood, or in brain heart infusion (BHI) broth (Oxoid) at 37°C in a microaerobic environment (6% O2, 6% CO2, 4% H2, and 84% N2). CV1178 was constructed by natural transformation of C. jejuni NCTC11168 with chromosomal DNA from a C. jejuni 81116 flaAB mutant , followed by selection for kanamycin resistance.
Stock cultures of INT 407 human embryonic intestinal epithelial cells were grown in minimal essential medium (MEM; Gibco) supplemented with 10% (vol/vol) fetal bovine serum (FBS; Gibco) and maintained at 37°C in a humidified, 5% CO2 incubator. Stock cultures of J774.1 murine macrophage-like cells were grown in RPMI1640 (Gibco) supplemented with 10% (vol/vol) FBS and maintained at 37°C in a humidified, 5% CO2 incubator.
Gentamicin protection assay
Adherence and invasion/phagocytosis assays were performed with monolayers of INT407 epithelial cells or J774.1 macrophage cells growing in MEM (INT407) or RPMI (J774.1) supplemented with 10% FBS at 37°C in a humidified microaerobic atmosphere containing 5% CO2. Approximately 4 × 107 bacterial cells in MEM or RPMI were centrifuged at 400 rpm onto a monolayer consisting of 4 × 105 host cells and incubated for 1 h. The actual inocula were enumerated by plate count. To determine adherence, the monolayers were washed three times with 0.9% NaCl, and host cells were lysed by adding 0.1% Triton X-100. Adhered bacteria were enumerated by plate count. To determine invasion/phagocytosis, the infected monolayers were incubated in MEM or RPMI containing 100 μg ml-1 gentamicin for 2 h at 37°C microaerobic-5% CO2 atmosphere to kill extracellular bacteria. The monolayers were washed three times with 0.9% NaCl, host cells were lysed with 0.1% Triton X-100, and internalized bacteria were enumerated by plate count. For the macrophage assay, the data from four individual experiments in duplicate was normalized to the values for the wild type, due to large variations between individual experiments. For the viability-control assay, a monolayer consisting of 4 × 105 INT407 cells was incubated in 1 ml MEM supplemented with 10% FBS at 37°C in 5% CO2. After 1 h, 900 μl medium was transferred to an empty well and approx. 4 × 107 bacterial cells were added and incubated for 1 h at 37°C in a humidified microaerobic atmosphere containing 5% CO2. Subsequently, Triton X-100 was added to 0.1% and bacteria enumerated by plate count.
Secreted C. jejuni proteins were detected essentially as described in  and  with minor modifications. Briefly, C. jejuni NCTC11168, LB1281 and KB1025 were cultured O/N in Mueller-Hinton (MH) broth on MH agar containing 0.1% sodium deoxycholate (Sigma) to stimulate expression of the cia genes . Bacteria were harvested in RPMI1640 without methionine (R7513, Sigma), pelleted at 8000 × g, and washed twice in RPMI1640. Six milliliters of a bacterial suspension containing approximately 109 CFU was labeled with L-35S-methionine (Perkin Elmer Life Sciences) at a concentration of 50 μCi ml-1, and incorporation of 35S-methionine was allowed for 30 min at 37°C under microaerobic conditions. Subsequently, the bacterial suspensions were incubated microaerobically at 37°C for 30 min with 128 μg ml-1 chloramphenicol to stop protein synthesis. The suspensions were then incubated with 1% FBS (Gibco) for 30 min at 37°C under microaerobic conditions to stimulate protein secretion. Subsequently, the bacteria were harvested at 8000 × g, and the supernatants, containing the secreted proteins, were filtrated through a 0.2 μm filter, and proteins were concentrated by adding five volumes of ice-cold 1 mM HCl-acetone followed by incubation O/N at -20°C. The precipitated proteins were sedimented at 13,000 × g and resuspended in water, followed by dialysis (6-8000 MWCO) to remove un-incorporated 35S-methionine. The secreted proteins and whole-cell lysates were separated by SDS-PAGE and the dried gels were exposed to a phosphorimager screen (PerkinElmer) to detect labeled proteins.
Surface associated proteins were extracted essentially as described by McCoy et al. . Briefly, C. jejuni cells were harvested from blood agar plates, and the pellet was resuspended in 0.2 M glycine-HCl pH 2.2 and incubated at room temperature for 10 min followed by removal of cells at 6,000 × g. Protein content in the extracts was quantified by densitometri of Amido Black stained proteins with BSA as standard, and equal amounts were separated by SDS-PAGE and stained with Coomassie Brillant Blue.
Dr. Mark Reuter is thanked for providing chromosomal DNA from the C. jejuni flaAB mutant. We sincerely appreciate the expert technical assistance of Christel Galschiøt Buerholt and Jan Pedersen. This study was financially supported by the Faculty of Life Sciences, University of Copenhagen.
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