Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis

Abstract

Background: The CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.

Results: CpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the λcI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain.

Conclusions: This interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway activation and repression.

Figure 1

BACTH analysis of CpxA-CpxA interactions. Full-length CpxA_1-458 was translationally fused to the N-terminus of CyaA_1-224 (T25 – dark green shade) creating a CpxA_1-458-T25 hybrid used as the ‘bait’. Full-length CpxA_1-458 was also translationally fused to the N-terminus of CyaA_225-399 (T18 – magenta shade) giving rise to a ‘prey’ CpxA_1-458-T18 hybrid. Based on CpxA divisions into sensor input (CpxA_1-156, cadet blue shade), HAMP – signal transmission (CpxA_157-240, soft yellow shade), Dhp – dimerization and histidine phosphorylation (CpxA_241-310, metallic gold shade) and histidine kinase catalysis (CpxA_311-458, grey shade) domains, an additional six ‘prey’ CpxA-T18 hybrids were constructed; CpxA_1-156-T18, CpxA_1-240-T18, CpxA_1-310-T18, CpxA_157-310-T18, CpxA_187-458-T18 and CpxA_311-458-T18. BACTH interaction analysis of ‘bait’ and ‘prey’ hybrids was quantified via measurement of β-galactosidase activity and is represented as units/mg dry weight of host E. coli BTH101 bacteria (left column; black font). As an internal positive control, we used the provided constructs expressing T18-Zip and T25-Zip that yielded 1547.0 ± 121.2 units of β-galactosidase activity /mg dry weight of bacteria. This was equivalent to ~83.8 fold more enzymatic activity produced compared to bacteria co-expressing only T18 and T25 (18.5 ± 3.9 units of β-galactosidase activity). The fold change in enzymatic activity caused by CpxA-CpxA interactions relative to this negative control is indicated in parentheses to the right. A level of β-galactosidase activity at least 3-fold higher than the negative control was considered to indicate a positive interaction (*). Data is presented as the mean (± standard error of the mean) of at least four independent experiments performed in triplicate.

Figure 2

Regions of CpxR interacting with CpxA as monitored by BACTH analysis. Full-length CpxA_1-458 was translationally fused to the N-terminus of CyaA_225-399 (T18 – magenta shade) giving rise to a ‘bait’ CpxA_1-458-T18 hybrid. Full-length CpxR_1-232 was translationally fused to the C-terminus of CyaA_1-224 (T25 – dark green shade) giving rise to a T25-CpxR_1-232 ‘prey’ hybrid. CpxR was also divided into N-terminal (pistachio green), internal linker (orange) and C-terminal (sky blue) domains. Based on these divisions, additional ‘prey’ T25 hybrids were similarly constructed that consisted of only the N-terminus without linker (T25-CpxR_1-117) or with linker (T25-CpxR_1-132) and the C-terminus without linker (T25-CpxR_132-232) or with linker (T25-CpxR_117-232). CpxR_1-132 was also fused to the N-terminus of T25 giving the CpxR_1-132-T25 hybrid. Full-length or near full-length CpxR ‘prey’ T25 constructs were also generated consisting of T25-CpxR_D51A or T25-CpxR_D51E lacking the phosphorylated aspartate residue as well as T25-CpxR_Δ11–24, T25-CpxR_Δ117–132 and T25-CpxR_Δ188–209 lacking the putative N-terminal coiled-coil, internal linker and C-terminal winged helix-turn-helix regions respectively. BACTH interaction analysis of ‘bait’ and ‘prey’ hybrids was quantified via measurement of β-galactosidase activity and is represented as units/mg dry weight of host E. coli BTH101 bacteria (left column; black font). The internal positive control based upon constructs expressing T18-Zip and T25-Zip yielded 1603.6 ± 146.4 units of β-galactosidase activity/mg dry weight of bacteria. This was equivalent to ~20.2 fold more enzymatic activity produced compared to bacteria co-expressing only T18 and T25 (79.2 ± 6.2 units of β-galactosidase activity). The fold change in enzymatic activity caused by CpxA-CpxR interactions relative to this negative control is indicated in parentheses to the right. The asterisks (*) indicates a positive interaction. Data is presented as the mean (± standard error of the mean) of at least four independent experiments performed in triplicate

Figure 3

Regions of CpxA interacting with CpxR as monitored by BACTH analysis. Full-length CpxR_1-232 was translationally fused to the C-terminus of CyaA_1-224 (T25 – dark green shade) creating a T25-CpxR_1-232 hybrid used as the ‘bait’. As in Figure 1, the same seven CpxA variants translationally fused to the N-terminus of CyaA_225-399 (T18 – magenta shade) were used as ‘prey’ hybrids, that is CpxA_1-458-T18, CpxA_1-156-T18, CpxA_1-240-T18, CpxA_1-310-T18, CpxA_157-310-T18, CpxA_187-458-T18 and CpxA_311-458-T18. BACTH interaction analysis of ‘bait’ and ‘prey’ hybrids was quantified via measurement of β-galactosidase activity and is represented as units/mg dry weight of host E. coli BTH101 bacteria (left column; black font). The internal positive control based upon the constructs expressing T18-Zip and T25-Zip yielded 1778.1 ± 120.3 units of β-galactosidase activity/mg dry weight of bacteria. This was ~32.8 fold more enzymatic activity produced compared to bacteria co-expressing only T18 and T25 (54.2 ± 9.0 units of β-galactosidase activity). The fold change in enzymatic activity caused by CpxR-CpxA interactions relative to this negative control is indicated in parentheses to the right. The asterisks (*) indicates a positive interaction. Data is presented as the mean (± standard error of the mean) of at least four independent experiments performed in triplicate.

Figure 4

BACTH analysis of CpxR-CpxR interactions. Full-length CpxR_1-232 was translationally fused to the C-terminus of CyaA_1-224 (T25 – dark green shade) creating a T25-CpxR_1-232 hybrid used as the ‘bait’. Full-length CpxR_1-232 was translationally fused to the N-terminus of CyaA_225-399 (T18 – magenta shade) giving rise to a CpxR_1-232-T18 ‘prey’ hybrid. Based upon divisions of CpxR into N-terminal (pistachio green), internal linker (orange) and C-terminal (sky blue) domains, additional ‘prey’ T18 hybrids were constructed that consisted of only the N-terminus without linker (CpxR_1-117-T18) or with linker (CpxR_1-132-T18) and the C-terminus without linker (CpxR_132-232-T18) or with linker (CpxR_117-232-T18). BACTH interaction analysis of ‘bait’ and ‘prey’ hybrids was quantified via measurement of β-galactosidase activity and is represented as units/mg dry weight of host E. coli BTH101 bacteria (left column; black font). The internal positive control based upon the constructs expressing T18-Zip and T25-Zip yielded 1521.9 ± 150.6 units of β-galactosidase activity/mg dry weight of bacteria. This has ~14.6 fold more enzymatic activity than bacteria co-expressing only T18 and T25 (104.6 ± 12.9 units of β-galactosidase activity). The fold change in enzymatic activity caused by CpxR-CpxR interactions relative to this negative control is indicated in parentheses to the right. The asterisks (*) indicates a positive interaction. Data is presented as the mean (± standard error of the mean) of at least four independent experiments performed in triplicate.

Figure 5

N-terminal CpxR dimerization in BACTH assays is enhanced by inclusion of the internal CpxR linker. The N-terminal domain of CpxR either with linker (A) or without linker (B) were translationally fused to the N-terminus of CyaA_225-399 (T18 – magenta shade) creating the ‘bait’ CpxR_1-132-T18 and CpxR_1-117-T18 hybrids respectively. As ‘prey’ hybrids, the CpxR N-terminus without linker (CpxR_1-117-T25) or with linker (CpxR_1-132-T25) and the C-terminus without linker (CpxR_132-232-T25) or with linker (CpxR_117-232-T25) were fused to the N-terminus of CyaA_1-224 (T25 – dark green). Interactions between ‘bait’ and ‘prey’ hybrids were again quantified via measurement of β-galactosidase activity (left columns; black font). Measurement of the interaction between T18-Zip and T25-Zip yielded 1449.1 ± 113.2 units of β-galactosidase activity was ~17.3 fold more than the enzymatic activity produced by negative-control bacteria co-expressing only T18 and T25 (83.8 ± 16.3 β-galactosidase activity units). The fold change in enzymatic activity caused by CpxR-CpxR interactions relative to this negative control is indicated in parentheses to the right. The asterisks (*) indicates a positive interaction. Data is presented as the mean (± standard error of the mean) of at least four independent experiments performed in triplicate.

Figure 6

Regions of CpxR interacting with N-terminal CpxR as monitored by BACTH analysis. The N-terminal domain of CpxR either with linker was translationally fused to the N-terminus of CyaA_225-399 (T18 – magenta shade) creating the ‘bait’ CpxR_1-132-T18 hybrid. As already used in Figure 2, a selection of full-length or near full-length CpxR variants translationally fused to the C-terminus of CyaA_1-224 (T25 – dark green shade) were used as ‘prey’ hybrids; that is T25-CpxR_D51A and T25-CpxR_D51E lacking the phosphorylated aspartate residue as well as T25-CpxR_Δ11–24, T25-CpxR_Δ117–132 and T25-CpxR_Δ188–209 lacking the putative N-terminal coiled-coil, internal linker and C-terminal winged helix-turn-helix regions respectively. We also employed the T25-CpxR_1-232 hybrid containing wild type CpxR sequence. BACTH interaction analysis of ‘bait’ and ‘prey’ hybrids was quantified via measurement of β-galactosidase activity and is represented as units/mg dry weight of host E. coli BTH101 bacteria (left column; black font). The internal positive control based upon constructs expressing T18-Zip and T25-Zip yielded 1495.1 ± 237.3 units of β-galactosidase activity/mg dry weight of bacteria. This was on average ~25.6 fold more enzymatic activity produced compared to bacteria co-expressing only T18 and T25 (58.3 ± 14.1 units of β-galactosidase activity). The fold change in enzymatic activity caused by CpxR-CpxR interactions relative to this negative control is indicated in parentheses to the right. The asterisks (*) indicates a positive interaction. Data is presented as the mean (± standard error of the mean) of at least four independent experiments performed in triplicate.

Figure 7

Reconstitution of the λcI repressor function through CpxR homodimerization. Fusion of CpxR_n variants to the C-terminus of λcI_1-131 (auburn brown shade) were generated. When expressed in E. coli JM109, growth was assessed by spotting 5 μl of 10-fold serially diluted (10⁰, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴ and 10⁻⁵) exponentially grown cultures onto LB agar containing 10 μg/ml tetracycline (Tet) and 0.1 mM IPTG (A). The assay was controlled through the expression of inactive λcI_1-131 alone [vector; pKWY2428] or the dimerization-competent fusion λcI_1-131-YycF_120-235 [YycF(120C); pKWY-YycF(120C)] [36]. Protein lysates were also fractionated on 12% acrylamide SDS-PAGE and analysed by western blotting (B). Fusions of λcI_1-131 to the N-terminus of CpxR_n variants were detected with rabbit polyclonal anti-CpxR antiserum. Samples were also probed with antiserum raised in rabbit and specific for chloramphenicol acetyltransferase (anti-CAT) to confirm the loading of an equal quantity of protein in each lane. The asterisks (*) highlight unknown protein bands that cross-react non-specifically with antibodies in the sera or, in some cases may represent a degradation product of the recombinant fusion proteins. Shown to the left is the approximate mobility of molecular weight standards (PageRuler^TM Plus Prestained Protein Ladder, Thermo Scientific).