Mycobacterial chaperonins in cellular proteostasis: Evidence for chaperone function of Cpn60.1 and Cpn60.2‐mediated protein folding

Abstract Mycobacterium tuberculosis encodes two chaperonin proteins, MtbCpn60.1 and MtbCpn60.2, that share substantial sequence similarity with the Escherichia coli chaperonin, GroEL. However, unlike GroEL, MtbCpn60.1 and MtbCpn60.2 purify as lower‐order oligomers. Previous studies have shown that MtbCpn60.2 can functionally replace GroEL in E. coli, while the function of MtbCpn60.1 remained an enigma. Here, we demonstrate the molecular chaperone function of MtbCpn60.1 and MtbCpn60.2, by probing their ability to assist the folding of obligate chaperonin clients, DapA, FtsE and MetK, in an E. coli strain depleted of endogenous GroEL. We show that both MtbCpn60.1 and MtbCpn60.2 support cell survival and cell division by assisting the folding of DapA and FtsE, but only MtbCpn60.2 completely rescues GroEL‐depleted E. coli cells. We also show that, unlike MtbCpn60.2, MtbCpn60.1 has limited ability to support cell growth and proliferation and assist the folding of MetK. Our findings suggest that the client pools of GroEL and MtbCpn60.2 overlap substantially, while MtbCpn60.1 folds only a small subset of GroEL clients. We conclude that the differences between MtbCpn60.1 and MtbCpn60.2 may be a consequence of their intrinsic sequence features, which affect their thermostability, efficiency, clientomes and modes of action.

(a) Schematic illustrating GroEL-normal and GroEL-depleted cells of the E. coli MGM100 strain.In MGM100, the chromosomal groES-groEL operon is regulated by the arabinoseinducible P BAD promoter (McLennan and Masters, 1998).GroEL production in MGM100 cells requires growth in arabinose-supplemented medium, and growth in glucose-supplemented medium is accompanied by an approximately 90 % reduction in cellular GroEL levels (McLennan and Masters, 1998).GroES-GroEL complexes ( )   Overnight cultures of the chaperonin expression strains (V, E, C1 and C2) were sub-cultured in LB-glucose-DAP medium, grown to 0.4 -0.5 OD 600 units and induced with 100 μM IPTG for 5 h.Cells were harvested by centrifugation, and total protein extracts were prepared and resolved on 10% SDS-polyacrylamide gels.The gels were stained with Coomassie Brilliant Blue R-250 (CBB) and analysed for chaperonin production.
(a) MS analysis of chaperonin proteins.Overexpressed protein bands corresponding to ~ 60 kDa were excised, digested with Trypsin and subjected to MS analysis as described in (Shevchenko et al., 2007).Peptide MS signatures were used to identify the proteins through Mascot Server (Perkins et al., 1999).(d) IPTG-independent groEL expression from leaky P trc promoter of pEcoESL.Aliquots of uninduced and induced cells harbouring the pEcoESL plasmid were collected and lysed.Total protein extracted from the cells were resolved on an SDS-polyacrylamide gel to check for leaky expression of the plasmid-borne groES and groEL genes in the absence of IPTG induction.

Figure S3: Effect of GroEL depletion on E. coli cell morphology and size.
Overnight cultures of the E. coli strains MG1655 and MGM100 were sub-cultured for 5 h in LB-arabinose and LB-glucose-DAP.Cultures were diluted 100-fold, and examined by phase contrast microscopy at 1000X for cell morphology and size.Cell lengths and widths were measured for 100 cells using the line measurement tool in Fiji image processing software (Schindelin et al., 2012), and subjected to statistical analyses using GraphPad Prism (www.graphpad.com).Statistical significance at P < 0.05.**** P < 0.0001, ns P > 0.05.).The dilutions were spotted onto LB-agar plates supplemented with 0.2% L-arabinose or 0.2% D-glucose, the plates incubated at 37 °C and analysed for colony formation.
(b) Normalised overnight cultures of E. coli MGM100 were sub-cultured into either LBarabinose or LB-glucose medium to an initial absorbance of 0.05 OD units, and culture growth was monitored at 37 °C by OD 600 measurement at 1 h intervals.
(c) Total protein extracts were prepared from culture samples collected at intervals of 1 h, resolved on a 10% SDS-polyacrylamide gel followed by immunoblotting (IB) with anti-GroEL antibody.(d) MS analysis of the MetE protein band.The MetE protein band was excised from the gel, subjected to in-gel proteolysis, the peptides were then extracted and subjected to MS analysis (Shevchenko et al., 2007).Protein identity was confirmed with the help of Mascot Server (Perkins et al., 1999).Bar graph illustrating global sequence similarity (grey) and identity (white) computed from pairwise sequence alignments using the EMBOSS Needle tool (Needleman and Wunsch, 1970) for protein sequences retrieved from the NCBI Protein database (Sayers et al., 2022).

Data
Protein   Cultures of the chaperonin expression strains (V, E, C1, C2) were grown in LB-glucose-IPTG, in parallel with the GroEL-normal culture (V/arabinose).The cultures were induced with 100 μM IPTG and incubated at 37 °C.Growth curves were obtained by periodic measurement of OD 600 every 10 mins.The curves were then fitted using the Gompertz model and the Gompertz equations were subsequently used to estimate critical growth parameters for each of the cultures.Data represents mean ± SD of three biological replicates.Strains: V -MGM100(pTrc99a), E -MGM100(pEcoESL), C1 -MGM100(pMtbCpn60.1),C2 -MGM100(pMtbCpn60.2).

Figure S1 :
Figure S1: Schematic illustration of the chaperonin expression strains, expression conditions and cellular chaperonin levels (adapted from Kumar et al., 2021).
are shown to indicate cellular levels of GroES and GroEL.(b) Schematic illustrating construction of the chaperonin expression strains.Strains V, E, C1 and C2 were constructed by transforming the E. coli MGM100 strain with chaperonin expression plasmids, pTrc99a (black), pEcoESL (green), pMtbCpn60.1 (orange) and pMtbCpn60.2(blue), respectively.(c) Schematic illustrating the growth conditions for chaperonin production and the corresponding levels of chaperonins produced in each strain.The strains were grown in glucose-supplemented growth medium to repress the chromosomal groES-groEL operon and the plasmid-borne chaperonin genes were induced with 100 μM IPTG.Cellular chaperonin levels were highest in the GroES-GroEL producing E strain, followed by the MtbCpn10-MtbCpn60.2 producing C2 strain, and the lowest in the MtbCpn10-Cpn60.1 producing C1 strain.Chaperonin complexes ( ) are shown in each strain to indicate cellular levels of the chaperonin proteins (GroES/MtbCpn10 and GroEL/MtbCpn60.1/MtbCpn60.2).

( b )
Protein gel and Western blot depicting MtbCpn60.1 expression in MGM100 (pMtbCpn1).MtbCpn60.1 expression was confirmed by probing lysates of IPTG-induced cells with the custom synthesized anti-MtbCpn60.1 antibody.(c) Cellular levels of overexpressed chaperonins.The protein levels of GroEL and MtbCpn60.2were quantified by relative densitometric analysis of the CBB stained gel.The bars represent the chaperonin band intensity relative to the sum of intensities of all protein bands in the respective lanes.

Figure S5 :
Figure S5: Effect of cellular GroEL levels on E. coli growth and proliferation.Overnight culture of the groES-groEL overexpressing strain (E / AI) was subcultured in arabinose-supplemented medium, and grown in parallel with the GroEL-normal (V / A) and GroEL-depleted (V / G) cultures of the E. coli MGM100 (pTrc99a) strain.The cultures were induced with 100 μM IPTG for 10 h and OD 600 measured every 20 minutes.(a) Semi-log plots illustrating growth curves of strains expressing groES-groEL at different levels.Data points represent mean OD600 values of three biological replicates plotted on a logarithmic y-axis and curves represent best fit of the Gompertz growth model.(b) -(e) Comparison of growth parameters of strains expressing groES-groEL at different levels.The Gompertz equation was used to compute growth parameters illustrated as bar

Figure S6 :
Figure S6: Effect of GroES-GroEL depletion on MetK folding and function.Parallel cultures of the E. coli MGM100 strain were grown in LB-arabinose or LB-glucose-DAP medium (to yield normal or depleted levels of GroEL respectively).(a)GroEL depletion associated changes in levels of endogenous MetK and MetE.Samples were collected from GroEL-depleted MGM100 culture at intervals of 1 h, followed by extraction of total protein.Total protein extracts were resolved on SDS-polyacrylamide gels, which were subjected to Coomassie Brilliant Blue (CBB) staining for MetE and immunoblotting (IB) for GroEL and MetK.(b) GroEL-depletion-associated changes in MetK solubility.Samples were collected from both GroEL-normal and GroEL-depleted MGM100 cultures after 5 h of incubation, followed by extraction of total protein, fractionation of the extracts into soluble (S) and insoluble (I) fractions, resolution of the fractions on SDS-polyacrylamide gels and immunoblotting with anti-

Figure S7 :
Figure S7: Sequence similarity between E. coli and M. tuberculosis chaperonin protein homologs.