Antibiotic resistance has increased global deaths by 8% between 1990 and 2021 and is expected to be a leading cause of death by 2050 [1]. Based on projections, 11.1 million deaths due to Antimicrobial Resistance could be prevented if a treatment is devised to effectively inhibit Gram-negative B – Lactam bacteria that have undergone these mutations [1]. However, the lack of an effective antibiotic treatment is causing β– Lactam antibiotic resistance to be an unnecessary top cause of disease burden/death. This is why it is imperative to get down to the molecular mechanisms behind antibiotic resistance, so something can be done about this catastrophe. Now, I know I am just a Georgia Tech student. However, I hope to shed some light on this issue today, so I can communicate to the general public and to the non-scientists out there that this really is a solvable problem. It really is not as dire as it seems. So, sit back and relax, as I take you through a journey, looking into the history of β-Lactam Antibiotics, β-Lactam Antibiotic resistance, and mutations that have occurred that have allowed Gram-negative bacteria to become so powerful and what we as people can do about this!
β-Lactam Antibiotics, such as penicillin, have been used to treat a wide variety of bacterial infections due to the diversity of mechanisms with which they can work, such as their ability to promote the breakdown of the peptidoglycan layer of the cell wall via the targeting of binding proteins, transport proteins that transport peptidoglycan precursors across the cell wall, phospholipids that normally decrease permeability, etc [2]. Recently, however, the overreliance on β-Lactam Antibiotics has led to the development of resistance mechanisms in β-Lactam bacteria to counteract the effects of these antibiotics, the main one being the evolution of β-Lactamase enzymes [4]. This is due to evolutionary and selective pressure, which cause Gram-negative bacteria to develop resistance mechanisms to evade the effects of antibiotic treatment, especially though the development of mutations in their lactamase enzymes [4,5]. Resistance mechanisms in β-Lactam bacteria take the form of Standard beta-lactamase (SBL) and extended spectrum beta-lactamase (ESBL) mutations.
The two mutations differ primarily in their substrate specificity and the complexity of their mutations. SBL mutations enable SBLs to hydrolyze first-generation β-Lactam Antibiotics, whereas they fail to effectively enable hydrolysis of second or third-generation beta-lactamases [6, 7]. On the other hand, β-Lactam bacteria, with more complex mutations that help to increase the number of substrate– binding sites and the size of the active site, allow for the hydrolysis of a broader and more diverse range of antibiotics, increasing catalytic efficiency and conformational flexibility [7]. The result is more formidable antibiotic resistance, which poses a significantly greater threat compared to standard mutations [8]. Examples of β-Lactamase enzymes with these ESBL mutations are the TEM, KPC, and CTX-M enzymes. The danger with these mutations is that they spread easily through plasmids, which replicate independently of an organism’s chromosomal DNA and can transfer genes to other bacterial cells via horizontal gene transfer [9]. This means that mutations conferring resistance can quickly spread to multiple bacterial cells making it very difficult for antibiotic treatment to be successful [9,10]. Based on projections, 11.1 million deaths due to Antimicrobial Resistance could be prevented if a treatment is devised to effectively inhibit Gram-negative B – Lactam bacteria that have undergone these mutations [1]. This makes it imperative to better understand the molecular mechanisms behind what makes these Amino Acid mutations so advantageous in the TEM, KPC, and CTX-M enzymes so that more effective treatments to tackle antibiotic resistance can be devised. This paper focuses on the molecular mechanisms behind such mutations that enable Gram-negative bacteria to better bind to the side chains of extended-spectrum β-Lactam Antibiotics to help achieve that goal.
As alluded to earlier, mutations in Gram-negative bacterial proteins leading to resistance began with standard spectrum β-lactamase (SBL) mutations (S70 → S70G, E166 → E166N, K73 → K73H). The S70G mutation enabled β-lactamase enzymes to conduct a nucleophilic attack on the β-lactam ring, while the E166N and K73H mutations helped stabilize the transition state of this reaction with the side chains of β-lactam antibiotics, such as penicillin [11]. However, at this point, the worst-case scenario had not yet materialized because the active site of the β-lactamase enzymes could not accommodate extended-spectrum β-lactam antibiotics with bulky oxyimino side chains at the C7 position. These side chains were too large, and the active sites lacked the conformational flexibility to accommodate them [11]. The binding affinity (Km) was too high, and catalytic efficiency was too low for hydrolysis to occur. Extended-spectrum β-lactamase (ESBL) mutations, however, overcame this limitation by enhancing the ability of the bacteria to bind extended-spectrum β-lactam side chains. ESBL mutations, such as G238S, R164S, and E104K, expanded the active site through structural changes in the omega loop, allowing for new interactions between the enzyme and side chains, such as electrostatic interactions and hydrogen bonding [11, 12]. This significantly increased catalytic efficiency and decreased Km. Various gain-of-function mutations enabled β-lactamases to degrade β-lactam side chains more effectively, increasing the likelihood of significant antibiotic resistance [12].
For example, the G238S and R164S mutations in TEM enhance degradation by significantly increasing the conformational flexibility of the β-lactamase active site, by promoting the formation of Hydrogen bonding networks, allowing for the effective accommodation of the bulky R1 and R2 side chains of second-generation βlactam antibiotics, such as cefotaxime and ceftazidime [12]. This increased flexibility improves the binding affinity between the active site and the side chains, thereby increasing the acylation rate of cefotaxime and ceftazidime [12, 13]. As a result, catalytic efficiency and turnover rate also increase. Other mutations, such as E104K and E240K, further increase catalytic efficiency by enhancing electrostatic interactions between the β-lactamase active site and the β-lactam side chains, thus reducing aggregation and promoting proper folding [13, 14]. This phenomenon of enhanced hydrolysis due to increased flexibility is also seen in KPC β-lactamase, where the V240G and H274Y mutations improve the accommodation of the ceftazidime side chain by increasing the conformational flexibility of the β3 strand and introducing a tyrosine residue that participates in pi-stacking interactions with positively charged residues on the β-lactam side chain, further increasing catalytic turnover and facilitating hydrolysis [15].
Some mutations enhance the hydrolysis of β-lactam side chains by stabilizing the β-lactamase enzyme, enabling it to withstand other mutations that could otherwise destabilize it. For example, the M182T mutation introduces a polar side chain with a hydroxyl group, which can participate in hydrogen bond network formation [16]. This helps compensate for destabilizing effects caused by mutations such as G238S and E104K, by promoting correct protein folding and reducing the likelihood of significant aggregation [16]. Similarly, the A237T mutation introduces a polar side chain that facilitates hydrogen bonding and dipole–dipole interactions [16, 17]. In metalloβ-lactamases, such as New Delhi Metallo-β-Lactamase (NDM-1) and Verona-integron encoded metallo-βlactamase 2 (VIM-2), mutations like D124N, H189Y, H120R, and H263Y can enhance metal ion coordination within the active site, such as zinc binding, thus increasing overall enzyme stability and activity. These stabilizing mutations can offset the negative effects of destabilizing mutations, such as R244S, which weakens enzyme stability by disrupting ionic and hydrogen bond networks with the introduction of smaller polar side chains, and S130G in TEM-1 β-lactamase, which disrupts protein folding by affecting hydrophobic interactions in the core, breaking hydrogen bonding networks, and impairing the stabilization of enzyme loop regions due to the loss of a polar group after mutation [18, 19].
Finally, other mutations enhance the hydrolysis and degradation process by promoting the formation of new bonds, bond networks, and intermolecular forces between residues in the β-lactamase enzyme active site and the β-lactam side chains. Initially, wild-type CTX-M β-lactamase enzymes were ineffective at hydrolyzing the βlactam side chains of certain β-lactam antibiotics, such as ceftazidime, because the predominant mutations only allowed for the accommodation of smaller β-lactam side chains, such as those found on cefotaxime [11]. However, mutations that wild-type CTX-M β-lactamase enzymes later underwent, such as D240G and P167S, enhanced the fit of the aminothiazole ring of ceftazidime into the β-lactamase active site and altered the steric conformation of proline from cis to trans, respectively, allowing for the formation of electrostatic interactions and new hydrogen bonds with the β-lactam side chains [11, 20]. This occurs because Proline has an extremely rigid cyclic structure, so by mutating to Serine, its flexibility increases, enabling it to form more bonds. This ultimately increases catalytic efficiency and enhances hydrolysis [21].
Overall, these results suggest that key mutations, such as R164S & G238S in TEM, P167S & D240G in CTX-M, etc. work to induce resistance to β-lactam antibiotics, by promoting increased conformational flexibility, increasing the stabilization of the β-lactamase enzyme, and enabling the formation of new bonds between the active site residues on the enzyme, and the side chains of the antibiotics. The questions that remain are how these mutations can ultimately induce multi-drug resistance, either to different classes of antibiotics or different types of β-lactam bacteria, and what methods can be harnessed to specifically target β – Lactamase enzymes and slow down the rate at which they undergo mutations.
References
1.Naghavi M, Vollset SE, Ikuta KS, Swetschinski LR, Gray AP, Wool EE, et al. Global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050. Lancet. 2024; 1-28.
2. Mora-Ochomogo M, Lohans CT. β-Lactam antibiotic targets and resistance mechanisms: from covalent inhibitors to substrates. RSC Med Chem. 2021; 12(10): 1623–1639.
3. Papp-Wallace KM, Mack AR, Taracila MA, Bonomo RA. Resistance to novel β-lactam-β-lactamase inhibitor combinations: the “price of progress.” Infect Dis Clin North Am. 2020; 34(4): 773–819.
4. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022; 399(10325): 629–655.
5. Oz T, Guvenek A, Yildiz S, Karaboga E, Tamer YT, Mumcuyan N, Ozan VB, Senturk GH, Cokol M, Yeh P, Toprak E. Strength of selection pressure is an important parameter contributing to the complexity of antibiotic resistance evolution. Mol Biol Evol. 2014; 31(9): 2387–2401.
6. Shitta G, Makanjuola O, Adefioye O, Olowe OA. Extended spectrum beta-lactamase (ESBL), blaTEM, blaSHV, and blaCTX-M resistance genes in community- and healthcare-associated Gram-negative bacteria from Osun State, Nigeria. Infect Disord Drug Targets. 2021; 21(4): 595–602.
7. Russ D, Glaser F, Shaer Tamar E, Yelin I, Baym M, Kelsic ED, Zampaloni C, Haldimann A, Kishony R. Escape mutations circumvent a tradeoff between resistance to a β-lactam and resistance to a β-lactamase inhibitor. Nat Commun. 2020; 11(1): 2029: 1–9.
8. Shaikh S, Fatima J, Shakil S, Rizvi SM, Kamal MA. Antibiotic resistance and extended spectrum betalactamases: types, epidemiology, and treatment. Saudi J Biol Sci. 2015; 22(1): 90–101.
9. Tao S, Chen H, Li N, Wang T, Liang W. The spread of antibiotic resistance genes in vivo model. Can J Infect Dis Med Microbiol. 2022; 2022: 3348695.
10. Pfeifer E, Bonnin RA, Rocha EPC. Phage-plasmids spread antibiotic resistance genes through infection and lysogenic conversion. mBio. 2022; 13(5): e0185122.
11. Hussain HI, Aqib AI, Seleem MN, Shabbir MA, Hao H, Iqbal Z, Kulyar MF, Zaheer T, Li K. Genetic basis of molecular mechanisms in β-lactam resistant gram-negative bacteria. Microb Pathog. 2021; 158: 105040.
12. Palzkill T. Structural and mechanistic basis for extended-spectrum drug-resistance mutations in altering the specificity of TEM, CTX-M, and KPC β-lactamases. Front Mol Biosci. 2018; 5: 1–19.
13. Soeung V, Lu S, Hu L, Judge A, Sankaran B, Venkataram Prasad BV, Palzkill T. A drug-resistant βlactamase variant changes the conformation of its active-site proton shuttle to alter substrate specificity and inhibitor potency. J Biol Chem. 2020; 295(52): 18239–18255.
14. Zhou HX, Pang X. Electrostatic interactions in protein structure, folding, binding, and condensation. Chem Rev. 2018; 118(4): 1691–1741.
15. Avci FG, Altinisik FE, Vardar Ulu D, Ozkirimli Olmez E, Sariyar Akbulut B. An evolutionarily conserved allosteric site modulates beta-lactamase activity. J Enzyme Inhib Med Chem. 2016; 31(sup3): 33–40.
16. Grigorenko VG, Krivitskaya AV, Khrenova MG, Rubtsova MY, Presnova GV, Andreeva IP, Serova OV, Egorov AM. Saturation mutagenesis and molecular modeling: the impact of methionine 182 substitutions on the stability of β-lactamase TEM-1. Int J Mol Sci. 2024; 25(14): 76-91.
17. Blázquez J, Negri MC, Morosini MI, Gómez-Gómez JM, Baquero F. A237T as a modulating mutation in naturally occurring extended-spectrum TEM-type beta-lactamases. Antimicrob Agents Chemother. 1998; 42(5): 1042–1044.
18. Palica K, Deufel F, Skagseth S, Di Santo Metzler GP, Thoma J, Rasmussen AA, Valkonen A, Sunnerhagen P, Leiros HKS, Andersson H, Erdelyi M. α-Aminophosphonate inhibitors of metallo-β-lactamases NDM-1 and VIM-2. RSC Med Chem. 2023; 14: 2277–2300.
19. Schroeder WA, Locke TR, Jensen SE. Resistance to beta-lactamase inhibitor protein does not parallel resistance to clavulanic acid in TEM beta-lactamase mutants. Antimicrob Agents Chemother. 2002; 46(11): 3568–3573.
20. Turner J, Muraoka A, Bedenbaugh M, Childress B, Pernot L, Wiencek M, Peterson YK. The chemical relationship among beta-lactam antibiotics and potential impacts on reactivity and decomposition. Front Microbiol. 2022; 13: 807955.
21. Kim D, Kim S, Kwon Y, Kim Y, Park H, Kwak K, Lee H, Lee JH, Jang KM, Kim D, Lee SH, Kang LW. Structural insights for β-lactam antibiotics. Biomol Ther. 2023; 31(2): 141–147.
