Properties of the HtrA Protease From Bacterium Helicobacter pylori Whose Activity Is Indispensable for Growth Under Stress Conditions

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrAHp) belongs to the well conserved family of serine proteases. HtrAHp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrAHp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed ΔhtrA mutant strain, we found that bacteria deprived of HtrAHp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrAHp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrAHp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (Tm) values of above 85°C. Moreover, HtrAHp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrAHp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope

Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E, and the mRNA 50 cap

Initiation is the rate-limiting step during mRNA 50 cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition

Characterization of the molecular chaperone ClpB from the pathogenic spirochaete Leptospira interrogans.

Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that an AAA+ chaperone ClpB (a member of the Hsp100 family) from L. interrogans (ClpBLi) is not only essential for survival of Leptospira under the thermal and oxidative stresses, but also during infection of a host. The aim of this study was to provide further insight into the role of ClpB in the pathogenic spirochaetes and explore its biochemical properties. We found that a non-hydrolysable ATP analogue, ATPγS, but not AMP-PNP induces the formation of ClpBLi hexamers and stabilizes the associated form of the chaperone. ADP also induces structural changes in ClpBLi and promotes its self-assembly, but does not produce full association into the hexamers. We also demonstrated that ClpBLi exhibits a weak ATPase activity that is stimulated by κ-casein and poly-lysine, and may mediate protein disaggregation independently from the DnaK chaperone system. Unexpectedly, the presence of E. coli DnaK/DnaJ/GrpE did not significantly affect the disaggregation activity of ClpBLi and ClpBLi did not substitute for the ClpBEc function in the clpB-null E. coli strain. This result underscores the species-specificity of the ClpB cooperation with the co-chaperones and is most likely due to a loss of interactions between the ClpBLi middle domain and the E. coli DnaK. We also found that ClpBLi interacts more efficiently with the aggregated G6PDH in the presence of ATPγS rather than ATP. Our results indicate that ClpB's importance during infection might be due to its role as a molecular chaperone involved in reactivation of protein aggregates

Reactivation of the aggregated substrates in the presence of ClpB_Li and ClpB_Ec.

<p>The reactivation of aggregated enzymes, G6PDH (A) and Fda (B) in the presence of DnaK/DnaJ/GrpE (KJE) from <i>E</i>. <i>coli</i> without ClpB and with ClpB_Ec or ClpB_Li. The native activity of G6PDH or Fda determined before the chemical denaturation or the heat treatment at 55°C, respectively, corresponds to 100%; the fraction of the enzyme activity remaining after the denaturation and also corresponding to the reactivation extent in the absence of chaperones (control) is marked by the broken line. (C) The effect of ClpB_Li and ClpB_Ec on the reactivation of β-galactosidase sequestered into IBs (VP1LAC) isolated from <i>E</i>. <i>coliΔclpB</i> mutant cells. A statistically significant difference in the β-galactosidase activity regain in the absence and presence of ClpB_Li assessed by the paired t-test (using GraphPad Prism software) is indicated as **, p<0.01. The results are presented as the average of three (A, C) or four (B) independent experiments with the standard deviations indicated.</p

Interaction of ClpB_Li with the aggregated G6PDH.

<p>(A) ClpB_Li was incubated with aggregates of G6PDH (Agg) in the presence of 5 mM ATP or ATPγS and without nucleotides. The solutions were passed through a 0.1-μm filter. Subsequently, the fractions retained on the filters were solubilized with an SDS buffer and analyzed by the Coomassie blue-stained 0.1%SDS-10%PAGE gel. A representative result from three independent experiments is shown. (B) Bands corresponding to ClpB_Li were analyzed with Sigma Gel software. Results are presented as the average of three independent experiments with standard deviations indicated. The amount of ClpB_Li detected in the absence of the aggregates is indicated with the broken line.</p

Structural characteristics of ClpB_Li used in this study.

<p>(A) Comparison of the domain organization of ClpB from <i>L</i>. <i>interrogans</i> and <i>E</i>. <i>coli</i>. Bacterial ClpB proteins are composed of the following domains: N-terminal domain (ND), nucleotide binding domain 1 (NBD1), middle coiled-coil domain (MD), and nucleotide binding domain 2 (NBD2). The functions of the domains are indicated at the top. The amino acid residue numbers are shown for each chaperone and the amino acid sequence identity between ClpB_Ec and ClpB_Li is indicated for each domain. (B) CD spectra of ClpB_Li at 20°C (folded form) and 75°C (unfolded form) are shown. The CD signal was expressed as mean molar residue ellipticity (θ). (C) Temperature-induced changes in the CD signal at 222 nm for ClpB_Li.</p

Summary of sedimentation coefficients for ClpB_Li.

<p>Summary of sedimentation coefficients for ClpB_Li.</p

Proteolytic sensitivity of ClpB_Li and ClpB_Ec in the absence and presence of nucleotides.

<p>ClpB (1 μM) was incubated at 37°C for the indicated periods with 5 ng of trypsin in the absence or presence of 5 mM nucleotides. The degradation products were resolved by 0.1%SDS-12.5%PAGE and visualized by Coomassie-blue staining. Representative results from three experiments are shown. The positions of standard molecular mass markers (M) (in kDa), PageRuler prestained Protein Ladder (ThermoScientific), are shown on the left.</p

ATPase activity of ClpB_Li and ClpB_Ec.

<p>The rate of ATP hydrolysis was determined at 37°C in the absence of other proteins (basal activity), in the presence of κ-casein (0.1 mg/ml), poly-lysine (0.04 mg/ml) (polyLys), or aggregated G6PDH (2.1 μM) (aggG6PDH). The average values from three independent experiments are shown with the standard deviations.</p

Nucleotide-induced oligomerization of ClpB_Li.

<p>Shown are the sedimentation coefficient distributions <i>c</i>(<i>s</i>_20,w) for 1.2 mg/ml ClpB_Li in the absence of nucleotides (A), in the presence of the indicated nucleotide at 2 mM concentration (B-D), and in the low-salt buffer without nucleotides for 3 mg/ml ClpB_Li (E). Sedimentation velocity data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181118#pone.0181118.g002" target="_blank">Fig 2</a> were analyzed with a continuous sedimentation coefficient distribution <i>c</i>(<i>s</i>) model. The distributions were transformed to standard conditions.</p