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Treatment of human challenge and MDR strains of Neisseria gonorrhoeae with LpxC inhibitors

Treatment of human challenge and MDR strains of Neisseria gonorrhoeae with LpxC inhibitors Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 J Antimicrob Chemother 2018; 73: 2064–2071 doi:10.1093/jac/dky151 Advance Access publication 2 May 2018 Treatment of human challenge and MDR strains of Neisseria gonorrhoeae with LpxC inhibitors 1,2 1 1,2 Constance M. John , Dongxiao Feng and Gary A. Jarvis * 1 2 Center for Immunochemistry, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA; Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA *Corresponding author. Tel: !1-415-221-4810, ext. 26303; E-mail: gary.jarvis@ucsf.edu orcid.org/0000-0002-7576-3702 Received 12 March 2018; accepted 29 March 2018 Objectives: Inhibitors of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), which cat- alyses the second step in the biosynthesis of lipid A, have been developed as potential antibiotics for Gram-negative infections. Our objectives were to determine the effect of LpxC inhibition on the in vitro survival and inflammatory potential of Neisseria gonorrhoeae. Methods: Survival of four human challenge strains was determined after treatment with two LpxC inhibitors for 2 and 4 h. To confirm results from treatment and assess their anti-inflammatory effect, the expression of TNF-a by human THP-1 monocytic cells infected with bacteria in the presence of the LpxC inhibitors was quantified. Cytotoxicity of inhibitors for THP-1 cells was evaluated by release of lactate dehydrogenase. Survival of five MDR strains was determined after 2 h of treatment with an LpxC inhibitor and the effect of co-treatment on MICs of ceftriaxone and azithromycin was examined. Results: The inhibitors had bactericidal activity against the four human challenge and five MDR strains with one compound exhibiting complete killing at 5 mg/L after either 2 or 4 h of treatment. Treatment of gonococci infecting THP-1 monocytic cells reduced the levels of TNF-a probably owing to reduced numbers of bacteria and a lower level of expression of lipooligosaccharide. Neither inhibitor exhibited cytotoxicity for THP-1 cells. The MIC of azithromycin was slightly lowered by sublethal treatment of two MDR strains with an LpxC inhibitor. Conclusions: Our in vitro results demonstrated promising efficacy of LpxC inhibition of N. gonorrhoeae that war- rants further investigation particularly owing to the rise in MDR gonorrhoea. non-specific. The lack of apparent symptoms in women can lead Introduction to untreated infections and serious complications including pelvic Gonorrhoea represents a growing burden of disease worldwide. inflammatory disease, infertility, ectopic pregnancies, spontan- The WHO estimates there are .106 million cases of gonorrhoea eous abortions, preterm birth and neonatal conjunctivitis causing annually and the reported incidence of gonococcal infection in scarring and blindness. countries with good surveillance has been increasing. For example, Neisseria gonorrhoeae is increasingly MDR with a real prospect cases of gonorrhoea increased by 90% in Australia from 2009 to that untreatable gonorrhoea could soon emerge. Lack of a vaccine 2 3 2014 and by 11% in the USA from 2014 to 2015. Gonorrhoea for gonorrhoea further escalates this problem. Surveys of resist- affects countries of all income levels with the highest rates ance in the population are the basis for treatment guidelines and reported in some African countries where there are 50 and when resistance to an antibiotic is.5%, its use is no longer recom- 100 new infections per 1000 women and men, respectively, each mended. Currently only ceftriaxone and azithromycin are recom- year. In addition, a growing number of studies have shown that mended for first-line therapy and clinical isolates resistant to both 8,9 gonococcal infection can facilitate susceptibility to and transmis- antibiotics have been reported in Denmark, Canada and Japan. 4–6 sion of HIV. Thus, there is an urgent need for new antibiotics for gonococcal In men, infection manifests most commonly as urethritis, but infections. as many as 40% of urogenital infections may be asymptomatic. The enzymes and the genes encoding them that are required Untreated urethral infection can lead to epididymitis, urethral stric- for biosynthesis of the lipid A moiety of Gram-negative bacteria ture and reduced fertility in men. Gonorrhoea is asymptomatic in have been well characterized in Escherichia coli and are thought to more than half of women and when present, symptoms can seem present targets for the development of new antibiotics. The first Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2018. This work is written by US Government employees and is in the public domain in the US. 2064 Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 Treatment of N. gonorrhoeae with LpxC inhibitors JAC 35,36 antimicrobials. The 35/02 and 59/03 strains are isolates from the USA step of lipid A biosynthesis is thermodynamically unfavourable with reduced susceptibility to ceftriaxone and cefixime. and catalysed by the product of the lpxA gene that transfers an R-3-hydroxymyristoyl moiety to the third position of the glucosa- mine ring of UDP-3-O-N-acetylglucosamine. UDP-3-O-(R-3- Antimicrobial compounds hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a The LpxC inhibitors, PF-5081090 and PF-04753299, were obtained from zinc metalloamidase that catalyses the second and more thermo- Sigma–Aldrich (St Louis, MO, USA) through its partnership with Pfizer Global dynamically favourable step of lipid A biosynthesis in which an ace- Research and Development (New London, CT, USA). tyl moiety is cleaved from the 2-N of the lipid A precursor molecule to produce a free amino group that is immediately acylated with Bacterial survival assays 12,13 an additional R-3-hydroxymyristoyl fatty acid by LpxD. Owing Bacteria were grown overnight on Difco GC agar (Becton, Dickinson and Co., to the critical role of the enzyme in lipid A biosynthesis and its lack Sparks, MD, USA) containing 1% BBL Isovitalex Enrichment (Becton, Dickinson of homology with mammalian proteins, inhibitors of LpxC have and Co.). After harvesting, bacteria were diluted in warm PBS and OD at been developed as potential antibiotics for the treatment of Gram- 590 nm was used to dilute serially the stocks to a concentration of 4%10 negative infections. bacteria per mL. Total reaction volumes were 200 lL with 180 lLofRPMI 1640 medium with 10% fetal bovine serum (FBS) (Gibco, ThermoFisher, The first description of small molecule LpxC inhibitors, to the Pittsburgh, PA, USA), inoculated with 4000 bacteria in 10 lL of RPMI 1640 and best of our knowledge, was by scientists at Merck in 1996. Since 15–19 with 10 lL of increasing concentrations of the LpxC inhibitors dissolved in then other groups have described inhibitors, many of which RPMI 1640 or of the vehicle only, which was 1% DMSO in RPMI 1640. After in- contain a hydroxamate or another substituent that can bind the cubation of the tubes at 37 C for 2 and 4 h, aliquots were plated in duplicate catalytic zinc cation of LpxC and a hydrophobic tail that is designed for each concentration tested. The plates were incubated overnight and then to mimic the hydroxymyristate moiety of the substrate. LpxC bacterial colonies were counted. Survival was expressed as the percentage of inhibitors have been shown to be bactericidal for pathogenic bacteria detected as cfu from aliquots of LpxC-treated bacteria compared Gram-negative organisms including E. coli, Klebsiella pneumoniae, with the cfu in aliquots of the vehicle-only treated bacterial suspensions. Pseudomonas aeruginosa and Haemophilus influenzae.Based on the relatively numerous reports of bactericidal activity against Effect of inhibition of LpxC on TNF-a expression induced Gram-negative organisms, we hypothesized that blocking the bio- by gonococcal infection of THP-1 cells synthesis of lipid A with an LpxC inhibitor could be efficacious for The human THP-1 monocytic cell line was cultured in RPMI 1640 with 10% the treatment of gonococcal infections either by being bactericidal FBS (growth medium) at 37 Cin a 5% CO atmosphere. THP-1 cells were for the bacteria or by modulation of the host response to infection. activated in growth medium containing 10 ng/mL phorbol myristate acet- Inhibition of purified recombinant LpxC from Neisseria meningi- ate (Sigma–Aldrich) for 18 h. After removing the medium and washing with tidis has been described. However, there have been no reports to PBS, the cells were trypsinized and plated in 96-well plates at 1%10 cells date on the effect of LpxC inhibitors on survival or inflammatory per well in growth medium. Aliquots of medium containing MS11mkA, MS11mkC, FA1090 A23a or FA1090 1-81-S2 gonococci were added to wells potential of either N. meningitidis or N. gonorrhoeae. In this study, to achieve an moi of 1. Serial dilutions were used to prepare solutions con- we evaluated the effect of LpxC inhibition on well-characterized taining the LpxC inhibitors in RPMI 1640 medium from stock solutions isolates of N. gonorrhoeae that have been used in human chal- (5 mg/mL in DMSO) and then 5 lL aliquots were added to the wells to lenge studies and on five strains that exhibit MDR. achieve final concentrations of 0.04, 1.0 or 25 mg/L. Cells in control wells were treated with vehicle only containing 1% DMSO. After incubating the plates at 37 Cin 5% CO for 18 h, cell culture supernatants were transferred Materials and methods 2 to another 96-well plate and frozen at #80 C. For the detection of TNF-a in Bacterial strains aliquots of the supernatants, an ELISA (Human TNF-a Ready-Set-Go; eBioscience, San Diego, CA, USA) was performed per the instructions of the The MS11 and FA1090 strains of N. gonorrhoeae have been used in most ex- vendor using a Thermomax (Molecular Devices, Sunnyvale, CA, USA) plate perimental human studies of gonococcal infections over the last 25 years. reader with detection at 450 nm. MS11, a porin serotype PIB-9 strain, was isolated from a patient with anter- 22,23 ior urethritis in 1970. The MS11mk variant A strain descended from MS11 and was used in human challenge studies from which the variant C Cytotoxicity of LpxC inhibitors for THP-1 cells strain was isolated. The a-chain of the oligosaccharide portion of the lip- Release of lactate dehydrogenase (LDH) into cell culture supernatants was ooligosaccharide (LOS) of MS11mkA is a lactose moiety whereas the analysed to determine if the LpxC inhibitors were cytotoxic to the THP-1 MS11mkC expresses a lacto-N-neotetraose a-chain moiety that can be sub- 38–40 cells. Cytotoxicity of polymyxin B (Sigma–Aldrich) for the human THP-1 25,26 stituted with one or more N-acetyllactosamine groups. FA1090 A23a, a cells was assayed as a positive control. After activation by treatment with porin serotype PIB-3 strain, was isolated in the 1970s from the endocervix phorbol myristate acetate (10 ng/mL) for 18 h, the THP-1 cells were plated of a patient who probably had a disseminated infection. FA1090 has been (1%10 per well) in 96-well plates in Opti-MEM medium with 1% FBS (Gibco). 28–30 widely used in human challenge studies and in one of these studies Once cells adhered to the wells, aliquots of the LpxC inhibitors in cell culture the 1-81-S2 strain was isolated. medium were added to create final concentrations of 0.008, 0.04, 0.2, 1.0, The FA6140 gonococcal strain is a clinical isolate that is resistant to 5.0 or 25.0 mg/L. Aliquots of polymyxin B in RPMI 1640 were added to the 32 8 penicillin. F89, classified as WHO Y, was isolated in France from a patient wells to achieve final concentrations of 5.0, 25 and 125 mg/L. Control cells with a urethral infection in 2010 and was found to have resistance to cef- were treated with vehicle only. The plates were incubated for 37 Cin5% triaxone and most other antibiotics tested. H041, classified as WHO X, is a CO for 18 h. The relative levels of LDH released from dying cells into cell cul- highly ceftriaxone-resistant isolate from the throat of a patient in Japan in ture supernatants were determined using a kit (Roche, Mannheim, 2009 that is considered an XDR strain owing to its extensive resistance to Germany) with detection using a Thermomax plate reader at 490 nm. 2065 Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 John et al. CH CH Effect of treatment of human challenge strains with HO HO LpxC inhibitors on inflammatory signalling of human HN HN THP-1 monocytes OO S OO S N LOS is a major component of the outer membrane of N. gonorrhoeae CH CH and is highly inflammatory. Previous work has shown that reducing phosphoethanolaminylation of the lipid A of gonococcal LOS dimin- ishes both the inflammatory signalling and the fitness of the bac- 28,41,42 teria in vivo. We theorized that the LpxC inhibitors could modulate inflammatory signalling induced in human THP-1 mono- cytic cells by killing the bacteria but also by reducing the amount of LOS expressed on the bacterial outer membrane or that was released in membrane blebs from dead or living bacteria. To deter- mine the effect of LpxC inhibition on inflammatory signalling, we OCH incubated THP-1 cells with bacteria at an moi of 1 in the presence of Figure 1. Structures of the two LpxC inhibitors PF-5081090 (left) and the LpxC inhibitors at 0.04, 1 and 25 mg/L. As shown in Figure 3, PF-04753299 (right). treatment with either inhibitor at all concentrations tested resulted in a statistically significant decrease in TNF-a expression (P, 0.01 for all comparisons). Effect of LpxC inhibition on MICs for MDR strains To determine the effect of co-treatment of the five MDR strains, absorbance LpxC inhibitors are not cytotoxic for human THP-1 cells at 590 nm based on comparison with a 0.5 McFarland turbidity standard To determine whether the LpxC inhibitors were cytotoxic at the (bioMe ´ rieux, Durham, NC, USA) was used to suspend the appropriate num- concentrations that were bactericidal and reduced inflammatory ber of bacteria in 396 lL of RPMI 1640 medium with 10% FBS in microcentri- signalling in THP-1 cells, we analysed the relative amount of LDH fuge tubes. To achieve a final concentration of PF-04753299 of 0.2 mg/L, released from the cells following 18 h of treatment with the inhibi- 4 lL aliquots of solutions of PF-04753299 in RPMI 1640 (20 mg/L) or vehicle tors. Results showed that cell death was not significantly increased only were added and the tubes were incubated at 37 Cfor 2 h. The MICs of by treatment with either inhibitor at the concentration range of ceftriaxone and azithromycin for the MDR strains were determined using 0.008–25 mg/L tested (Figure 4; P. 0.05 for all comparisons). By the Etest method (bioMe ´ rieux) according to the instructions of the vendor. comparison, use of polymyxin B as a positive control resulted in a significant increase in LDH (P, 0.001) following treatment of THP- 1 cells with 125 mg/L polymyxin B, which is a concentration similar Statistical analyses to the MICs of polymyxin B for strains MS11 and FA1090 that have SigmaPlot version 12.5 (Systat Software, San Jose, CA, USA) was used to 42,44 been previously reported to be 50 and 100 mg/L, respectively. perform statistical analyses. Multigroup comparisons were performed using Thus, the effective concentration range of the LpxC inhibitors for one-way analysis of variance with Bonferroni’s post hoc tests. Significance gonococci does not induce cytotoxicity for THP-1 cells, whereas was defined as P, 0.05 for all comparisons. that for polymyxin B approaches cytotoxic concentrations. Results Activity of LpxC inhibitors against MDR strains Activity of LpxC inhibitors against human challenge The five MDR strains were tested with PF-04753299, which was the strains more efficacious of the two inhibitors. All of the MDR strains were completely killed by incubation with PF-04753299 at 5mg/L and The structures of the two LpxC inhibitors that were tested are bacterial survival was ,50% after incubation at 1 mg/L for 2 h shown in Figure 1. Complete killing of all four strains was observed (Figure 5). The susceptibility of the MDR strains was similar be- after 4 h of treatment with PF-5081090 at a concentration of tween strains at the concentrations tested in general except at 25 mg/L and after 2 or 4 h of treatment with PF-04753299 at 1.0 mg/L where, e.g..30% of the FA6140 bacteria and,5% of the 5mg/L (Figure 2). Only the MS11mkC isolate survived treatment H041 bacteria survived. The MDR strains had comparable suscepti- with 25 mg/L PF-5081090 for the shorter 2 h period at a minimum bility with the human challenge strains to 2 h of treatment with PF- level of 0.4% survival. The survival of the two FA1090 strains was 04753299 (Figure 2b). significantly reduced by 4 h of treatment with PF-04753299 at all five concentrations tested (P, 0.001), whereas the MS11 strains Effect of LpxC inhibition on MICs for MDR strains were resistant to the lowest concentration of inhibitors tested (0.008 mg/L). The two FA1090 strains tended to be more suscep- Co-treatment of the MDR strains with a sublethal concentration tible to either inhibitor at virtually all concentrations of 0.2 mg/L (0.2 mg/L) of PF-04753299 and ceftriaxone and azithromycin was compared with the MS11 strains. Increasing the length of treat- explored using Etest strips. As shown in Table 1,inthe absence of ment from 2 to 4 h reduced the survival of the FA1090 bacteria to the inhibitor the MICs of the antibiotics for the MDR strains were 32,33,35,37 a greater degree compared with the MS11 strains. Overall, PF- similar to values previously reported for these strains. 04753299 tended to be a more efficacious bactericidal compound In the presence of the inhibitor, no changes were observed in the than PF-5081090. susceptibility of the strains to ceftriaxone, which is a bactericidal 2066 Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 Treatment of N. gonorrhoeae with LpxC inhibitors JAC (a) (b) 160 160 MS11mkA 140 140 MS11mkC FA1090 A23a 120 120 FA1090 1-81-S2 100 100 80 80 60 60 40 40 20 20 0 0 0.008 0.04 0.2 1 5 25 0.008 0.04 0.2 1 5 25 PF-5081090 (mg/L) PF-04753299 (mg/L) (c) (d) 160 160 140 140 120 120 100 100 80 80 60 60 40 40 0.008 0.04 0.2 1 5 25 0.008 0.04 0.2 1 5 25 PF-5081090 (mg/L) PF-04753299 (mg/L) Figure 2. Bactericidal activity of the two LpxC inhibitors against the four human challenge isolates of N. gonorrhoeae. Increasing concentrations (0.0, 0.008, 0.04, 1.0, 5.0 or 25 mg/L) of the inhibitors were incubated with the gonococci and survival was determined after 2 and 4 h of treatment. Bars represent the mean+ SEM of triplicate experiments that were each performed in duplicate. MS11mkA MS11mkC FA1090 A23a FA1090 1-81-S2 400 400 200 200 0 0 0 0.04 125 0 0.04 125 PF-5081090 (mg/L) PF-04753299 (mg/L) Figure 3. Levels of TNF-a in supernatants of cultures of activated human THP-1 cells infected with human gonococcal isolates at an moi of 1 in the presence of increasing concentrations (0.0, 0.04, 1.0 or 25 mg/L) of LpxC inhibitors after 18 h. Bars represent the mean+ SEM of quadruplicate experiments. TNF-α (pg/mL) Percentage survival after 4 h Percentage survival after 2 h TNF-α (pg/mL) Percentage survival after 4 h Percentage survival after 2 h Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 John et al. 160 160 *** 140 140 120 120 100 100 100 80 80 60 60 60 40 40 40 20 20 20 0 0 0 0.008 0.04 0.2 1 5 25 0.008 0.04 0.2 1 5 25 5 25 125 PF-5081090 (mg/L) PF-04753299 (mg/L) Polymyxin B (mg/L) Figure 4. Relative levels of LDH in culture supernatants of activated human THP-1 cells in the presence of increasing concentrations of LpxC inhibitors (0.008–25 mg/L) or polymyxin B (5–125 mg/L) after 18 h. Bars represent the mean+ SEM of quadruplicate experiments. ***P, 0.001 compared with control. Table 1. MICs for MDR strains with and without treatment with an LpxC inhibitor FA6140 F89 MIC (mg/L) HO41 35/02 PF-04753299! PF-04753299! 59/03 Strain ceftriaxone ceftriaxone azithromycin azithromycin FA6140 0.032 0.032 0.19 0.125 F89 1.0 1.0 0.25 0.25 H041 1.5 1.5 0.25 0.25 35/02 0.064 0.064 0.25 0.19 59/03 0.064 0.064 0.38 0.38 MICs were determined using the Etest method, determined after treat- ment with PF-04753299 (0.2 mg/L) or vehicle for 2 h. 0.008 0.04 0.2 1 5 25 PF-04753299 (mg/L) Figure 5. Bactericidal activity of PF-04753299 against five MDR strains of N. gonorrhoeae. Increasing concentrations of PF-04753299 (0.0, 0.008, previous comparison of treatment of a panel of 106 recent clinical 0.04, 1.0, 5.0 or 25 mg/L) were incubated with the gonococci and survival isolates of Gram-negative bacteria species including P. aeruginosa was determined after 2 h. Bars represent the mean+ SEM of triplicate and K. pneumoniae with PF-5081090 and PF-04753299 found that experiments that were each performed in duplicate. the former was more efficacious. The structures of LpxC orthologues vary significantly between agent. However, a modest decrease in the MIC of azithromycin, bacterial species. Differences in the C-termini have been shown to which is a bacteriostatic drug, was found with co-treatment of the alter the susceptibility to proteolytic cleavage and enzymatic FA6140 and 35/02 strains. Furthermore, no antagonism from co- degradation, which could affect the rate of LPS or LOS biosyn- treatment causing a decrease in susceptibility to either drug was thesis and, thus, sensitivity to LpxC inhibitors. There is 57% iden- observed. tity and 79% similarity of the primary sequence of the LpxC enzymes from P. aeruginosa and E. coli, but only 32% identity and 51% similarity of the Aquifex aeolicus LpxC enzyme to that of Discussion E. coli. The structure of N. gonorrhoeae LpxC has 49% identity Here we show that biphenyl alkyl sulphonamide hydroxamic de- and 71% similarity to that of E. coli. Potent LpxC inhibitors have rivative LpxC inhibitors are bactericidal for human challenge and been described, but the MICs for Gram-negative species can vary 15,47,50,51 MDR strains of N. gonorrhoeae. The concentrations at which PF- more than an order of magnitude. Furthermore, our 04753299 was bactericidal for the gonococcal isolates were com- results show that unlike a number of other Gram-negative species parable with the concentrations that were bactericidal for E. coli, that were tested with both inhibitors, N. gonorrhoeae is more P. aeruginosa and K. pneumoniae strains that had MIC values of 90 susceptible to PF-04753299 than PF-5081090. The differences in 2, 4 and 16 mg/L, respectively, following treatment periods of susceptibility could be owing to differences between bacterial spe- 15,17 16–20 h. However, compared with these species the gono- cies in the structures of the LpxC enzyme and highlight the poten- cocci were somewhat more susceptible to PF-04753299 as tial importance of testing in the species of interest. treatment with it at a concentration of 5 mg/L was completely We found that treatment with LpxC inhibitors at concentrations bactericidal to all strains after treatment for only 2 or 4 h. of 0.04 mg/L or greater significantly reduced expression levels of We found that overall the gonococci were less susceptible to TNF-a in an in vitro model of infection using a human THP-1 mono- PF-5081090 compared with PF-04753299. Interestingly, a cytic cell line. Induction of inflammatory signalling would be Percentage survival after 2 h LDH OD (percentage of control) LDH OD (percentage of control) LDH OD (percentage of control) 490 Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 Treatment of N. gonorrhoeae with LpxC inhibitors JAC largely due to LOS molecules in the outer membrane or in outer of LOS on N. gonorrhoeae that survived LpxC inhibitor treatment membrane blebs released from bacteria. It is likely that the should reduce bacterial fitness and virulence. Reducing TNF-a ex- reductions in cytokine levels observed were due to reduced num- pression in gonococcal infections in women potentially could pre- bers of bacteria as well as reduced levels of LOS. Our results indi- vent or limit damage to the fallopian tube and the sloughing of the 65–67 ciliated cells that leads to infertility. The lower inflammatory cate that LpxC is a promising target for the development of new potential is a hallmark of commensal Neisseria species relative to antibiotics for treating gonorrhoea as LpxC inhibitors exhibit both the pathogenic species, and surviving N. gonorrhoeae treated bactericidal and anti-inflammatory activities for N. gonorrhoeae. with an LpxC inhibitor could have a commensal-like, non-patho- Differences between the FA1090 and MS11 isolates have been genic relationship with its human host. reported including the serum sensitivity of and presence of a gen- There is synergism in the clinical and epidemiological occur- etic island in MS11 and serum resistance of and absence of a gen- 52,53 rence of HIV-1 and N. gonorrhoeae, which facilitates HIV-1 etic island in FA1090. Furthermore, whereas MS11 does expression. The heptose monosaccharides that are absent in express lactoferrin-binding proteins A and B and utilizes lactoferrin, 52,54 mammalian glycomes but present in the gonococcal LOS were FA1090 strains do not. A review of published studies revealed found to drive HIV-1 expression and innate immune responses. that MS11mkC has an ID for urethral infection that is nearly 2 Furthermore, recent data show that vaginal inflammation facili- log less than FA1090 and similar differences in infectivity have tates the transmission of HIV. Thus, treatment of gonorrhoea been observed in the mouse model of female genital infection, with an LpxC inhibitor that not only kills the bacteria but also although the reason for the differences is unclear. However, the reduces its expression of LOS and capacity for inflammatory sig- expression level of the multiple transferable resistance (Mtr) efflux nalling potentially could limit the transmission of HIV by coinfected pump was found to be higher in MS11 compared with FA1090. individuals. MS11 has a naturally occurring mutation in the mtr locus that Ideally, new antimicrobial agents for gonorrhoea would have leads to the expression of a consensus element that increases the activity against other coinfecting sexually transmitted pathogens promotion of mtrCDE transcription and, thus, resistance to some such as Chlamydia trachomatis. Small molecule inhibitors of antimicrobial compounds and possibly host cationic antimicro- LpxC were not bactericidal for C. trachomatis, but did block LPS syn- bial peptides such as LL-37. Increased expression of the Mtr ef- thesis and the transition of reticulate bodies to elementary bodies, flux pump probably explains the reduced susceptibility of MS11 a form that is required for the invasion of mammalian cells. compared with FA1090. Overall, our results showing the bactericidal effects on human Our results showing that the susceptibility of the MDR gonococ- challenge isolates and MDR strains of N. gonorrhoeae indicate that cal strains to PF-04753299 was very similar to that of the human further studies of LpxC inhibitors as treatments for gonococcal challenge strains are encouraging for potential clinical applica- infections are warranted, particularly as there is an urgent need for tions. The data imply that mechanisms conferring resistance to new antibiotics for gonorrhoea because of the rise in MDR strains. LpxC inhibition and to antibiotics such as cephalosporins, fluoroqui- Furthermore, owing to the likely ability of LpxC inhibitors to reduce nolones and macrolides differ and, furthermore, indicate that MDR the expression of LOS on viable gonococci and, thus, impair bacter- gonorrhoea may be treatable with an LpxC inhibitor. ial fitness, testing in an in vivo model of gonococcal infection po- We tested combination treatment with ceftriaxone and azith- tentially could reveal efficacy that would be even greater than that romycin as these two agents are recommended as dual therapy 58 observed in in vitro experimentation. for gonorrhoea in current treatment guidelines. More combina- tions of antimicrobials with LpxC inhibitors for N. gonorrhoeae should be tested as co-treatment with PF-04753299 showed that there was a modest decrease in the MICs of azithromycin for two Acknowledgements of the MDR strains. This is paper number 118 from the Center for Immunochemistry. The lipid A component of LOS is highly inflammatory and the We are grateful to Alison K. Criss (Department of Microbiology, anti-inflammatory effects of LpxC inhibitors could be beneficial in Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, treating gonorrhoea. This postulate is supported by the finding USA) and Robert A. Nicholas (Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA) for providing the MDR strains. that an LpxC inhibitor that was not bactericidal for Acinetobacter baumannii, which can survive in the absence of LOS expression, nonetheless, protected mice from infection by reducing inflamma- tion and facilitating phagocytosis. Furthermore, treatment with Funding the PF-5081090 LpxC inhibitor increased the susceptibility of A. This work was supported by Merit Review Award BX000727 from the baumannii to other antibiotics by increasing the permeability of Research Service of the United States Department of Veterans Affairs (G. the bacteria. N. meningitidis and Moraxella catarrhalis can survive A. J.). G. A. J. is the recipient of a Senior Research Career Scientist Award in the absence of lipid A although this was produced by directed from the Research Service of the United States Department of Veterans 61,62 in vitro mutation, whereas A. baumannii LOS-deficient organ- Affairs. isms can arise after polymyxin B treatment and have been iso- 63,64 lated, albeit rarely, in the clinic. Human and animal studies show that LptA-catalysed phos- phoethanolaminylation of the lipid A confers a significant survival Transparency declarations advantage to N. gonorrhoeae. 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Treatment of human challenge and MDR strains of Neisseria gonorrhoeae with LpxC inhibitors

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Oxford University Press
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Copyright © 2022 British Society for Antimicrobial Chemotherapy
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0305-7453
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1460-2091
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10.1093/jac/dky151
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Abstract

Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 J Antimicrob Chemother 2018; 73: 2064–2071 doi:10.1093/jac/dky151 Advance Access publication 2 May 2018 Treatment of human challenge and MDR strains of Neisseria gonorrhoeae with LpxC inhibitors 1,2 1 1,2 Constance M. John , Dongxiao Feng and Gary A. Jarvis * 1 2 Center for Immunochemistry, Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA; Department of Laboratory Medicine, University of California, San Francisco, CA 94143, USA *Corresponding author. Tel: !1-415-221-4810, ext. 26303; E-mail: gary.jarvis@ucsf.edu orcid.org/0000-0002-7576-3702 Received 12 March 2018; accepted 29 March 2018 Objectives: Inhibitors of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), which cat- alyses the second step in the biosynthesis of lipid A, have been developed as potential antibiotics for Gram-negative infections. Our objectives were to determine the effect of LpxC inhibition on the in vitro survival and inflammatory potential of Neisseria gonorrhoeae. Methods: Survival of four human challenge strains was determined after treatment with two LpxC inhibitors for 2 and 4 h. To confirm results from treatment and assess their anti-inflammatory effect, the expression of TNF-a by human THP-1 monocytic cells infected with bacteria in the presence of the LpxC inhibitors was quantified. Cytotoxicity of inhibitors for THP-1 cells was evaluated by release of lactate dehydrogenase. Survival of five MDR strains was determined after 2 h of treatment with an LpxC inhibitor and the effect of co-treatment on MICs of ceftriaxone and azithromycin was examined. Results: The inhibitors had bactericidal activity against the four human challenge and five MDR strains with one compound exhibiting complete killing at 5 mg/L after either 2 or 4 h of treatment. Treatment of gonococci infecting THP-1 monocytic cells reduced the levels of TNF-a probably owing to reduced numbers of bacteria and a lower level of expression of lipooligosaccharide. Neither inhibitor exhibited cytotoxicity for THP-1 cells. The MIC of azithromycin was slightly lowered by sublethal treatment of two MDR strains with an LpxC inhibitor. Conclusions: Our in vitro results demonstrated promising efficacy of LpxC inhibition of N. gonorrhoeae that war- rants further investigation particularly owing to the rise in MDR gonorrhoea. non-specific. The lack of apparent symptoms in women can lead Introduction to untreated infections and serious complications including pelvic Gonorrhoea represents a growing burden of disease worldwide. inflammatory disease, infertility, ectopic pregnancies, spontan- The WHO estimates there are .106 million cases of gonorrhoea eous abortions, preterm birth and neonatal conjunctivitis causing annually and the reported incidence of gonococcal infection in scarring and blindness. countries with good surveillance has been increasing. For example, Neisseria gonorrhoeae is increasingly MDR with a real prospect cases of gonorrhoea increased by 90% in Australia from 2009 to that untreatable gonorrhoea could soon emerge. Lack of a vaccine 2 3 2014 and by 11% in the USA from 2014 to 2015. Gonorrhoea for gonorrhoea further escalates this problem. Surveys of resist- affects countries of all income levels with the highest rates ance in the population are the basis for treatment guidelines and reported in some African countries where there are 50 and when resistance to an antibiotic is.5%, its use is no longer recom- 100 new infections per 1000 women and men, respectively, each mended. Currently only ceftriaxone and azithromycin are recom- year. In addition, a growing number of studies have shown that mended for first-line therapy and clinical isolates resistant to both 8,9 gonococcal infection can facilitate susceptibility to and transmis- antibiotics have been reported in Denmark, Canada and Japan. 4–6 sion of HIV. Thus, there is an urgent need for new antibiotics for gonococcal In men, infection manifests most commonly as urethritis, but infections. as many as 40% of urogenital infections may be asymptomatic. The enzymes and the genes encoding them that are required Untreated urethral infection can lead to epididymitis, urethral stric- for biosynthesis of the lipid A moiety of Gram-negative bacteria ture and reduced fertility in men. Gonorrhoea is asymptomatic in have been well characterized in Escherichia coli and are thought to more than half of women and when present, symptoms can seem present targets for the development of new antibiotics. The first Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy 2018. This work is written by US Government employees and is in the public domain in the US. 2064 Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 Treatment of N. gonorrhoeae with LpxC inhibitors JAC 35,36 antimicrobials. The 35/02 and 59/03 strains are isolates from the USA step of lipid A biosynthesis is thermodynamically unfavourable with reduced susceptibility to ceftriaxone and cefixime. and catalysed by the product of the lpxA gene that transfers an R-3-hydroxymyristoyl moiety to the third position of the glucosa- mine ring of UDP-3-O-N-acetylglucosamine. UDP-3-O-(R-3- Antimicrobial compounds hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a The LpxC inhibitors, PF-5081090 and PF-04753299, were obtained from zinc metalloamidase that catalyses the second and more thermo- Sigma–Aldrich (St Louis, MO, USA) through its partnership with Pfizer Global dynamically favourable step of lipid A biosynthesis in which an ace- Research and Development (New London, CT, USA). tyl moiety is cleaved from the 2-N of the lipid A precursor molecule to produce a free amino group that is immediately acylated with Bacterial survival assays 12,13 an additional R-3-hydroxymyristoyl fatty acid by LpxD. Owing Bacteria were grown overnight on Difco GC agar (Becton, Dickinson and Co., to the critical role of the enzyme in lipid A biosynthesis and its lack Sparks, MD, USA) containing 1% BBL Isovitalex Enrichment (Becton, Dickinson of homology with mammalian proteins, inhibitors of LpxC have and Co.). After harvesting, bacteria were diluted in warm PBS and OD at been developed as potential antibiotics for the treatment of Gram- 590 nm was used to dilute serially the stocks to a concentration of 4%10 negative infections. bacteria per mL. Total reaction volumes were 200 lL with 180 lLofRPMI 1640 medium with 10% fetal bovine serum (FBS) (Gibco, ThermoFisher, The first description of small molecule LpxC inhibitors, to the Pittsburgh, PA, USA), inoculated with 4000 bacteria in 10 lL of RPMI 1640 and best of our knowledge, was by scientists at Merck in 1996. Since 15–19 with 10 lL of increasing concentrations of the LpxC inhibitors dissolved in then other groups have described inhibitors, many of which RPMI 1640 or of the vehicle only, which was 1% DMSO in RPMI 1640. After in- contain a hydroxamate or another substituent that can bind the cubation of the tubes at 37 C for 2 and 4 h, aliquots were plated in duplicate catalytic zinc cation of LpxC and a hydrophobic tail that is designed for each concentration tested. The plates were incubated overnight and then to mimic the hydroxymyristate moiety of the substrate. LpxC bacterial colonies were counted. Survival was expressed as the percentage of inhibitors have been shown to be bactericidal for pathogenic bacteria detected as cfu from aliquots of LpxC-treated bacteria compared Gram-negative organisms including E. coli, Klebsiella pneumoniae, with the cfu in aliquots of the vehicle-only treated bacterial suspensions. Pseudomonas aeruginosa and Haemophilus influenzae.Based on the relatively numerous reports of bactericidal activity against Effect of inhibition of LpxC on TNF-a expression induced Gram-negative organisms, we hypothesized that blocking the bio- by gonococcal infection of THP-1 cells synthesis of lipid A with an LpxC inhibitor could be efficacious for The human THP-1 monocytic cell line was cultured in RPMI 1640 with 10% the treatment of gonococcal infections either by being bactericidal FBS (growth medium) at 37 Cin a 5% CO atmosphere. THP-1 cells were for the bacteria or by modulation of the host response to infection. activated in growth medium containing 10 ng/mL phorbol myristate acet- Inhibition of purified recombinant LpxC from Neisseria meningi- ate (Sigma–Aldrich) for 18 h. After removing the medium and washing with tidis has been described. However, there have been no reports to PBS, the cells were trypsinized and plated in 96-well plates at 1%10 cells date on the effect of LpxC inhibitors on survival or inflammatory per well in growth medium. Aliquots of medium containing MS11mkA, MS11mkC, FA1090 A23a or FA1090 1-81-S2 gonococci were added to wells potential of either N. meningitidis or N. gonorrhoeae. In this study, to achieve an moi of 1. Serial dilutions were used to prepare solutions con- we evaluated the effect of LpxC inhibition on well-characterized taining the LpxC inhibitors in RPMI 1640 medium from stock solutions isolates of N. gonorrhoeae that have been used in human chal- (5 mg/mL in DMSO) and then 5 lL aliquots were added to the wells to lenge studies and on five strains that exhibit MDR. achieve final concentrations of 0.04, 1.0 or 25 mg/L. Cells in control wells were treated with vehicle only containing 1% DMSO. After incubating the plates at 37 Cin 5% CO for 18 h, cell culture supernatants were transferred Materials and methods 2 to another 96-well plate and frozen at #80 C. For the detection of TNF-a in Bacterial strains aliquots of the supernatants, an ELISA (Human TNF-a Ready-Set-Go; eBioscience, San Diego, CA, USA) was performed per the instructions of the The MS11 and FA1090 strains of N. gonorrhoeae have been used in most ex- vendor using a Thermomax (Molecular Devices, Sunnyvale, CA, USA) plate perimental human studies of gonococcal infections over the last 25 years. reader with detection at 450 nm. MS11, a porin serotype PIB-9 strain, was isolated from a patient with anter- 22,23 ior urethritis in 1970. The MS11mk variant A strain descended from MS11 and was used in human challenge studies from which the variant C Cytotoxicity of LpxC inhibitors for THP-1 cells strain was isolated. The a-chain of the oligosaccharide portion of the lip- Release of lactate dehydrogenase (LDH) into cell culture supernatants was ooligosaccharide (LOS) of MS11mkA is a lactose moiety whereas the analysed to determine if the LpxC inhibitors were cytotoxic to the THP-1 MS11mkC expresses a lacto-N-neotetraose a-chain moiety that can be sub- 38–40 cells. Cytotoxicity of polymyxin B (Sigma–Aldrich) for the human THP-1 25,26 stituted with one or more N-acetyllactosamine groups. FA1090 A23a, a cells was assayed as a positive control. After activation by treatment with porin serotype PIB-3 strain, was isolated in the 1970s from the endocervix phorbol myristate acetate (10 ng/mL) for 18 h, the THP-1 cells were plated of a patient who probably had a disseminated infection. FA1090 has been (1%10 per well) in 96-well plates in Opti-MEM medium with 1% FBS (Gibco). 28–30 widely used in human challenge studies and in one of these studies Once cells adhered to the wells, aliquots of the LpxC inhibitors in cell culture the 1-81-S2 strain was isolated. medium were added to create final concentrations of 0.008, 0.04, 0.2, 1.0, The FA6140 gonococcal strain is a clinical isolate that is resistant to 5.0 or 25.0 mg/L. Aliquots of polymyxin B in RPMI 1640 were added to the 32 8 penicillin. F89, classified as WHO Y, was isolated in France from a patient wells to achieve final concentrations of 5.0, 25 and 125 mg/L. Control cells with a urethral infection in 2010 and was found to have resistance to cef- were treated with vehicle only. The plates were incubated for 37 Cin5% triaxone and most other antibiotics tested. H041, classified as WHO X, is a CO for 18 h. The relative levels of LDH released from dying cells into cell cul- highly ceftriaxone-resistant isolate from the throat of a patient in Japan in ture supernatants were determined using a kit (Roche, Mannheim, 2009 that is considered an XDR strain owing to its extensive resistance to Germany) with detection using a Thermomax plate reader at 490 nm. 2065 Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 John et al. CH CH Effect of treatment of human challenge strains with HO HO LpxC inhibitors on inflammatory signalling of human HN HN THP-1 monocytes OO S OO S N LOS is a major component of the outer membrane of N. gonorrhoeae CH CH and is highly inflammatory. Previous work has shown that reducing phosphoethanolaminylation of the lipid A of gonococcal LOS dimin- ishes both the inflammatory signalling and the fitness of the bac- 28,41,42 teria in vivo. We theorized that the LpxC inhibitors could modulate inflammatory signalling induced in human THP-1 mono- cytic cells by killing the bacteria but also by reducing the amount of LOS expressed on the bacterial outer membrane or that was released in membrane blebs from dead or living bacteria. To deter- mine the effect of LpxC inhibition on inflammatory signalling, we OCH incubated THP-1 cells with bacteria at an moi of 1 in the presence of Figure 1. Structures of the two LpxC inhibitors PF-5081090 (left) and the LpxC inhibitors at 0.04, 1 and 25 mg/L. As shown in Figure 3, PF-04753299 (right). treatment with either inhibitor at all concentrations tested resulted in a statistically significant decrease in TNF-a expression (P, 0.01 for all comparisons). Effect of LpxC inhibition on MICs for MDR strains To determine the effect of co-treatment of the five MDR strains, absorbance LpxC inhibitors are not cytotoxic for human THP-1 cells at 590 nm based on comparison with a 0.5 McFarland turbidity standard To determine whether the LpxC inhibitors were cytotoxic at the (bioMe ´ rieux, Durham, NC, USA) was used to suspend the appropriate num- concentrations that were bactericidal and reduced inflammatory ber of bacteria in 396 lL of RPMI 1640 medium with 10% FBS in microcentri- signalling in THP-1 cells, we analysed the relative amount of LDH fuge tubes. To achieve a final concentration of PF-04753299 of 0.2 mg/L, released from the cells following 18 h of treatment with the inhibi- 4 lL aliquots of solutions of PF-04753299 in RPMI 1640 (20 mg/L) or vehicle tors. Results showed that cell death was not significantly increased only were added and the tubes were incubated at 37 Cfor 2 h. The MICs of by treatment with either inhibitor at the concentration range of ceftriaxone and azithromycin for the MDR strains were determined using 0.008–25 mg/L tested (Figure 4; P. 0.05 for all comparisons). By the Etest method (bioMe ´ rieux) according to the instructions of the vendor. comparison, use of polymyxin B as a positive control resulted in a significant increase in LDH (P, 0.001) following treatment of THP- 1 cells with 125 mg/L polymyxin B, which is a concentration similar Statistical analyses to the MICs of polymyxin B for strains MS11 and FA1090 that have SigmaPlot version 12.5 (Systat Software, San Jose, CA, USA) was used to 42,44 been previously reported to be 50 and 100 mg/L, respectively. perform statistical analyses. Multigroup comparisons were performed using Thus, the effective concentration range of the LpxC inhibitors for one-way analysis of variance with Bonferroni’s post hoc tests. Significance gonococci does not induce cytotoxicity for THP-1 cells, whereas was defined as P, 0.05 for all comparisons. that for polymyxin B approaches cytotoxic concentrations. Results Activity of LpxC inhibitors against MDR strains Activity of LpxC inhibitors against human challenge The five MDR strains were tested with PF-04753299, which was the strains more efficacious of the two inhibitors. All of the MDR strains were completely killed by incubation with PF-04753299 at 5mg/L and The structures of the two LpxC inhibitors that were tested are bacterial survival was ,50% after incubation at 1 mg/L for 2 h shown in Figure 1. Complete killing of all four strains was observed (Figure 5). The susceptibility of the MDR strains was similar be- after 4 h of treatment with PF-5081090 at a concentration of tween strains at the concentrations tested in general except at 25 mg/L and after 2 or 4 h of treatment with PF-04753299 at 1.0 mg/L where, e.g..30% of the FA6140 bacteria and,5% of the 5mg/L (Figure 2). Only the MS11mkC isolate survived treatment H041 bacteria survived. The MDR strains had comparable suscepti- with 25 mg/L PF-5081090 for the shorter 2 h period at a minimum bility with the human challenge strains to 2 h of treatment with PF- level of 0.4% survival. The survival of the two FA1090 strains was 04753299 (Figure 2b). significantly reduced by 4 h of treatment with PF-04753299 at all five concentrations tested (P, 0.001), whereas the MS11 strains Effect of LpxC inhibition on MICs for MDR strains were resistant to the lowest concentration of inhibitors tested (0.008 mg/L). The two FA1090 strains tended to be more suscep- Co-treatment of the MDR strains with a sublethal concentration tible to either inhibitor at virtually all concentrations of 0.2 mg/L (0.2 mg/L) of PF-04753299 and ceftriaxone and azithromycin was compared with the MS11 strains. Increasing the length of treat- explored using Etest strips. As shown in Table 1,inthe absence of ment from 2 to 4 h reduced the survival of the FA1090 bacteria to the inhibitor the MICs of the antibiotics for the MDR strains were 32,33,35,37 a greater degree compared with the MS11 strains. Overall, PF- similar to values previously reported for these strains. 04753299 tended to be a more efficacious bactericidal compound In the presence of the inhibitor, no changes were observed in the than PF-5081090. susceptibility of the strains to ceftriaxone, which is a bactericidal 2066 Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 Treatment of N. gonorrhoeae with LpxC inhibitors JAC (a) (b) 160 160 MS11mkA 140 140 MS11mkC FA1090 A23a 120 120 FA1090 1-81-S2 100 100 80 80 60 60 40 40 20 20 0 0 0.008 0.04 0.2 1 5 25 0.008 0.04 0.2 1 5 25 PF-5081090 (mg/L) PF-04753299 (mg/L) (c) (d) 160 160 140 140 120 120 100 100 80 80 60 60 40 40 0.008 0.04 0.2 1 5 25 0.008 0.04 0.2 1 5 25 PF-5081090 (mg/L) PF-04753299 (mg/L) Figure 2. Bactericidal activity of the two LpxC inhibitors against the four human challenge isolates of N. gonorrhoeae. Increasing concentrations (0.0, 0.008, 0.04, 1.0, 5.0 or 25 mg/L) of the inhibitors were incubated with the gonococci and survival was determined after 2 and 4 h of treatment. Bars represent the mean+ SEM of triplicate experiments that were each performed in duplicate. MS11mkA MS11mkC FA1090 A23a FA1090 1-81-S2 400 400 200 200 0 0 0 0.04 125 0 0.04 125 PF-5081090 (mg/L) PF-04753299 (mg/L) Figure 3. Levels of TNF-a in supernatants of cultures of activated human THP-1 cells infected with human gonococcal isolates at an moi of 1 in the presence of increasing concentrations (0.0, 0.04, 1.0 or 25 mg/L) of LpxC inhibitors after 18 h. Bars represent the mean+ SEM of quadruplicate experiments. TNF-α (pg/mL) Percentage survival after 4 h Percentage survival after 2 h TNF-α (pg/mL) Percentage survival after 4 h Percentage survival after 2 h Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 John et al. 160 160 *** 140 140 120 120 100 100 100 80 80 60 60 60 40 40 40 20 20 20 0 0 0 0.008 0.04 0.2 1 5 25 0.008 0.04 0.2 1 5 25 5 25 125 PF-5081090 (mg/L) PF-04753299 (mg/L) Polymyxin B (mg/L) Figure 4. Relative levels of LDH in culture supernatants of activated human THP-1 cells in the presence of increasing concentrations of LpxC inhibitors (0.008–25 mg/L) or polymyxin B (5–125 mg/L) after 18 h. Bars represent the mean+ SEM of quadruplicate experiments. ***P, 0.001 compared with control. Table 1. MICs for MDR strains with and without treatment with an LpxC inhibitor FA6140 F89 MIC (mg/L) HO41 35/02 PF-04753299! PF-04753299! 59/03 Strain ceftriaxone ceftriaxone azithromycin azithromycin FA6140 0.032 0.032 0.19 0.125 F89 1.0 1.0 0.25 0.25 H041 1.5 1.5 0.25 0.25 35/02 0.064 0.064 0.25 0.19 59/03 0.064 0.064 0.38 0.38 MICs were determined using the Etest method, determined after treat- ment with PF-04753299 (0.2 mg/L) or vehicle for 2 h. 0.008 0.04 0.2 1 5 25 PF-04753299 (mg/L) Figure 5. Bactericidal activity of PF-04753299 against five MDR strains of N. gonorrhoeae. Increasing concentrations of PF-04753299 (0.0, 0.008, previous comparison of treatment of a panel of 106 recent clinical 0.04, 1.0, 5.0 or 25 mg/L) were incubated with the gonococci and survival isolates of Gram-negative bacteria species including P. aeruginosa was determined after 2 h. Bars represent the mean+ SEM of triplicate and K. pneumoniae with PF-5081090 and PF-04753299 found that experiments that were each performed in duplicate. the former was more efficacious. The structures of LpxC orthologues vary significantly between agent. However, a modest decrease in the MIC of azithromycin, bacterial species. Differences in the C-termini have been shown to which is a bacteriostatic drug, was found with co-treatment of the alter the susceptibility to proteolytic cleavage and enzymatic FA6140 and 35/02 strains. Furthermore, no antagonism from co- degradation, which could affect the rate of LPS or LOS biosyn- treatment causing a decrease in susceptibility to either drug was thesis and, thus, sensitivity to LpxC inhibitors. There is 57% iden- observed. tity and 79% similarity of the primary sequence of the LpxC enzymes from P. aeruginosa and E. coli, but only 32% identity and 51% similarity of the Aquifex aeolicus LpxC enzyme to that of Discussion E. coli. The structure of N. gonorrhoeae LpxC has 49% identity Here we show that biphenyl alkyl sulphonamide hydroxamic de- and 71% similarity to that of E. coli. Potent LpxC inhibitors have rivative LpxC inhibitors are bactericidal for human challenge and been described, but the MICs for Gram-negative species can vary 15,47,50,51 MDR strains of N. gonorrhoeae. The concentrations at which PF- more than an order of magnitude. Furthermore, our 04753299 was bactericidal for the gonococcal isolates were com- results show that unlike a number of other Gram-negative species parable with the concentrations that were bactericidal for E. coli, that were tested with both inhibitors, N. gonorrhoeae is more P. aeruginosa and K. pneumoniae strains that had MIC values of 90 susceptible to PF-04753299 than PF-5081090. The differences in 2, 4 and 16 mg/L, respectively, following treatment periods of susceptibility could be owing to differences between bacterial spe- 15,17 16–20 h. However, compared with these species the gono- cies in the structures of the LpxC enzyme and highlight the poten- cocci were somewhat more susceptible to PF-04753299 as tial importance of testing in the species of interest. treatment with it at a concentration of 5 mg/L was completely We found that treatment with LpxC inhibitors at concentrations bactericidal to all strains after treatment for only 2 or 4 h. of 0.04 mg/L or greater significantly reduced expression levels of We found that overall the gonococci were less susceptible to TNF-a in an in vitro model of infection using a human THP-1 mono- PF-5081090 compared with PF-04753299. Interestingly, a cytic cell line. Induction of inflammatory signalling would be Percentage survival after 2 h LDH OD (percentage of control) LDH OD (percentage of control) LDH OD (percentage of control) 490 Downloaded from https://academic.oup.com/jac/article/73/8/2064/4991921 by DeepDyve user on 16 July 2022 Treatment of N. gonorrhoeae with LpxC inhibitors JAC largely due to LOS molecules in the outer membrane or in outer of LOS on N. gonorrhoeae that survived LpxC inhibitor treatment membrane blebs released from bacteria. It is likely that the should reduce bacterial fitness and virulence. Reducing TNF-a ex- reductions in cytokine levels observed were due to reduced num- pression in gonococcal infections in women potentially could pre- bers of bacteria as well as reduced levels of LOS. Our results indi- vent or limit damage to the fallopian tube and the sloughing of the 65–67 ciliated cells that leads to infertility. The lower inflammatory cate that LpxC is a promising target for the development of new potential is a hallmark of commensal Neisseria species relative to antibiotics for treating gonorrhoea as LpxC inhibitors exhibit both the pathogenic species, and surviving N. gonorrhoeae treated bactericidal and anti-inflammatory activities for N. gonorrhoeae. with an LpxC inhibitor could have a commensal-like, non-patho- Differences between the FA1090 and MS11 isolates have been genic relationship with its human host. reported including the serum sensitivity of and presence of a gen- There is synergism in the clinical and epidemiological occur- etic island in MS11 and serum resistance of and absence of a gen- 52,53 rence of HIV-1 and N. gonorrhoeae, which facilitates HIV-1 etic island in FA1090. Furthermore, whereas MS11 does expression. The heptose monosaccharides that are absent in express lactoferrin-binding proteins A and B and utilizes lactoferrin, 52,54 mammalian glycomes but present in the gonococcal LOS were FA1090 strains do not. A review of published studies revealed found to drive HIV-1 expression and innate immune responses. that MS11mkC has an ID for urethral infection that is nearly 2 Furthermore, recent data show that vaginal inflammation facili- log less than FA1090 and similar differences in infectivity have tates the transmission of HIV. Thus, treatment of gonorrhoea been observed in the mouse model of female genital infection, with an LpxC inhibitor that not only kills the bacteria but also although the reason for the differences is unclear. However, the reduces its expression of LOS and capacity for inflammatory sig- expression level of the multiple transferable resistance (Mtr) efflux nalling potentially could limit the transmission of HIV by coinfected pump was found to be higher in MS11 compared with FA1090. individuals. MS11 has a naturally occurring mutation in the mtr locus that Ideally, new antimicrobial agents for gonorrhoea would have leads to the expression of a consensus element that increases the activity against other coinfecting sexually transmitted pathogens promotion of mtrCDE transcription and, thus, resistance to some such as Chlamydia trachomatis. Small molecule inhibitors of antimicrobial compounds and possibly host cationic antimicro- LpxC were not bactericidal for C. trachomatis, but did block LPS syn- bial peptides such as LL-37. Increased expression of the Mtr ef- thesis and the transition of reticulate bodies to elementary bodies, flux pump probably explains the reduced susceptibility of MS11 a form that is required for the invasion of mammalian cells. compared with FA1090. Overall, our results showing the bactericidal effects on human Our results showing that the susceptibility of the MDR gonococ- challenge isolates and MDR strains of N. gonorrhoeae indicate that cal strains to PF-04753299 was very similar to that of the human further studies of LpxC inhibitors as treatments for gonococcal challenge strains are encouraging for potential clinical applica- infections are warranted, particularly as there is an urgent need for tions. The data imply that mechanisms conferring resistance to new antibiotics for gonorrhoea because of the rise in MDR strains. LpxC inhibition and to antibiotics such as cephalosporins, fluoroqui- Furthermore, owing to the likely ability of LpxC inhibitors to reduce nolones and macrolides differ and, furthermore, indicate that MDR the expression of LOS on viable gonococci and, thus, impair bacter- gonorrhoea may be treatable with an LpxC inhibitor. ial fitness, testing in an in vivo model of gonococcal infection po- We tested combination treatment with ceftriaxone and azith- tentially could reveal efficacy that would be even greater than that romycin as these two agents are recommended as dual therapy 58 observed in in vitro experimentation. for gonorrhoea in current treatment guidelines. More combina- tions of antimicrobials with LpxC inhibitors for N. gonorrhoeae should be tested as co-treatment with PF-04753299 showed that there was a modest decrease in the MICs of azithromycin for two Acknowledgements of the MDR strains. This is paper number 118 from the Center for Immunochemistry. The lipid A component of LOS is highly inflammatory and the We are grateful to Alison K. Criss (Department of Microbiology, anti-inflammatory effects of LpxC inhibitors could be beneficial in Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, treating gonorrhoea. This postulate is supported by the finding USA) and Robert A. Nicholas (Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA) for providing the MDR strains. that an LpxC inhibitor that was not bactericidal for Acinetobacter baumannii, which can survive in the absence of LOS expression, nonetheless, protected mice from infection by reducing inflamma- tion and facilitating phagocytosis. Furthermore, treatment with Funding the PF-5081090 LpxC inhibitor increased the susceptibility of A. This work was supported by Merit Review Award BX000727 from the baumannii to other antibiotics by increasing the permeability of Research Service of the United States Department of Veterans Affairs (G. the bacteria. N. meningitidis and Moraxella catarrhalis can survive A. J.). G. A. J. is the recipient of a Senior Research Career Scientist Award in the absence of lipid A although this was produced by directed from the Research Service of the United States Department of Veterans 61,62 in vitro mutation, whereas A. baumannii LOS-deficient organ- Affairs. isms can arise after polymyxin B treatment and have been iso- 63,64 lated, albeit rarely, in the clinic. Human and animal studies show that LptA-catalysed phos- phoethanolaminylation of the lipid A confers a significant survival Transparency declarations advantage to N. gonorrhoeae. 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Journal

Journal of Antimicrobial ChemotherapyOxford University Press

Published: Aug 1, 2018

Keywords: antibiotics; azithromycin; tumor necrosis factors; lipid a; monocytes; bacteria; catalysis; acetylglucosamine; lipid-linked oligosaccharides

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