Functional Differences between E. coli and ESKAPE Pathogen GroES/GroEL

Abstract

As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coli groESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity. IMPORTANCE The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins. It was, therefore, shocking to discover ESKAPE pathogen GroES/GroEL formed mixed-complex chaperonins in the presence of E. coli GroES/GroEL, leading to loss of organism viability in some cases. Complete replacement of E. coli groESL with ESKAPE groESL restored organism viability, but produced an elongated phenotype, suggesting differences in chaperonin function, including client specificity and/or refolding cycle rates. These data offer important mechanistic insight into these remarkable machines, and the new strains developed allow for the synthesis of homogeneous chaperonins for biochemical studies and to further our efforts to develop chaperonin-targeted antibiotics.

Allosteric differences dictate GroEL complementation of E. coli

“…25 We found no statistical difference between GroEL coli and GroEL A. baumannii regarding K m , K cat /K m , ATP concentration for the first allosteric transition from tense to relaxed state (positive allostery between subunits within same GroEL ring), Hill coefficient for positive allostery (cooperativity), and Hill coefficient for negative allostery (cooperativity) (Figure 4A,B and Table 1). GroEL K. pneumoniae and GroEL E. cloacae , which rescue LG6, but display abnormal phenotypes in their respective E. coli knock-in strains, 25 were found to have similar K cat /K m and positive allostery Hill slope values compared to GroEL coli . GroEL E. faecium and GroEL P. aeruginosa , which form nonfunctional GroEL heterooligomers when co-expressed with GroEL coli , differ from GroEL coli when comparing K m , first transition point, and second transition points (negative allostery between GroEL rings transitioning from tense to relaxed state).…”

“…Due to monomeric translation of GroEL subunits, co-expression of GroEL monomers from different species within the same strain generates mature tetradecamers comprised of both species of GroEL monomers (Figure 1). 25,[55][56][57][58] Heterooligomeric GroEL, made up of GroEL K. pneumoniae/coli , GroEL A. baumannii/coli , or GroEL E. cloacae/coli (but not GroEL P. aeruginosa/coli , GroEL E. faecium/coli , or GroEL S. aureus/coli ), were able to rescue the LG6 E. coli strain (Table 1). A pulldown experiment of heterooligomeric GroEL P. aeruginosa/coli revealed a fully assembled tetradecamer, devoid of ATPase and refolding activity.…”

“…Functional replacement of GroEL coli with GroEL from other organisms in E. coli has been reported, 12,22 although organism rescue is not a universal trend. 23,24 To pursue GroESL ESKAPE as a potential antibiotic target in these medically relevant pathogens, we attempted to express these chaperonins in E. coli 25 for further purification and high throughput molecule screening in a biochemical assay.…”

“…However, in more stringent systems, all GroESL ESKAPE except for groESL S. aureus was able to rescue E. coli without groESL coli present. 25 Pull-down experiments indicated that when GroEL ESKAPE was co-expressed with GroEL coli , the purified GroEL tetradecamers contained monomers from both species (Figure 1). Of the GroESL ESKAPE which could not rescue LG6, it was found the respective heterooligomeric GroELs were devoid of ATPase activity.…”

The Functional Differences between the GroEL Chaperonin of Escherichia coli and the HtpB Chaperonin of Legionella pneumophila Can Be Mapped to Specific Amino Acid Residues

“…Deviatio n = 43.13, n = 14), and this group contained the aa position with the highest ET rank (146.41) of the protein folding-related aa. This variability could explain why HtpB and GroEL (among other chaperonins) are not functionally interchangeable [ 53 , 54 , 55 ]. The areas where the protein folding-related aa reside are delineated in Figure 2 B.…”