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Deep sequencing analysis of CRISPR-escaping plasmid transconjugants in Enterococcus faecalis

Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged that are replete with mobile genetic elements (MGEs). Some E. faecalis strains possesses CRISPR-Cas systems, which reduce the conjugation frequency of pheromone-responsive plasmids. However, many transconjugants still arise, and we have demonstrated in previous studies that E. faecalis can transiently maintain both a functional CRISPR-Cas system and a CRISPR-Cas target. In this study, we used serial passage and deep sequencing to analyze CRISPR array dynamics over time in transconjugants which possess both a functional CRISPR-Cas system and a CRISPR-Cas target. In the presence of antibiotic selection for the plasmid, we found that plasmids ultimately escape CRISPR defense via the emergence of compromised CRISPR-Cas defense in host populations. As a consequence, these populations have enhanced abilities to acquire a second antibiotic resistance plasmid. In the absence of antibiotic selection, plasmids are lost from wild-type but not Δcas9 host populations over time. We conclude that the adaptive immune system of E. faecalis becomes compromised under antibiotic selection for MGEs, generating populations with enhanced abilities to undergo horizontal gene transfer.


Introduction
Enterococcus faecalis is a Gram-positive bacterium that normally colonizes the gastrointestinal mediated primarily by plasmids and integrative conjugative elements (5-7). One of the most after 1 day of passage in the presence of erythromycin and identified a mutation resulting in an 146 Ala749Thr substitution. Ala749 occurs within the RuvC nuclease domain in T11RF Cas9 and is 147 conserved in the model Streptococcus pyogenes Cas9 (32). Due to the critical catalytic function of the RuvC domain, we hypothesize that the Ala749Thr substitution confers a loss of Cas9 149 function. We tested the ability of the BHI-and erythromycin-passaged WT4 populations to 150 interfere with plasmid transfer using derivatives of the PRP, pCF10 (45), that are targeted by 151 CRISPR3 spacers 1, 6, and 7. These experiments revealed that the Ala749 mutation indeed 152 renders these populations deficient for CRISPR-Cas activity (Fig 2).  (Table 2). In contrast to WT populations, CRISPR3 arrays for the T11RFΔcas9(pAM714) 165 transconjugants were unchanged (Fig 1d and Table 2). We chose the Δ4 population as a 166 representative of the T11RFΔcas9(pAM714) transconjugant populations for future analyses.

168
To investigate the possibility of mutations within cas genes, we performed whole genome WT1, WT2, and WT3 populations ( Table 3). All of the mutations led to nonsynonymous changes some of the populations possessed variation in one or more genes, none of which are predicted to have an impact on the phenotypes observed in our study (STable2). No mutations were 175 identified in the S6 protospacer or PAM region of the repB gene in pAM714. However, we did 176 identify a mutation within repB, not associated with the protospacer or PAM, in the WT2 177 population (STable2). The sequencing also allowed us to investigate the possibility of Tn917 with selection (Fig 3e-g). For WT1, depletion of S6 was not detected after 14 days passage with 193 selection (Fig 3c), consistent with our Sanger sequencing results (  Fig S1a), or any internal spacer within the CRISPR3 201 array (from spacer 1 to spacer 21; or S1 to S21; Fig S1a). The terminal repeat following S21 in the 202 CRISPR3 array is divergent from the direct repeat sequence, so references containing 5'- ). In total, 484 references were constructed. Wild-type CRISPR3 alleles were represented 206 by the reference sequences where 5'-SxRS(x+1)-3' (0 ≤ x <21) and 5'-S21TRST-3' and mutant 207 alleles were represented by 5'-SxRSy-3' (y ≠ x+1). For the control population, which was BHI-208 passaged T11RF lacking pAM714, all but 28 (Day 1) and 4 (Day 14) of the 484 possible alleles 209 were represented in our amplicon reads. We resolved the most abundant mutant CRISPR3 210 alleles in transconjugant populations by mapping amplicon reads to wild-type and mutant 211 CRISPR3 references (Fig 4). Of the five T11RF transconjugant populations analyzed, all other 212 than WT1 possessed at least one mutant CRISPR3 allele after 14 days of passage with 213 antibiotic selection, and each of those alleles lacked S6 (Table 2).

215
We tested the ability of the Day 14 WT5 population to prevent the acquisition of pCF10 and 216 pCF10 derivatives that are targeted by CRISPR3 spacers 1, 6 and 7. Based on deep 217 sequencing data (Table 2), we expected that the WT5 population would interfere with transfer of 218 pCF10 bearing spacer 1, but not pCF10 bearing spacers 6 or 7. Compared to the BHI-passaged 219 WT5 population that significantly reduced the acquisition of each of the pCF10 derivatives, the 220 erythromycin-passaged WT5 population was unable to interfere with pCF10 bearing targets of Preference for spacer deletion over spacer inversion events. Spacer deletion events due to 224 plasmid-CRISPR conflict have been previously reported (33,37). However, the occurrence of 225 spacer inversions, where a leader distal spacer becomes more leader proximal, have not been 226 extensively studied. Due to the high sequence depth achieved in our study, we were able to 227 observe both events and monitor their dynamics for the duration of the serial passaging 228 experiments. Among our 484 artificial references, the spacer deletion group is represented by 229 the mutant alleles where a spacer has been removed from its natural position (5'-SxRSy-3' 230 where y > x+1 and 5'-SxTRST-3' where x < 21), while the spacer inversion group is represented 231 by the mutant alleles where a terminal spacer has become more leader-proximal (5'-SxRSy-3' 232 where y < x). We calculated spacer deletion and spacer inversion rates for each spacer as 233 described in Materials and Methods.

235
In Day 1 and Day 14 BHI-passaged T11RF amplicons, we observed slightly higher spacer 236 deletion rates than spacer inversion rates for leader end spacers, suggesting that spacers at the 237 leader end are more readily deleted than inverted in naturally occurring CRISPR3 arrays (Fig   238   5a). On average, Day 14 T11RF showed slightly lower rates for deletion and inversion events 239 than Day 1 T11RF. However, the deletion and inversion rates were similar at spacers S9-S15 for 240 Day 1 and Day 14 T11RF populations, indicating an equal chance of spacer deletion and 241 inversion (Fig 5a). Terminal end spacers are associated with decreased deletion and inversion 242 rates compared to leader end spacers. Interestingly, the inversion rates of terminal end spacers 243 are slightly higher than the rates for leader end spacers. We hypothesize that the increased 244 spacer inversion rates for terminal spacers is due to a greater number of homologous The deletion and inversion rates of five T11RF(pAM714) transconjugants and the Δ4 population 249 from Day 1 and Day 14 erythromycin passages were evaluated in the same manner as the 250 T11RF population. The distribution of deletion and inversion rates in the Δ4 and WT1 251 populations (Fig 5b and c) were similar to T11RF (Fig 5a). We did observe an elevated deletion 252 rate at S5 for WT1 at Day 1, consistent with deep sequencing results which detected alleles

263
T11RF(pAM714) transconjugants lost S6 to resolve the conflict between CRISPR-Cas and its 264 target. The loss of S6 was often coupled with the loss of surrounding spacers, ranging from S1 to 265 S18 (Table 2). From our amplicon sequencing data, we noticed that the rearrangements 266 associated with shortened CRISPR3 arrays occurred between repeat-spacer junctions leaving 267 behind perfectly intact repeat-spacer-repeat sequences that are still functional for CRISPR 268 interference (Fig 2). This observation led us to hypothesize that either homologous 269 recombination or slippage during DNA replication plays a role in eliminating spacers from the 270 array, as has also been hypothesized by other groups (33,34,37,38).

272
To study if homologous recombination had an impact on spacer loss, we constructed an in-273 frame deletion of recA in T11RF, generating strain T11RFΔrecA, and used it as a recipient in conjugation reactions with the donor OG1SSp(pAM714). We confirmed the presence of a wild 275 type CRISPR3 array in the recA mutant with Sanger sequencing. Across three independent 276 trials, only 12 transconjugant colonies were obtained. We selected two transconjugants 277 (recA.TC1 and recA.TC2) for 14 day serial passaging with continuous erythromycin selection.

294
For these studies, we used the well-characterized human oral isolate OG1RF in combination 295 with pKH12-derived plasmids (Table 1) that were described by Hullahalli et al (39). pKH12 is a 296 chloramphenicol resistant shuttle vector that is mobilizable by the oriT sequence derived from were each transformed into electrocompetent OG1RF. The initial integrity of the CRISPR1-Cas 304 and CRISPR2 arrays, regardless of the plasmid transformed, was confirmed to be wild-type by 305 Sanger sequencing.

307
Three transformants for each plasmid were selected to be used in serial passaging 308 experiments. Each transformant was passaged in plain BHI medium and BHI medium  It has been well documented that antibiotic use contributes to the dissemination of antibiotic 324 resistance plasmids and the emergence of MDR organisms. However, bacteria encode genome 325 defense systems such as CRISPR-Cas to reduce plasmid acquisition. The interactions of CRISPR-Cas systems and naturally occurring resistance plasmids are poorly understood, which 327 is of particular concern in the opportunistic pathogen E. faecalis due to its propensity to engage 328 in intra-and interspecies HGT.

330
We previously demonstrated that the CRISPR3-Cas system of the natively drug sensitive E.

331
faecalis isolate T11RF significantly reduced acquisition of the PRP pAM714 in a cas9-and 332 spacer 6-dependent mechanism (32). The plasmid pAM714 is a derivative of the model PRP

340
In our previous study, we observed a substantial number of T11RF(pAM714) transconjugants, 341 despite CRISPR3-Cas activity. Based on previous data in other organisms (33, 34, 38), we 342 hypothesized that conjugation occurred into T11RF recipient cells with pre-existing CRISPR-343 inactivating mutations. However, recent data from our lab on another CRISPR-Cas system in E.

344
faecalis and using different model strains indicated that this hypothesis was incomplete.

345
Specifically, we observed spacer deletion mutants emerging over time in E. faecalis populations 346 having CRISPR-Cas and a plasmid target in intracellular conflict (39). To attain deeper 347 resolution, in the present study we tracked six distinct pAM714 transconjugant lineages that 348 were predicted to experience intracellular conflict due to the presence of an active CRISPR-Cas 349 system and its plasmid target. Our results describe multiple possible outcomes for conflict 350 resolution between plasmids and CRISPR, with the outcome dependent on the presence or emergence of cas9 mutation (WT1), mutant CRISPR arrays with internal spacer deletions (WT5 353 and WT6), or both (WT2 and WT3) during passage with selection; and 3) plasmid loss in the 354 absence of selection. Importantly, we did not observe spacer adaptation against the plasmid 355 during passage in the absence of selection; this in in contrast to other studies where spacer 356 acquisition against non-selected plasmids was observed (23). Rather, we hypothesize that 357 plasmid-containing cells are at a competitive disadvantage due to intracellular CRISPR/plasmid 358 conflict (39) and are depleted from the population over time, or, a slow-acting CRISPR-Cas 359 system progressively scrubs the plasmid from the population over time.

361
We used erythromycin resistance as a marker for pAM714 carriage. We acknowledge that the 362 erythromycin marker is encoded within Tn917, which could theoretically integrate into the 363 chromosome and escape CRISPR targeting of the pAM714 backbone. However, erythromycin 364 resistance was lost from five T11RF lineages passaged in the absence of selection, but not from 365 the Δcas9 transconjugants. This indicates that Tn917 was lost, along with the pAM714 366 backbone, from the T11RF lineages in a CRISPR-dependent mechanism. Moreover, our 367 analysis of the whole genome sequencing from 8 independent populations found no evidence 368 for Tn917 hopping under erythromycin selection.

370
The plasmid pAM714 encodes a toxin-antitoxin system (44,48,49). The system encodes a 371 stable toxin that will kill daughter cells that have not inherited a plasmid copy; an unstable 372 antitoxin is encoded from the same locus that blocks toxin translation in cells with proper 373 plasmid segregation. However, in our study, we observed a gradual decrease of erythromycin-374 resistant cells when we passaged T11RF(pAM714) in BHI for 14 days, indicating that the toxin-We performed high-coverage Illumina sequencing to assess the dynamics of the entire set of 378 repeats and spacers that make up the CRISPR3 array. We determined that natural 379 heterogeneity in CRISPR3 exists at a low frequency in wild-type T11RF populations. We reason 380 that heterogeneity could result from slippage during DNA replication and/or recombination 381 between CRISPR3 direct repeat sequences, consistent with previous research that proposed 382 that heterogeneity exists within CRISPR arrays in bacterial populations (33,34,38).

383
Furthermore, our results demonstrate that spacer deletion can occur in the absence of recA, 384 which implicates DNA replication slippage in the emergence of mutant CRISPR alleles.

385
However, the fact that we observed the occurrence of spacer inversions indicates that 386 homologous recombination does play a role in the inherent allelic diversity of CRISPR arrays. It 387 is likely that both recombination and replication slippage contribute to the emergence of 388 heterogeneous CRISPR alleles. We do not have an estimate of which process has a greater 389 effect, nor whether additional stresses beyond antibiotic selection could influence rates for each.

391
There were consequences to the resolution of CRISPR/plasmid conflicts in E. faecalis populations.

392
In one case, in which pAM714 conjugated into a recipient with a pre-existing cas9 mutation (WT4), 393 the population could no longer impede entry of PRPs bearing CRISPR targets. For another 394 population, WT5, its susceptibility to future plasmid transfer events was dependent upon whether it 395 was passaged with erythromycin. The WT5 population passaged without erythromycin could 396 impede entry of PRPs bearing CRISPR targets at levels comparable to the wild-type control.

397
However, the WT5 population passaged with erythromycin, in which multiple CRISPR deletion 398 alleles arose, lost the ability to defend against PRPs bearing certain spacer targets. In effect, 399 antibiotic selection for pAM714 selected for the emergence of mutants with compromised CRISPR-400 Cas. We also observed that the acquisition frequencies of pCF10 and its derivatives were higher for 401 all populations possessing pAM714 (WT5-Erm, WT4-BHI, and WT4-Erm). We infer that pAM714 402 enhances pCF10 conjugation frequency via an unknown mechanism. This was an unanticipated additional outcome of the enforced maintenance of pAM714, and it remains to be determined model strains (T11RF and OG1RF), three CRISPR loci (CRISPR3-Cas, and CRISPR1-Cas and

486
The basic variants and fixed ploidy variants were combined for each sequencing sample and 487 subjected to manual inspection. The variants that were detected in the T11 genome from all 488 samples were inferred to be variants in our parent T11 stock and were manually removed. The 489 variants that were detected in pAD1 genome from all transconjugant samples were inferred to 490 be variants in our pAM714 stock, hence were also manually removed. Next, variants within the 491 CRISPR3 array were removed as we analyzed CRISPR3 alleles using a different approach  were generated for the CRISPR3 amplicon. Considering that the read length is 150 bp, we 517 manually split each read into two subsequences (one subsequence was 75 bp; with the 518 remainder of the read being the second subsequence) to enhance mapping efficiency, allowing 519 for retrieval of maximal sequence information. The split amplicon sequencing reads were 520 mapped to the 5'-SxRSy-3' and 5'-SxTRST-3' references using stringent mapping parameters in 521 CLC Genomics Workbench (Qiagen). The stringent mapping parameters require 100% of each 522 mapped read to be ≥95% identical to one unique reference. Thus, the sequencing reads from 523 different CRISPR alleles will be distinguished. These amplicon mapping results were applied to 524 the calculation of forward spacer deletion and backward spacer rearrangement rates.

526
To further evaluate the mapping efficiency, the unmapped reads from initial mapping to the T11 527 CRISPR3 reference (STable3, step 1) were subjected to additional quality control analysis. The 528 unmapped reads were mapped to the 484 manually created spacer[x]-repeat-spacer [y] possible references (CRISPR3 region plus manually created references) using default mapping parameters, ignoring unspecific mapping (80% of each mapped read has at least 50% identity 533 to the reference sequence; STable3, step 3 mapping). The unmapped reads from step 3 were 534 mapped to all possible references using the default mapping parameters and randomly map 535 unspecific matching reads (STable3, step 4 mapping).