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Evolution, persistence, and host adaption of a gonococcal AMR plasmid that emerged in the pre-antibiotic era [1]

['Wearn-Xin Yee', 'Sir William Dunn School Of Pathology', 'University Of Oxford', 'Oxford', 'United Kingdom', 'Muhammad Yasir', 'Quadram Institute', 'Norwich', 'A. Keith Turner', 'David J. Baker']

Date: 2023-12

Plasmids are diverse extrachromosomal elements significantly that contribute to interspecies dissemination of antimicrobial resistance (AMR) genes. However, within clinically important bacteria, plasmids can exhibit unexpected narrow host ranges, a phenomenon that has scarcely been examined. Here we show that pConj is largely restricted to the human-specific pathogen, Neisseria gonorrhoeae. pConj can confer tetracycline resistance and is central to the dissemination of other AMR plasmids. We tracked pConj evolution from the pre-antibiotic era 80 years ago to the modern day and demonstrate that, aside from limited gene acquisition and loss events, pConj is remarkably conserved. Notably, pConj has remained prevalent in gonococcal populations despite cessation of tetracycline use, thereby demonstrating pConj adaptation to its host. Equally, pConj imposes no measurable fitness costs and is stably inherited by the gonococcus. Its maintenance depends on the co-operative activity of plasmid-encoded Toxin:Antitoxin (TA) and partitioning systems rather than host factors. An orphan VapD toxin encoded on pConj forms a split TA with antitoxins expressed from an ancestral co-resident plasmid or a horizontally-acquired chromosomal island, potentially explaining pConj’s limited distribution. Finally, ciprofloxacin can induce loss of this highly stable plasmid, reflecting epidemiological evidence of transient reduction in pConj prevalence when fluoroquinolones were introduced to treat gonorrhoea.

Plasmids are extrachromosomal elements that disseminate antimicrobial resistance (AMR) among bacteria. Neisseria gonorrhoeae is a leading cause of sexually transmitted disease and a concern due to increasing AMR. It contains a restricted repertoire of plasmids, including a conjugative plasmid, pConj, which disseminates plasmid-mediated AMR. We show that, in contrast to broad host range plasmids, pConj is largely restricted to and adapted to N. gonorrhoeae, and has been remarkably conserved since it first emerged over 80 years ago. pConj is highly persistent, with no plasmid loss detected after 160 generations under standard laboratory conditions. We were unable to identify any chromosomal gene to account for the success of pConj. Instead, the lack of fitness costs and co-operative effects of maintenance systems result in its stable inheritance. Of note, pConj harbours an orphan VapD toxin that can be neutralised by VapX antitoxins expressed by a co-resident plasmid; this potential ‘split’ toxin:antitoxin system allows exquisite association of pConj with the gonococcus. Finally, we show that ciprofloxacin can induce pConj loss, mirroring the reduction in pConj carriage in the gonococcal population following introduction of this antibiotic for gonorrhoea, and paving the way for approaches to eliminate plasmid-mediated AMR in this important human pathogen.

Funding: The work was supported by an A*STAR NSS PhD scholarship awarded to WXY and the Wellcome Trust (award number 214374/Z/18/Z to CMT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Yee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We show that pConj is largely restricted to the gonococcus and track its evolution from when it first appeared over 80 years ago to now. Even though N. gonorrhoeae is a highly variable pathogen [ 24 ] with pConj present in multiple lineages, pConj has been remarkably conserved over this time except for acquisition of tetM and TA systems. The plasmid does not impose obvious fitness costs and is stably inherited, explaining the persistence of pConj after tetracycline was discontinued for treating gonococcal infection. Transposon insertion sequencing (TIS) indicates that pConj does not rely on any non-essential chromosomal gene for its maintenance. Instead, plasmid-encoded TA and partitioning systems act in concert to ensure pConj maintenance. Of note, we found that the orphan VapD encoded by pConj is part of a split TA system with VapX antitoxins encoded by other mobile genetic elements, pCryp or a horizontally-acquired Type IV secretion system (T4SS) genomic island, potentially explaining the restriction of pConj to Neisseria spp.. As the ParAB partitioning system is important for the vertical transmission of pConj, we tested whether ciprofloxacin, which can impair plasmid segregation through its effect on DNA gyrase [ 25 ], can be used to cure pConj. Importantly, exposure to ciprofloxacin enhances pConj loss from the gonococcus. This is consistent with the decrease in pConj prevalence that occurred when this antibiotic was introduced for treating gonococcal disease [ 26 , 27 ], and provides proof-in-principle that this highly stable and adapted plasmid can be eliminated from N. gonorrhoeae.

( A ) Map of pConj showing four regions involved in replication/partitioning (including parAB), mating bridge formation (trb), conjugation (tra) and the genetic load region which includes three toxin-antitoxin (TA) related loci. ( B ) Appearance of plasmids in N. gonorrhoeae based on WGS at PubMLST and previous reports; pConj first occurred in strains with pCryp. Years during which penicillin and tetracycline monotherapy were recommended in the USA are as shown. ( C ) pConj is present in four species of Neisseria; only species with ≥40 WGS are included. pConj was not detected in N. bergeri, N. cinerea and N. subflava. Only N. gonorrhoeae carried tetM + pConj. ( D ) There are two phylogenetic groups of pConj, depending on the presence of ε:ζ2 or ε:ζ3 TA systems. Sequences were aligned independently of tetM and the surrounding transposon. Each dot represents an isolate, colour-coded according to year of isolation. ( E ) Despite pConj’s conservation, the plasmid is found in multiple lineages across the gonococcal population. Each dot represents an isolate, colour-coded according to pConj carriage.

pConj encodes a predicted partitioning system, consisting of a ParA ATPase and ParB DNA binding protein, while the genetic load (GL) region of the plasmid encodes two putative epsilon-zeta (ε:ζ) type II TA systems ( Fig 1A ) [ 20 ]. ε:ζ2 shares ~40% amino acid sequence identity with a characterised streptococcal ε:ζ system [ 21 ], while some gonococci carry ε:ζ3 instead of ε:ζ2 [ 9 ]. The GL region also encodes an uncharacterised orphan VapD toxin which lacks a cognate VapX antitoxin found in vapXD TA systems [ 22 , 23 ]. None of these systems have been characterised in the gonococcus, although ε:ζ1 is a functional TA system in Escherichia coli [ 21 ].

Plasmids deploy strategies to ensure their stable inheritance in bacteria. Partitioning systems segregate plasmids to the poles of dividing bacteria so that each daughter cell contains a plasmid following division [ 18 ]. In addition, Toxin:Antitoxin (TA) systems encode a toxin and a cognate antitoxin, and promote plasmid maintenance through post-segregational killing (PSK). In Type II TA systems, daughter cells which fail to inherit a plasmid are killed through the unopposed activity of the toxin once the protein antitoxin has been degraded [ 19 ].

Here we describe the evolution and maintenance of a narrow host plasmid in Neisseria gonorrhoeae. N. gonorrhoeae (the gonococcus) causes gonorrhoea, a serious threat to sexual and maternal health, and a co-factor for HIV infection [ 6 ]. The bacterium has evolved resistance against most available antibiotics so has been classified as a priority pathogen by the World Health Organisation and Centers for Disease Control and Prevention [ 7 , 8 ]. pCryp is an almost ubiquitous 4.2 kb plasmid of unknown function [ 9 ], while pbla (3.2–9.3 kb) and pConj (39–42 kb, markerless or carrying tetM from Tn916) led to the discontinuation of penicillin and tetracycline for treating gonococcal disease, respectively [ 8 , 10 ]. pConj is conjugative and disseminates itself and pbla [ 11 – 13 ] so is central to the spread of AMR. pConj could acquire further elements conferring resistance against other antibiotics including macrolides [ 14 ], while only one or two amino acid changes are needed for pbla to encode an extended spectrum beta-lactamase (ESBL) [ 15 ]. These changes would undermine currently recommended therapies against the gonococcus [ 16 , 17 ]. Therefore, it is crucial to understand how pConj is maintained in gonococcal populations, as this could inform approaches to combat AMR in this important human pathogen.

Plasmids are extrachromosomal elements which confer beneficial traits on bacteria including antimicrobial resistance (AMR) [ 1 ]. Plasmids can be transferred and maintained in different, unrelated species [ 2 ], and such broad host range plasmids have been extensively studied [ 2 ]. Narrow host range AMR plasmids are found in pathogenic bacteria including Klebsiella spp. and Acinetobacter spp. [ 3 – 5 ]; despite their clinical relevance, little is known about the mechanisms of their host restriction and maintenance.

Results

TIS does not reveal host genes for pConj maintenance The adaptation and restriction of pConj to the gonococcus led us to hypothesise that the plasmid relies on host-specific genes for its maintenance. To identify chromosomal genes involved in pConj maintenance, we constructed a library of FA1090 mutants by in vitro transposon mutagenesis of FA1090 chromosomal DNA followed by uptake of mutagenised DNA via transformation. pConj was then introduced into the library. The resulting library containing >100,000 unique insertion sites (UIS) was grown with or without tetracycline in duplicate cultures for 56 generations; transposon insertion sites were determined using the Tradis-Xpress nucleotide sequencing method [33] after the 8th, 16th and 56th generation. The number of UIS diminished over time, with 183,919 UIS in the initial library, 81,655–89,336 UIS by the 8th generation and 54,708–70,973 UIS by the 16th generation. By the 56th generation, there were only between 5,815 and 6,442 UIS in the cultures, too few to identify genes involved in plasmid maintenance (S1 Dataset). The profile of insertions was highly consistent between cultures (S4 Fig). No chromosomal genes were found to contribute to plasmid maintenance after eight generations (S4 Table). Significant hits (i.e. enriched in cultures lacking tetracycline, log fold change < 0, q < 0.01) were only identified after 16 generations (Fig 3 and S4 Table). PPT PowerPoint slide

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TIFF original image Download: Fig 3. TIS does not identify chromosomal genes involved in pConj maintenance. Transposon insertion sites of significant hits at the 16th generation (q < 0.01, Bio-Tradis; log fold change < 0) visualised in Artemis; gene orientation is shown. Graphs show the distribution, numbers and orientation of transposon insertion mutant library obtained under both control and tetracycline conditions (two independent biological repeats). Each vertical line indicates a UIS, with the height reflecting the number of mutants at each site. Red and blue lines indicate transposon insertions in the forward and reverse direction, respectively. Blue lines are plotted in front of red, masking some red insertions. The maximum number of insertions displayed in each panel is marked individually. https://doi.org/10.1371/journal.pgen.1010743.g003 However, all hits were due to differences in one or two insertions per gene (Fig 3), which is characteristic of false positive hits [34]. To determine whether they were false positives, mutants Δneis1066, Δneis1845, Δneis2592, which gave the most significant q-values or highest counts per million (logCPM) identified by Tn-Seq were constructed in pC1GFP. Mutants were passaged for 16 generations, and plasmid loss was determined by detecting the absence of GFP. No plasmid loss was observed (LOD = 0.3%), confirming that TIS yielded only false positive hits. Therefore, after 16 generations we also inspected the distribution of transposon insertions in/around 16 genes encoding proteases and DNA replication machinery, which influence plasmid maintenance in other bacteria [35–38]. No differences were detected in the distribution or frequency of insertions around these genes (S4 Fig). Overall, these results suggest that pConj does not rely on a single non-essential chromosomal gene for its stable inheritance.

TA systems co-operate to maintain pConj As we did not identify any host genes contributing to pConj stability, we next examined plasmid genes. Initially, we determined whether the TA systems encoded by the GL region contribute to pConj stability. We replaced the entire GL region in pConj (neis2203 to vapD inclusive [20], Fig 4A) with gfp:kan, generating pCΔGL, and assessed plasmid loss after growing bacteria for 30–40 generations under non-selective conditions. Under these conditions, loss of pC1GFP (pConj with the GL region) was below the limit of detection while deletion of the GL region reduced pConj stability (loss of pCΔGL, 8.5% ±3.1, Fig 4B). PPT PowerPoint slide

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TIFF original image Download: Fig 4. TA systems cooperate to maintain pConj. (A) Schematic of the genetic load region. gfp:kan was used to monitor the presence of pConj. Genes and related functions are annotated based on PubMLST. (B) Replacement of the genetic load region with gfp:kan (pCΔGL) resulted in detectable plasmid loss. (C) pConj lacking individual TA systems (pCtΔvapD, pCt:ζ1K115A, pCtΔζ2) were stably maintained, whereas loss of combinations of TA systems (pCtΔvapD:ζ1 and pCtΔTA) resulted in plasmid loss within 40 generations (LOD = 0.3%). All assays consist of three independent repeats and results were analysed with one-way ANOVA with Sidak’s multiple comparisons, shown as mean ± SD; * p ≤ 0.05). https://doi.org/10.1371/journal.pgen.1010743.g004 To define which TA systems encoded in the GL region contribute to plasmid stability, we next deleted or inactivated the toxins individually (pCtΔvapD, pCt:ζ1K115A, pCtΔζ2) or in combination (pCtΔvapD:ζ1 and pCtΔTA, Fig 4C). trbC, which encodes the conjugative pilus, was removed from all plasmids to prevent pConj re-acquisition by conjugation [39]. No pConj loss was detected after growth for 30–40 generations following deletion of vapD or ζ2. However, inactivation of ζ1 led to low level plasmid loss (0.33% ±0.3) demonstrating that this TA system contributes to pConj maintenance (Fig 4C). Subsequent removal of vapD then ζ2 from pCtΔζ1 mutant led to stepwise increases in pConj loss (loss of pCtΔvapD:ζ1 and pCtΔTA, 3.2% ±0.4 and 5.6% ±1.6, respectively, p = 0.041 and p = 0.019, one-way ANOVA with Sidak’s multiple comparisons test, Fig 4C) indicating that vapD and ε:ζ2 also support the maintenance of pConj in the absence of other TA systems. Overall, our results demonstrate that pConj TA systems act redundantly to maintain the plasmid.

Interactions between mobile genetic elements involved in pConj maintenance To understand the mechanism by which VapD expressed by pConj (VapDpConj) contributes to plasmid maintenance, we examined whether VapDpConj is toxic. VapDpConj shares homology with Helicobacter pylori [40] and Haemophilus influenzae [22] VapD (34.8% and 34.4% amino acid similarity respectively), and is also predicted to be a dimer by AlphaFold (Fig 5A). VapDpConj expression in E. coli under an arabinose-inducible promoter [41] led to a marked reduction in bacterial survival (p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test, Fig 5B), demonstrating that VapDpConj is toxic. We next investigated if VapDpConj interacts with VapX homologues encoded elsewhere in the gonococcal genome. Of note, pCryp, which was in the first gonococcal isolate found with pConj, encodes a vapXD TA system (Fig 1B). pCryp, similar to pConj, is restricted to Neisseria spp., and is most common in N. gonorrhoeae (96.8% of isolates, 9,313/9,618, S1 Table). VapXD is also associated with a horizontally acquired chromosomal island containing a Type IV secretion system (T4SS) in some gonococci [9]. Due to the high prevalence of pCryp [9,42], we tested whether expression of vapXpCryp can prevent VapDpConj toxicity. Results demonstrate that VapXpCryp abrogates VapDpConj toxicity (p = 0.0069, Fig 5B), consistent with VapDpConj forming a split TA system with VapXpCryp. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Interaction between mobile genetic elements is associated with pConj carriage. (A) VapDpConj is homologous to other VapDs. Shading intensity is proportionate to percentage conservation. AlphaFold predicted structure of VapDpConj (red) has similar architecture to VapD from H. pylori (PDB 3UI3, dark blue) and H. influenzae (PDB 6ZN8, light blue). (B) VapDpConj is toxic in E. coli, and is neutralised by VapXpCryp. Viability of E. coli (CFU/ml) after induction with 0.2% of L-arabinose is shown. (C) pConj carriage in N. gonorrhoeae with vapX (n = 9,316) compared to isolates without vapX (n = 302) (D) VapDpConj toxicity in E. coli is neutralised by VapXNm. (E) pConj carriage in N. meningitidis isolates with vapX (n = 194) compared to isolates without vapX (n = 16,718). Toxin-antitoxin assays were carried out in three independent repeats, analysed with two-way ANOVA with Tukey’s multiple comparisons and shown as mean ± SD. In B and D the number of CFU at 360 min post-induction are shown. pConj carriage was analysed with Fisher’s exact test. ** p ≤ 0.01, **** p ≤ 0.0001. https://doi.org/10.1371/journal.pgen.1010743.g005 We also examined the association between pConj with VapX and found that pConj carriage is significantly higher in strains harbouring vapX (pConj in 31.2%, 2,914/9,316 of vapX-carrying strains) than in gonococci which do not carry vapX (12.5%, 38/302, p < 0.0001, Fisher’s exact test Fig 5C). Thus, vapX carrying strains are also more likely to carry pConj (odds ratio 3.1, 95% confidence interval 2.2–4.4, Woolf logit). We hypothesised that the association between pConj VapD and pCryp VapX might also be evident in other Neisseria spp. that carry pConj. Analysis of WGS suggested that N. cinerea, N. subflava and N. bergeri, which did not carry pConj (Fig 1C), had very low levels of vapX carriage (0/40 and 0/53 for N. cinerea and N. subflava respectively, and 1/60 for N. bergeri). For pConj-carrying species, although no isolates carrying vapX was identified for N. polysaccharea (0/61), vapX carriage in N. lactamica was 68.4% (377/551) and 1.1% (194/16,912) in N. meningitidis; vapX in both species are not found on pCryp. Specifically, vapX in N. meningitidis is usually found on a horizontally-acquired island encoding a Type IV secretion system [43] (T4SS, in 87.1%, or 169/194 of vapX-carrying strains), and less commonly on pCryp (10.3%, 20/194). The vapX allele on the T4SS was designated vapXNm, and differs from vapXpCryp by an insertion of three amino acid residues near the N-terminus. We confirmed that VapXNm also neutralises VapDpConj following expression in E. coli (p = 0.0013, Fig 5D). Furthermore, similar to N. gonorrhoeae, pConj carriage is significantly more frequent in meningococci harbouring vapX; pConj is present in 4.1% of meningococci with vapX (8/194 isolates) but only in 0.44% of strains lacking vapX (73/6,718 isolates, p < 0.0001, Fig 5E, odds ratio 9.8, 95% confidence interval 4.7–20, Baptista-Pike). Interestingly, vapD has been lost from 13 pConj-carrying meningococcal strains, and replaced by a 433 bp sequence within pConj (isolates 4313, 26256, 49535, 56700, 57863, 57509, 59291, 83101, 86009, 92506, 93290, 94878, and 116658; S1 Table). All pConj lacking vapD are found in vapX-negative strains. In contrast, every other pConj in N. meningitidis and N. gonorrhoeae contains vapD. Overall, these data indicate that VapX, encoded by pCryp or a chromosomal T4SS island, can form a split TA system with the orphan VapD expressed by pConj.

Interplay between TA and partitioning systems stably maintain pConj Apart from TA systems, partitioning systems can promote the maintenance of low copy number plasmids. We therefore examined the effect of the putative ParAB partitioning system on pConj maintenance (Fig 1A). We generated pConj lacking parB (pCtΔparB), and found that after only 6–9 generations approximately 15% of bacteria had lost the plasmid (Fig 6A). We reasoned that the dramatic impact of ParAB might be masked by the activity of TAs, so additionally inactivated the TA systems in this plasmid (generating pCtΔparBΔTA). This led to almost all bacteria losing pConj over only 6–9 generations (98% ±3.6, p = 0.0022 compared with pCtΔparB, Mann-Whitney test, Fig 6A), demonstrating that pConj TA and partitioning systems operate in concert to prevent plasmid loss. PPT PowerPoint slide

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TIFF original image Download: Fig 6. pConj is stably maintained by multiple mechanisms. (A) pConj is rapidly lost from strains lacking TA and/or partitioning systems (i.e. within 6–9 generations). Assays were carried out three times and analysed with Mann-Whitney test, LOD = 0.3%, shown as mean ± SD. ** p ≤ 0.01. Treatment of (B) FA1090 or (C) 60755 with ciprofloxacin promotes pConj loss. Bacteria were grown overnight on GCB before incubation in 0.5 MIC of ciprofloxacin or spectinomycin. Colonies (n ≥ 200; LOD = 0.5%) were tested for pConj carriage. Assays were carried out three times and analysed with student’s unpaired t-test, shown as mean ± SD. * p ≤ 0.05, *** p ≤ 0.001. (D) As pConj enters N. gonorrhoeae, interactions between pCryp and pConj via VapXD contribute to the stabilisation of pConj in the gonococcus as well as its maintenance. pConj encodes TA and partitioning systems–therefore, pConj has developed multiple fail-safe mechanisms to ensure it is maintained in N. gonorrhoeae. While pConj is stably maintained in N. gonorrhoeae, antibiotic use facilitated spread of both itself and pbla, resulting in the spread of plasmid-mediated antimicrobial resistance. Currently, despite the lack of tetracycline use, pConj persists in N. gonorrhoeae; as such, treatment regimens no longer suggest the use of penicillin or tetracycline. https://doi.org/10.1371/journal.pgen.1010743.g006

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