Diamond Annual Review 2019/20

14 15 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 liposome swelling assays, revealed that the OmpK36 WT+GD displays the same extent of resistance as the OmpK36 ST258 suggesting that the other mutations are not participating in the resistance mechanism. The crystal structure of the OmpK36 WT+GD at 2.0 Å resolution revealed an identical L3 conformation and stabilisation as the OmpK36 ST258 . The role of the salt-bridge in conferring carbapenem resistance was further investigated by generating an R127A substitution (OmpK36 ST258R127A ). The MIC and liposome swelling assays indicated that stabilisation of L3 by the salt-bridge is not contributing towards the Carbapenem resistance but only by the Gly-Asp insertion. Further functional data and mouse infection models showed that the L3 Gly-Asp insertion is detrimental to the fitness of the bacteria, potentially as a result of altered nutrient uptake. The functional and structural data have enabled the probing of the molecular mechanism of Carbapenem resistance mediated by outer membrane porins. Unlike other porins that display changes in their charge profile within the pore 4 , thus repulsing antibiotics from influxing through them, the K. pneumoniae resistant bacteria utilise a unique mechanism of pore constriction alone that gives them the advantage of becoming resistant to several antibiotics without the need to alter their charge profile for each class of antibiotics. This mechanism very likely contributes to the high severity and mortality of hospital-acquired Carbapenem-resistant infections. References: 1. Xu L. et al. Systematic review and meta-analysis of mortality of patients Macromolecular Crystallography Group Beamlines I03, I04 and I24 Investigating antibiotic resistance in Klebsiellapneumoniae bacteria Related publication: Wong J. L. C., RomanoM., Kerry L. E., Kwong H. -S., LowW. -W., Brett S. J., Clements A., Beis K. & Frankel G. OmpK36- mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo. Nat. Commun. 10 , 3957 (2019). DOI: 10.1038/s41467-019- 11756-y Publication keywords: Carbapenem resistance; Minimum inhibitory concentration; Outer membrane porin; Pore constriction; Liposome swelling assays; Mouse infectionmodel K lebsiella pneumoniae are common gut bacteria that can cause severe infections in other areas of the body. Such infections are usually acquired in hospital, and antibiotic-resistant strains account for a 40%mortality rate in hospital settings. More than 20,000 K. pneumoniae infections were recorded in UK hospitals in the past year. Carbapenems are used as antibiotics in hospitals when others have failed or are ineffective. However, K. pneumoniae is becoming increasingly resistant to Carbapenems and other antibiotics. Researchers at Imperial College London used Macromolecular Crystallography (MX) beamlines I03, I04 and I24 at Diamond Light Source to uncover one mechanism of antibiotic resistance in K. pneumoniae. The key lies in surface doorways known as pores. Resistant bacteria have much smaller pores, which blocks antibiotics from entering the bacteria. Understanding the molecular mechanismof Carbapenem resistance will be valuable tomedicinal chemists, who can use this information to designmore effective antibiotics. Interestingly, having smaller pores also makes the bacteria weaker, by restricting the amount of nutrients they can absorb. The resistant bacteria grow more slowly, but this disadvantage is outweighed by being able to avoid antibiotics, allowing them to maintain a high level of infection. These results suggest that the extensive use of Carbapenems in hospitals is a significant driver in the spread of these antibiotic-resistant ‘superbugs’. The study suggests that doctors should be more cautious in prescribing broad-spectrum antibiotics such as Carbapenems. Resistance to Carbapenems by K. pneumoniae and other Gram-negative bacteria is a global problem and hospital-acquired Carbapenem-resistant infection mortality is high 1 . Resistance to Carbapenems is a result of carbapenemase enzymes, which inactivate Carbapenems by hydrolysis. In addition to the enzymes, resistance can also emerge as a result of mutations to the outer membrane porins through which antibiotics diffuse to the bacterial periplasm. Porins play important physiological roles including the influx of small hydrophilic nutrients. The two major porins on the outer membrane of K. pneumoniae are OmpK35 and OmpK36. A major pathogenic K. pneumoniae clade is ST258 2 , which expresses a truncated / non-functional ompK35 and mutations in ompK36 which reduced solute diffusion inside the cell. In order to understand how the OmpK36 mutations (12 substitutions and 5 insertions) confer resistance to Carbapenems (Fig. 1), the crystal structures of the OmpK36 WT and OmpK36 ST258 variant were determined at 1.9 and 3.2 Å resolution, respectively 3 (Fig. 2). The overall structure of OmpK36 resembles other porins; it is a trimer that is composed of 16-stranded β-barrels. The OmpK36 ST258 structure can be superimposed on OmpK36 WT with an rmsd of 0.43 Å over 340 C α atoms. Two important structural components in porins are loops 3 and 4 (L3 and L4). L3 is not exposed at the cell surface but folds back into the barrel, forming a constriction zone half way inside the channel that contributes to the permeability properties, such as size exclusion limit and ion selectivity of the pore. L4 lies away from the pore and is involved in monomer trimerisation and subsequent stability. Comparison of the OmpK36 WT andOmpK36 ST258 revealed that a Gly-Asp insertion (at positions 113 and 114) in L3 has constricted the pore size by approximately 26% relative to the OmpK36 WT (Fig. 3). This constriction is a result of a more extended L3 inside the barrel that is stabilised by a salt-bridge between Asp114 and Arg127 at the barrel face of the pore (Fig. 2). To further validate the role of the Gly-Asp insertion in L3, a chimera was constructed, where the Gly-Asp insertion was introduced on the OmpK36 WT background (OmpK36 WT+GD ). The functional data, including minimum inhibitory concentration (MIC) and infected with carbapenem-resistant Klebsiella pneumoniae. Ann. Clin. Microbiol. Antimicrob. 6 , 18 (2017). DOI: 10.1186/s12941-017-0191-3 2. Peirano G. et al. Importance of clonal complex 258 and IncFK2-like plasmids among a global collection of Klebsiella pneumoniae with blaKPC. Antimicrob. Agents Chemother. 61 , e02610 (2017). DOI: 10.1128/AAC.02610-16 3. Wong, J. L. C. et al. OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo. Nat. Commun. 10 , 3957 (2019). DOI: 10.1038/s41467-019-11756-y 4. Lou, H. et al. Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli. PLoS One 6 , e25825 (2011). DOI: 10.1371/journal.pone.0025825 Funding acknowledgements: K.B. is supported by a grant for the Medical Research Council (MR/ N020103/1). J.L.C.W. is supported by a clinical training programme from the MRC (MRC CMBI Studentship award MR/R502376/1). G.F. is supported by an Investigator grant from theWellcome Trust. Corresponding authors: Prof Gad Frankel, Imperial College London, g.frankel@imperial.ac.uk and Dr Konstantinos Beis, Imperial College London, kbeis@imperial.ac.uk Figure 1: Minimum Inhibitory Concentration assays to assess the impact of mutations to antibiotics. OmpK35ST258 and OmpK36ST258 substitutions show increased resistance to several Carbapenems used to treat Gram-negative infections. Antibiotic key: IPM Imipenem, MEM Meropenem, ETP Ertapenem. Figure 2: Crystal structure of the OmpK36 ST258 porin. (left panel) The porin is shown in cartoon (green) and the mutations are shown in stick format (orange). (right panel) Close up view of the pore constriction as a result of the Gly-Asp insertion. The extended conformation is stabilised by a salt-bridge with Arg127. Figure 3: Pore diameter comparison. The OmpK36 monomer structure is shown as a cartoon. The face of the β-barrel has been removed to expose the pore and L3 (left panel). The minimal pore diameter graph (right panel) demonstrates a reduction in minimal pore diameter in both OmpK36 ST258 and OmpK36 WT+GD compared to OmpK36 WT . The calculated pore diameter of the clinical OmpK36 ST258 is 2.37 Å and the OmpK36 WT+GD chimera is 2.87 Å, whereas the OmpK36 WT is 3.2 Å.