Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Podsumowanie

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Streszczenie

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.

This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB's capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB's chaperone function in vivo.

Wprowadzenie

A common natural environment in which microbial pathogens experience acid-induced protein unfolding conditions is the mammalian stomach (pH range 1-4), whose acidic pH serves as an effective barrier against food-borne pathogens ¹. Protein unfolding and aggregation, which is caused by amino acid side chain protonation, affects biological processes, damages cellular structures and eventually causes cell death ^1,2. Since the pH of the bacterial periplasm equilibrates almost instantaneously with the environmental pH due to the free diffusion of protons through the porous outer membrane, periplasmic and inner membrane proteins of Gram-negative bacteria are the most vulnerable cellular components under acid-stress conditions ³. To protect their periplasmic proteome against rapid acid-mediated damage, Gram-negative bacteria utilize the acid-activated periplasmic chaperones HdeA and HdeB. HdeA is a conditionally disordered chaperone ^4,5: At neutral pH, HdeA is present as a folded, chaperone-inactive dimer. Upon a pH shift below pH 3, HdeA's chaperone function is quickly activated ^6,7. Activation of HdeA requires profound structural changes, including its dissociation into monomers, and the partial unfolding of the monomers ^6-8. Once activated, HdeA binds to proteins that unfold under acidic conditions. It effectively prevents their aggregation both during the incubation at low pH as well as upon pH neutralization. Upon return to pH 7.0, HdeA facilitates the refolding of its client proteins in an ATP-independent manner and converts back into its dimeric, chaperone-inactive conformation ⁹. Similarly, the homologous chaperone HdeB is also chaperone-inactive at pH 7.0. Unlike HdeA, however, HdeB's chaperone activity reaches its apparent maximum at pH 4.0, conditions under which HdeB is still largely folded and dimeric ¹⁰. Moreover, further lowering the pH causes the inactivation of HdeB. These results suggest that despite their extensive homology, HdeA and HdeB differ in their mode of functional activation allowing them to cover a broad pH range with their protective chaperone function. One other chaperone that has been implicated in the acid resistance of E. coli is the cytoplasmic Hsp31, which appears to stabilize unfolded client proteins until neutral conditions are restored. The precise mode of Hsp31's action, however, has remained enigmatic ¹². Given that other enteropathogenic bacteria such as Salmonella lack the hdeAB operon, it is very likely that other yet unidentified periplasmic chaperones might exist that are involved in acid resistance of these bacteria ¹¹.

The protocols presented here allow to monitor the pH-dependent chaperone activity of HdeB in vitro and in vivo ¹⁰ and can be applied to investigate other chaperones such as Hsp31. Alternatively, the complex network of transcription factors that control the expression of hdeAB can potentially be investigated by the in vivo stress assay. To characterize the chaperone function of proteins in vivo, different experimental setups can be applied. One route is to apply protein unfolding stress conditions and phenotypically characterize mutant strains that either overexpress the gene of interest or carry a deletion of the gene. Proteomic studies can be conducted to identify which proteins no longer aggregate under stress conditions when the chaperone is present, or the influence of a chaperone on a specific enzyme can be determined during stress conditions using enzymatic assays ^14-16. In this study, we chose to overexpress HdeB in an rpoH deletion strain, which lacks the heat shock sigma factor 32. RpoH controls the expression of all major E. coli chaperones and its deletion is known to increase sensitivity to environmental stress conditions that cause protein unfolding ¹⁵. The in vivo chaperone activity of HdeB was determined by monitoring its ability to suppress the pH sensitivity of the ΔrpoH strain. Altogether, the protocols presented here provide a fast and straightforward approach to characterize the activity of an acid-activated chaperone in vitro as well as in the in vivo context.

Protokół

1. Expression and Purification of Periplasmic HdeB

NOTE: HdeB was expressed in E. coli cells harboring the plasmid pTrc-hdeB¹⁰, and purified from the periplasm upon polymyxin lysis.

1. Prepare an overnight culture of E. coli cells harboring the plasmid pTrc-hdeB ¹⁰ in 30 ml LB containing 200 µg/ml ampicillin (LB_Amp). Inoculate four 1 L cultures of LB_Amp and grow them at 37 °C and 200 rpm until O.D._600nm of 0.7 is reached. Then, add 300 µM IPTG to induce the expression of HdeB and decrease the growth temperature to 30 °C.
2. After 5 hr of protein expression at 30 °C, harvest the cells by centrifugation at 8,000 x g for 5 min at 4 °C.
3. Wash the cell pellet with 100 ml buffer A (50 mM Tris/HCl, 50 mM NaCl, pH 7.5) and centrifuge the cells again at 8,000 x g for 5 min at 4 °C.
4. Subsequently, resuspend the cell pellet in 80 ml buffer A, containing 1 mg/ml polymyxin sulfate. For efficient disruption of the outer membrane, gently stir the suspension for 1 hr at 4 °C.
5. To remove the cytoplasmic fraction and cell debris, centrifuge the suspension for 20 min at 15,000 x g at 4 °C. This results in ~60 ml supernatant containing the soluble HdeB.
6. Dialyze the supernatant containing the periplasmic extract overnight against 150x volume of buffer B (20 mM Tris/HCl, 0.5 mM EDTA, pH 8.0) using a dialysis membrane with 6 kDa MW cut-off. Concentrate the proteins to 15 ml using centrifugal filter units with a molecular weight cut-off of 3 kDa. Filter the protein solution using a 0.2 µm pore filter.
7. Apply the protein onto an anion exchange chromatography column (column volume 5 ml) that has been equilibrated with 5 column volumes buffer B with a flow rate of 2.5 ml/min. Once the protein is loaded onto the column, wash the column with buffer B for 10 min at a flow rate of 2.5 ml/min. Elute HdeB with a linear gradient from 0 to 0.5 M NaCl in buffer B over a time period of 50 min with a flow rate of 2.5 ml/min ⁶.
8. Identify fractions containing HdeB using a 15% SDS-PAGE. Mix 20 µl sample with 5 µl 5x reduced SDS loading buffer. Load 10 µl onto the gel and run in Tris-glycine buffer (14.4 g/L glycine, 2.9 g/L Tris, 1 g/L sodium dodecyl sulfate, pH 8.3). Run the gel at 150 V until the bromophenol band has migrated close to the bottom of the gel (~45 min).
9. Pool all HdeB-containing fractions, dialyze at 4 °C overnight against 4 L HdeB storage buffer (50 mM Tris/HCl, 200 mM NaCl, pH 8.0), and concentrate the protein to approximately 300 µM using centrifugal filter units with a molecular weight cut-off of 3 kDa. Determine the concentration of HdeB at 280 nm using the extinction coefficient ε_280nm= 15,595 M^-1cm^-1. Prepare 100 µl aliquots and flash-freeze the aliquots in liquid nitrogen.
   NOTE: HdeB can be stored at -70 °C for at least 6 months.

2. Chaperone Activity Assay Using Thermally Unfolding Malate Dehydrogenase (MDH)

NOTE: The influence of purified HdeB on the aggregation of thermally unfolding porcine mitochondrial malate dehydrogenase (MDH) at different pH values was monitored as described below. All listed protein concentrations refer to the monomer concentration.

1. To prepare MDH, dialyze MDH at 4 °C overnight against 4 L buffer C (50 mM potassium phosphate, 50 mM NaCl, pH 7.5) and concentrate the protein to approximately 100 µM using centrifugal filter units with a molecular weight cut-off of 30 kDa.
   NOTE: Careful dialysis of MDH is required as MDH is delivered as ammonium sulfate solution.
2. To remove aggregates, centrifuge the protein for 20 min at 20,000 x g at 4 °C. Determine MDH concentration by absorbance at 280 nm (ε_{280 nm}= 7,950 M^-1 cm^-1). Prepare 50 µl aliquots of MDH and flash-freeze the aliquots for storage.
3. Place 1 ml quartz cuvette into a fluorescence spectrophotometer equipped with temperature controlled sample holders and stirrer. Set λ_ex/em to 350 nm.
4. Add appropriate volumes of pre-warmed (43 °C) buffer D (150 mM potassium phosphate, 150 mM NaCl) at the desired pH values (here: pH 2.0, pH 3.0, pH 4.0, and pH 5.0) to the cuvette and set the temperature in the cuvette holder to 43 °C. The total volume is 1,000 µl.
5. Add 12.5 µM HdeB (or alternatively the same volume of HdeB storage buffer for the buffer control) to the buffer, followed by the addition of 0.5 µM MDH. Begin monitoring light scattering. Incubate the reaction for 360 sec to allow sufficient unfolding of MDH.
6. Raise the pH to 7 by adding 0.16-0.34 volume of 2 M unbuffered K₂HPO₄ and continue recording light scattering for another 440 sec.
7. Set the extent of MDH aggregation that is recorded in the absence of the chaperone at a defined time point after neutralization (here after 500 sec, when maximal light scattering of MDH was observed) to 100%. Normalize HdeB's activity to the light scattering signal of MDH in the absence of HdeB at each indicated pH value.

3. Detection of HdeB-LDH Complex Formation by Analytical Ultracentrifugation (aUC)

NOTE: Sedimentation velocity experiments of HdeB alone or in complex with thermally unfolding lactate dehydrogenase (LDH) were performed using an analytical ultracentrifuge.

1. To prepare LDH, dialyze LDH at 4 °C overnight against 4 L buffer C (50 mM potassium phosphate, 50 mM NaCl, pH 7.5) and concentrate the protein to approximately 200 µM using centrifugal filter units with a molecular weight cut-off of 30 kDa.
   NOTE: Careful dialysis of LDH is required as LDH is delivered as ammonium sulfate solution.
2. To remove aggregates, centrifuge LDH for 20 min at 20,000 x g at 4 °C. Determine LDH concentration by absorbance at 280 nm (ε_{280 nm}= 43,680 M^-1 cm^-1). Prepare 50 µl aliquots of LDH and flash-freeze the aliquots for storage.
3. Incubate 3 µM LDH in the presence and absence of 30 µM HdeB in buffer D (pH 4 and 7, respectively) for 15 min at 41 °C.
   NOTE: Incubation of LDH at higher temperatures results in its complete aggregation, and no chaperone effect of HdeB can be observed.
4. Let the samples cool down to room temperature. Then, load samples into cells containing standard sector shaped 2-channel centerpieces with 1.2 cm path length. Load the cells into the ultracentrifuge and equilibrate to 22 °C for at least 1 hr prior to sedimentation.
5. Spin samples at 22 °C and 167,000 x g in the respective rotor for 12 hr, and monitor the sedimentation of the protein continuously at 280 nm. As previously demonstrated, the signal to noise ratio is improved when the transmitted light intensity of each channel is measured rather than absorbance. This also improves the quality of subsequent data fitting.
6. Conduct data analysis with SEDFIT (version 15.01b, December 2015), using the continuous c(s) distribution model ¹⁷. A tutorial describing how to use SEDFIT can be found in reference ¹⁸.
   1. Set the confidence level for the ME (Maximum Entropy) regularization to 0.7.
7. Calculate buffer density as well as viscosity using SEDNTERP ¹⁹. To estimate the amount of aggregated client protein, compare the integrals of sedimented LDH in pH 4 to pH 7 as a reference.
   NOTE: Integration of the sedimentation distribution plots can be done directly in SEDFIT. Alternative software to analyze sedimentation velocity data can be found in a recent review ²⁰.

4. Monitoring MDH Inactivation and Reactivation in the Presence of HdeB

NOTE: The influence of purified HdeB on the refolding of pH-unfolded MDH was determined by monitoring MDH activity upon neutralization.

1. Incubate 1 µM MDH in buffer D at the desired pH values (here: pH 2.0, pH 3.0, pH 4.0, and pH 5.0) for 1 hr at 37 °C in the absence or presence of 25 µM HdeB. Then, shift the temperature to 20° C for 10 min.
   NOTE: No MDH refolding was observed even in the presence of HdeB when MDH was incubated at temperatures higher than 37 °C.
2. To initiate refolding of acid denatured MDH, neutralize the samples to pH 7 by addition of 0.13-0.42 volume of 0.5 M sodium phosphate, pH 8.0.
3. After incubation for 2 hr at 20 °C, determine MDH activity by monitoring the decrease of NADH at 340 nm ⁹.
   NOTE: MDH catalyzes the NADH-dependent reduction of oxaloacetate into L-malate.
   1. Mix 50 µl of the incubation reaction with 950 µl of assay buffer (50 mM sodium phosphate, pH 8.0, 1 mM oxaloacetate, and 150 µM NADH).
      NOTE: The final concentration of MDH in the assay buffer should be 44 nM.
   2. Monitor the change in absorbance using a spectrophotometer, equipped with a Peltier temperature control block set to 20 °C.
   3. Report the MDH activity relative to 44 nM native MDH that has been kept at pH 7.0.

5. Effect of HdeB Overexpression on E. coli Survival under Acid Stress

NOTE: E. coli MG1655 genomic DNA was isolated using a published protocol ²¹.

1. Amplify hdeB from E. coli MG1655 by PCR using primers hdeB-BamHI-rev GGT GGT CTG GGA TCC TTA ATT CGG CAA GTC ATT and hdeB-EcoRI-fw GGT GCC GAA TTC AGG AGG CGC ATG AAT ATT TCA TCT CTC C.
2. Set up the PCR reaction in 50 µl as follows: 10 µl 5x polymerase buffer, 200 µM dNTPs, 0.5 µM primer JUD2, 0.5 µM primer JUD5, 150 ng genomic DNA MG1655, 0.5 µl DNA polymerase, add ddH₂O to 50 µl.
3. Perform amplification of hdeB as follows: Step 1: 5 min at 95 °C, 1 cycle; step 2: 30 sec at 95 °C, 30 sec at 55 °C, 30 sec at 72 °C, 40 cycles; Step 3: 10 min at 72 °C.
4. Clone resulting PCR fragment into the EcoRI and BamHI sites of plasmid pBAD18 using standard methods for restriction site cloning. Purify the plasmid using a plasmid purification kit according to manufacturer's instructions. Verify the resulting plasmid by sequencing ¹⁰.
5. Transform the plasmid expressing HdeB or the empty vector control pBAD18 into strain BB7224 (ΔrpoH) (genotype: F^-, λ^-, e14^-, [araD139]_B/r Δ(argF-lac)169 flhD5301 Δ(fruK-yeiR)725(fruA25) relA1 rpsL150(Sm^R) rbsR22 Δ(fimB-fimE)632(::IS1) ptsF25 zhf::Tn10(Tc^S) suhX401 deoC1 araD⁺ rpoH::kan⁺;¹⁶) using chemically competent cells.
   NOTE: This strain is temperature-sensitive.
6. After 45 sec heat-shock at 42 °C and prior to the plating, incubate cells at 30 °C and 200 rpm. Perform single colony streak-outs of the positive clones and incubate overnight at 30 °C. Prepare an overnight culture in 50 ml LB_Amp and cultivate the cells at 200 rpm and 30 °C.
7. Dilute overnight cultures 40-fold into 25 ml LB_Amp and grow the bacteria in the presence of 0.5% arabinose (Ara) at 30 °C and 200 rpm to an O.D._600nm = 1.0 to induce HdeB protein expression.
8. For the pH shift experiments, use LB_Amp+Ara to dilute the cells to O.D._600nm of 0.5 and adjust to the respective pH values (here: pH 2.0, pH 3.0, and pH 4.0) by adding appropriate volumes of 5 M HCl.
9. After the indicated time points (pH 2, 1 min; pH 3, 2.5 min; pH 4, 30 min), neutralize the cultures by addition of the appropriate volumes of 5 M NaOH.
10. Monitor the growth of the neutralized cultures in liquid culture for 12 hr at 30 °C using O.D. measurements.

Wyniki

HdeA and HdeB are homologous E. coli proteins, known to protect periplasmic proteins against acid stress conditions ¹⁰. Our work revealed that similar to HdeA, HdeB also functions as an acid activated molecular chaperone. However, in contrast to HdeA, HdeB functions at a pH that is still potentially bactericidal, but significantly higher than the pH optimum of HdeA ^6,9,10,22. To investigate the pH optimum of HdeB's chaperone activity in vitro, n...

Dyskusje

In order to study the mechanism of activation and chaperone function of HdeB, large quantities of HdeB have to be expressed and purified. A number of expression vector systems are available for the production of high levels of a target protein, including pTrc or pBAD vectors, both of which were used in this study. The promoters are readily accessible for E. coli RNA polymerase and thus allow strongly upregulated expression of HdeB in any E. coli strain. This aspect is especially relevant for in vivo...

Materiały

Name	Company	Catalog Number	Comments
NEB10-beta E. coli cells	New England Biolabs	C3019I
Ampicillin	Gold Biotechnology	A-301-3
LB Broth mix, Lennox	LAB Express	3003
IPTG	Gold Biotechnology	I2481C50
Sodium chloride	Fisher Scientific	S271-10
Tris	Amresco	0826-5kg
EDTA	Fisher Scientific	BP120-500
Polymyxin B sulfate	ICN Biomedicals Inc.	100565
0.2 µm pore sterile Syringe Filter	Corning	431218
HiTrap Q HP (CV 5 ml)	GE Healthcare Life Sciences	17-1153-01
Mini-Protean TGX, 15%	Bio-Rad	4561046
Malate dehydrogenase (MDH)	Roche	10127914001
Potassium phosphate (Monobasic)	Fisher Scientific	BP362-500
Potassium phosphate (Dibasic)	Fisher Scientific	BP363-1
F-4500 fluorescence spectrophotometer	Hitachi	FL25
Oxaloacetate	Sigma	O4126-5G
NADH	Sigma	N8129-100MG
Sodium phosphate monobasic	Sigma	S9390-2.5KG
Sodium phosphate dibasic	Sigma	S397-500
Lactate dehydrogenase (LDH)	Roche	10127230001
Beckman Proteome Lab XL-I analytical Ultracentrifuge	Beckman Coulter	392764
Centerpiece, 12 mm, Epon Charcoal-filled	Beckman Coulter	306493
AN-50 Ti Rotor, Analytical, 8-Place	Beckman Coulter	363782
Wizard Plus Miniprep Kit	Promega	A1470	used for plasmid purification (Protocol 5.1)
L-arabinose	Gold Biotechnology	A-300-500
Glycine	DOT Scientific Inc	DSG36050-1000
Fluorescence Cell cuvette	Hellma Analytics	119004F-10-40
Oligonucleotides	Invitrogen
Phusion High-Fidelity DNA polymerase	New England Biolabs	M0530S
dNTP set	Invitrogen	10297018
Hydrochloric Acid	Fisher Scientific	A144-212
Sodium Hydroxide	Fisher Scientific	BP359-500
Amicon Ultra 15 ml 3K NMWL	Millipore	UFC900324
Centrifuge Avanti J-26XPI	Beckman Coulter	393127
Varian Cary 50 spectrophotometer	Agilent Tech
Spectra/Por 1 Dialysis Membrane MWCO: 6 kDa	Spectrum Laboratories	132650
Amicon Ultra Centrifugal Filter Units 30K	Millipore	UFC803024
SDS	Fisher Scientific	bp166-500
Veriti 96-Well Thermal Cycler	Thermo Fisher	4375786