In Vivo Cross-Linking MS Reveals Conservation in OmpA Linkage to Different Classes of β-Lactamase Enzymes

Abstract

Molecular interactions between two different classes of β-lactamase enzymes and outer membrane protein A (OmpA) were studied by in vivo chemical cross-linking of a multi-drug-resistant strain of Acinetobacter baumannii AB5075. Class A β-lactamase blaGES-11 and Class D β-lactamase Oxa23, responsible for hydrolysis of different types of β-lactam antibiotics, were found to be cross-linked to similar lysine sites of the periplasmic domain of outer membrane protein OmpA, despite low sequence homology between the two enzymes. The findings from in vivo XL-MS suggest that the interacting surfaces between both β-lactamase enzymes and OmpA are conserved during molecular evolution, and the OmpA C-terminus domain serves an important function of anchoring different types of β-lactamase enzymes in the periplasmic space.

Graphical Abstract

INTRODUCTION

In vivo cross-linking (XL)-MS is a useful tool to study protein conformations, protein complex architectures, and molecular interactions under their native environment.¹ In the past decade, our laboratory has developed a class of lysine-targeting chemical cross-linker molecules, named protein interaction reporter (PIR) cross-linker,² and Real-time Analysis for Cross-linked peptide Technology (ReACT),³ as well as associated bioinformatic tools^4,5 for in vivo applications of XL-MS on a variety of biological systems including bacterial cells,^3,6,7 human cancer cells,^8,9 subcellular organelle such as mitochondria,¹⁰ and tissue samples.¹¹ In a previous study,¹² we applied in vivo XL-MS to a multi-drug-resistant (MDR) strain of Acinetobacter baumannii, AB5075, which is a contemporary clinical isolate from a patient in the U.S. military health care system and has recently been established as a model strain for the evaluation of pathogenesis and antimicrobial treatments.¹³ In vivo XL-MS of AB5075 revealed multiple novel molecular interactions among outer membrane and periplasmic proteins, especially the interactions between a key resistance factor, Class D β-lactamase Oxa-23 encoded on the chromosome, and several outer membrane porin proteins such as OmpA, OmpW and CarO. These results provided new structural insights on how the enzyme Oxa-23 is localized at the entry port of antibiotics and other molecules through interaction with the porin proteins.¹² Another study by Ambrosi et al.¹⁴ also showed that oxacillinases, including Oxa-23 and Oxa-51, copurified with outer membrane proteins such as OmpA, in other carbapenem-resistant A. baumannii strains, which provides independent validation of OmpA-β-lactamase interactions without using cross-linking reagents.

With increased in vivo XL-MS experiments using AB5075, the XL-MS protein interaction network of AB5075 was revealed with increasing detail, and we discovered that another import resistance factor, the Class A β-lactamase blaGES-11 encoded on the resistance island in one of the plasmids, also interacts with the C-terminal domain of the most abundant outer membrane protein OmpA. Both Oxa-23 and blaGES-11originate from the penicillin-binding proteins, the β-lactam antibiotic targets, and key enzymes involved in peptidoglycan biosynthesis.¹⁵ The two β-lactamase enzymes share some three-dimensional structural similarity and utilize a conserved serine residue as catalysis center despite low sequence identity.¹⁵ Composed of an N-terminal β-barrel domain spanning the cell outer membrane and C-terminal domain in the periplasmic space, OmpA is an integral membrane involved in a variety of biological processes, such as ion transport,^16,17 maintenance of cell envelope structural integrity,¹⁸ and invasion of host cells.^19,20 By comparing the cross-linked sites between the two β-lactamase enzymes and the OmpA C-terminal domain, we found that not only the three-dimensional folding of the enzymes but also their interaction surfaces with OmpA periplasmic domain are conserved, despite the striking primary sequence and β-lactam substrate differences. These findings have extended our knowledge on the interplay between β-lactamase enzymes and outer membrane porin proteins, as well as the conservation of interaction surfaces during molecular evolution. In addition, localization of multiple β-lactamase enzymes at the OmpA C-terminal domain could increase resistance to multiple antibiotics and be an important feature of MDR phenotypes.

EXPERIMENTAL SECTION

Agar Diffusion Assay.

Sensitivity of wild-type AB5075 and transposon insertion mutants against antibiotic drugs was evaluated by agar diffusion assay measurements. Overnight LB media A. baumannii cell cultures were diluted to OD₆₀₀ nm = 0.2 and then spread onto LB agar plates with sterile cotton swabs. The antibiotic disks (BBL Sensi-Disc, Becton, Dick-inson and Company, Franklin Lakes, NJ) including aztreonam (30 μg/mL), ceftazidime (30 μg/mL), ertapenem (10 μg/mL), and meropenem (10 μg/mL) were placed onto the bacterial lawn. Cell growth on an agar plate with an antibiotic disk was allowed for 24 h at 37 °C. The annular radius (mm) of the inhibition zone was measured for drug sensitivity comparison. The transposon insertion mutants were obtained from the Manoil Lab at the University of Washington.²¹

In Vivo Cross-Linking of AB5075 and Analysis of Cross-Linked Peptides.

In vivo cross-linking of live AB5075 cells using the PIR cross-linker and LC−MS analysis of cross-linked peptide pairs were similar as previously described.^6,12,22 Briefly, the harvested bacteria cells were cross-linked with biotin-aspartate proline-n-hydroxyphthalimide,^2,22 lysed, and digested by trypsin. The digested sample were desalted, fractionated, and affinity enriched prior to LC-ReACT analysis. Details of the sample processing, LC−MS instrument settings, and data analysis are described in the Supporting Information.

RESULTS AND DISCUSSION

From genomic analysis, the AB5075 genome harbors three unique β-lactamase genes, the Class A β-lactamase blaGES-11, Class D β-lactamase Oxa23, and putative β-lactamase HcpC, in addition to another 15 orthologous β-lactamase genes also present in ATCC19606, a traditional model A. baumannnii strain commonly used by many other researchers. By data-dependent MS analysis of the proteome of AB5075, we found among all the β-lactamase enzymes, Oxa23 and blaGES-11 are the most abundantly expressed, with the peptide-spectra match counts around 6-fold of the third most abundant β-lactamase. Our previous study showed that inactivation of AmpC or Oxa-69 in WT AB5075 by transposon insertion does not decrease the intrinsic resistance to meropenem and impenem.¹² Here, we further investigated β-lactam sensitivity of transposon mutants of Oxa-23 and blaGES-11 by agar disk diffusion assay (Figure 1). The results show that transposon mutant of Oxa23 exhibits reduced sensitivity to meropenem and ertapenem while retaining the resistance to aztreonam and ceftazidime; the transposon mutant of blaGES-11 exhibits increased sensitivity to aztreonam and ceftazidime but retains the resistance to meropenem and ertapenem. From these observations, we can conclude that AB5075 gains resistance to the extended spectrum of β-lactam antibiotics by producing a versatile family of β-lactamase enzymes that are complementary in hydrolyzing different classes of β-lactams.

Figure 1.

Antibiotic sensitivity comparison of WT AB5075 and transposon insertion mutants of blaGES-11 and Oxa23 in AB5075 by disc diffusion assay. The error bars denote standard deviation of measured annular diameter of growth inhibition zone from four biological replicates. Larger annular diameter of the growth inhibition zone indicates increased antibiotic sensitivity: MEM, meropenem, 10 μg/mL; ETP, ertapenem, 10 μg/mL; ATM, aztreonam, 30 μg/mL; CAZ, cefazidime, 30 μg/mL.

By in vivo XL-MS study, we found that blaGES-11 and Oxa23 are both extensively cross-linked to the periplasmic C-terminal OmpA domain. As an example for demonstration of PIR and ReACT applied in this study, Figure 2 shows the MS spectra corresponding to the identification of a cross-linked peptide pair between OmpA and Oxa23. All cross-linked peptide spectral matches for inter-protein crosslinks between two β-lactamases and OmpA C-terminus from three biological replicates of XL-ReACT MS experiments are summarized in Table S1. Table 1 lists the non-redundant interprotein cross-linked peptide pairs between the two β-lactamase enzymes and OmpA C-terminus. These results indicate that the two classes of β-lactamase enzymes are both localized in the periplasmic space between the outer membrane and the peptidoglycan layer of AB5075 through interaction with the C-terminal domain of OmpA. In spite of only 14% primary sequence identity, superimposition of crystal structures of Oxa23 (PDB: 4JF4) and blaGES-11 (PDB: 3V3R) reveals similarity in overall three dimensional folding of the two β-lactamase enzymes, rmsd = 2.4 Å) (Figure 3). Moreover, despite low conservation of lysine residues in blaGES-11 and Oxa-23, a highly similar interprotein linkage configuration with the OmpA C-terminal domain was observed. For instance, three pairs of lysine sites, K60 of Oxa23 and K25 of blaGES-11, K145 of Oxa23 and K149 of blaGES-11, and K104/K102 of Oxa23 and K88 of blaGES-11, located at similar relative positions within the three-dimensional surfaces of the two enzymes, as highlighted in Figure 3B, were found to be cross-linked to the same lysine sites on the OmpA C-terminus. Details of the cross-linking site interaction between the two β-lactamase enzymes and the OmpA C-terminal domain are depicted in Figure 4. Strikingly, these lysine residues located on the surface opposite to the antibiotic binding pocket (Figure 3B) were cross-linked to the same lysine sites in the OmpA C-terminal domain, despite very different local primary sequences. For example, K60 of Oxa23 located between strands β1 and β2 was identified to be cross-linked to K252 of OmpA, as was K25 in helix α1 of blaGES-11 (Figure 4A); K145 in helix α6 of Oxa23 and K149 in helix α6 of blaGE-11 were both cross-linked to K252 and K319 of OmpA (Figure 4B); K102 and K104 located in the α3−α4 loop of Oxa23, and K88 located in the P-loop of blaGES-11 were identified as being cross-linked to K252, K288, and K317 of OmpA (Figure 4C). From these site interactions defined by XL-MS, we can infer the relative spatial orientations of the two β-lactamase enzymes toward OmpA. The interacting surfaces between the two β-lactamase enzymes and OmpA C-terminus are opposite to the substrate binding pocket, thus allowing OmpA interaction compatible with free access of antibiotics with β-lactamase active sites, specifically the S-x-x-K motif conserved in both classes of enzymes (Figure 3B). In this way, blaGES-11 and Oxa23 are concentrated in the periplasmic space, which could deactivate β-lactam molecules upon entry into the cell and block their access to their target proteins, the penicillin binding proteins involved in the biosynthesis of the peptidoglycan layer. Moreover, interactions of both lactamase enzymes with the OmpA C-terminal domain are compatible with OmpA interaction with peptidoglycan which occurs via a pocket at the distal end of the domain (Figure 4).¹⁸ These results also suggest that, in addition to its role in stabilizing the outer membrane envelope by binding with peptidoglycan, the OmpA C-terminal domain has an important function of localizing and stabilizing β-lactamase enzymes in the periplasmic space between the cell outer membrane and peptidoglycan layer.

Figure 2.

ReACT spectra for identification of cross-linking between Oxa23 K104 and OmpA K252. (A) Full MS survey scan of precursor ion; (B) CID MS/MS scan that discovers the cross-linking mass relationship; (C, D) CID MS/MS/MS scans for sequence identification of the two linear peptides released from the cross-linked precursor ion. The cross-linked peptide regions are highlighted in purple in the crystal structures of Oxa23 (PDB: 4JF4) and OmpA C-terminal domain (PDB: 3TD3).

Table 1.

List of Interprotein Cross-Linked Peptide Pairs between Oxa23 (uniport ID: Q9L4P2) and OmpA (uniprot ID: W6RU67), blaGES-11 (uniprot ID: C5HUY1), and OmpA from in Vivo Cross-Linking Experiments^a

blaGES-11 peptide_OmpA peptide	Oxa-23 peptide_OmpA peptide
LTFK25TDLEK_ VAEK252LSEYPNATAR	K60INLYGNALSR_ VAEK252LSEYPNATAR
K149IGDSVSR_ VAEK252LSEYPNATAR K149IGDSVSR TK319EGR	IGLDLMQK145EVK_VAEK252LSEYPNATAR IGLDLMQK145EVK TK319EGR
K88LSYGPDMIVEWSPATER_VAEK252LSEYPNATAR	WK104GEK_ VAEK252LSEYPNATAR WK104GEKR_ VAEK252LSEYPNATAR TDINEIFK102WK_VAEK252LSEYPNATAR
K88LSYGPDMIVEWSPATER_ANSVK288SALVNEYNVDASR	TDINEIFK102WK_ANSVK288SALVNEYNVDASR
K88LSYGPDMIVEWSPATER_LSTQGFAWDQPIADNK317TK	WK104GEKR_ LSTQGFAWDQPIADNK317TK WK104GEK_ TK319EGR
LTFK25TDLEK_VFFDTNK235SNIKDQYKPEIAK LTFK25TDLEK_SNIKDQYKPEIAK248VAEK LTFK25TDLEK_ VAEK252LSEYPNATAR LTFK25TDLEK_ANSVK288SALVNEYNVDASR LTFK25TDLEK_LSTQGFAWDQPIADNK317TK LTFK25TDLEK_TK319EGR	VTPIQEVEFVSQLAHTQLPFSEK194VQANVK_SNIKDQYKPEIAK248VAEK VTPIQEVEFVSQLAHTQLPFSEK194VQANVK_ANSVK288SALVNEYNVDASR TGWAMDIK224PQVGWLTGWVEQPDGK_VAEK252LSEYPNATAR WK104GEK_SNIKDQYKPEIAK248VAEK TDINEIFK102WK_SNIKDQYK243PEIAKVAEK VTPIQEVEFVSQLAHTQLPFSEK194VQANVK_VAEK252LSEYPNATAR

^aThe gray shaded linkages are highlighted in Figure 4.

Figure 3.

Sequence (A) and crystal structure (B) alignment of blaGES-11(PDB: 3V3R) and Oxa23 complexed with meropenem (PDB: 4JF4). Lysine residues crosslinked to the OmpA C-terminus and located in similar regions of 3D structures are highlighted in blue. The active site Ser and Lys residues in the conserved S-xx-K motif in both Class A and D β-lactamase enzymes are highlighted in red in the structures. The four consecutive conserved residues (STFK) near the substrate binding pocket are highlighted in red in the backbone alignment. Sequence alignment between blaGES-11 (UniprotID: C5HUY1) and Oxa23 (Uniprot ID: Q9L4P2) was performed by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Figure 4.

Conserved lysine site linkage between the two β-lactamase enzymes (PDB: 4JF4, 3V3R) and the OmpA C-terminal domain (PDB:3TD3). (A) Oxa23 K60 and blaGES-11 K25 are both crosslinked to OmpA K252; (B) Oxa23 K145 and blaGES-11 K149 are both crosslinked to OmpA K252 and OmpA K319; (C) blaGES-11 K88 is crosslinked to OmpA K252, K288, K317, and this same OmpA linkage was observed with K102/K104 of Oxa23. The OmpA N-terminal structure model (a.a. 23–191, Uniprot ID: W6RU67) was generated by Phyre2 using the intensive modeling mode.²⁶ The residues for noncovalent interaction with peptidoglycan in the OmpA CTD domain, Asp268, Arg283, Ala284, are highlighted in orange.¹⁸

CONCLUSIONS

In vivo XL-MS of the multi-drug-resistant strain of A. baumannii, AB5075, provides new insight on how different classes of β-lactamase enzymes interact with the periplasmic domain of outer membrane protein OmpA. Despite little sequence homology, the Class D β-lactamase Oxa23 and Class A β -lactamase blaGES-11 have similar interacting surfaces with OmpA, which is revealed by conservation of cross-linking sites between the enzymes and OmpA C-terminal domain. These results also indicate that OmpA serves an important role in anchoring different types of β-lactamase enzymes to protect the bacterial cells from different classes of β-lactams. This finding is consistent with previous results indicating deletion of OmpA results in a significant decrease in minimum inhibitory concentration of multiple antibiotics in A. baumannii.²³ Finally, our finding also may help explain the conclusion by Kwon et al. that OmpA contributes to the antimicrobial resistance of A. baumannii through the C-terminal domain.²⁴ Deletion of the OmpA C-terminal domain not only impairs OmpA-peptidoglycan binding but also adversely affects the ability to localize β-lactamase enzymes at the site of this porin involved in antibiotic permeation.²⁵ Through this study, we also demonstrated that general utility of in vivo XL-MS strategy exists for discovery of intermolecular linkage conservation of protein interaction partners, even for enzymes with considerable sequence divergence. Conserved linkage at common interactor sites may serve as a means of the future to not only discover common interactors but also reveal conserved structural features and molecular interfaces in a way not previously possible.