Protozoan predation enhances stress resistance and antibiotic tolerance in Burkholderia cenocepacia by triggering the SOS response

Abstract Bacterivorous protists are thought to serve as training grounds for bacterial pathogens by subjecting them to the same hostile conditions that they will encounter in the human host. Bacteria that survive intracellular digestion exhibit enhanced virulence and stress resistance after successful passage through protozoa but the underlying mechanisms are unknown. Here we show that the opportunistic pathogen Burkholderia cenocepacia survives phagocytosis by ciliates found in domestic and hospital sink drains, and viable bacteria are expelled packaged in respirable membrane vesicles with enhanced resistance to oxidative stress, desiccation, and antibiotics, thereby contributing to pathogen dissemination in the environment. Reactive oxygen species generated within the protozoan phagosome promote the formation of persisters tolerant to ciprofloxacin by activating the bacterial SOS response. In addition, we show that genes encoding antioxidant enzymes are upregulated during passage through ciliates increasing bacterial resistance to oxidative radicals. We prove that suppression of the SOS response impairs bacterial intracellular survival and persister formation within protists. This study highlights the significance of protozoan food vacuoles as niches that foster bacterial adaptation in natural and built environments and suggests that persister switch within phagosomes may be a widespread phenomenon in bacteria surviving intracellular digestion.

cenocepacia ΔrecA mutant was complemented as follows.The coding region of recA was PCR amplified with primers FA219Fw and FA220Rv from gDNA of K56-2 and cloned into NdeI/XbaI sites of plasmid pSCrhaB2plus [2].The resulting plasmid, named pFA107, was introduced into K56-2 ΔrecA strain by tri-parental mating as described above.
Transconjugants harbouring the complementing plasmid were selected on trimethoprim and gentamicin containing plates and confirmed by PCR.This strain was named as FA108.
The lexA(Ind-) mutant strain was obtained by gene replacement.To generate the lexA(S119A) mutation cassette, two fragments containing the 500 bp upstream region with the complete lexA CDS (locus tag K562_12117, accession number NZ_CP053300) and the 500 bp downstream region were PCR amplified with Phusion HF DNA polymerase (Thermo Scientific) from K56-2 gDNA using primers FA225Fw-FA226Rv and FA227Fw-FA228Rv, respectively.The tetracycline cassette was PCR amplified from pDAI-SceI-SacB using primers FA223Fw and FA224Rv.Then, the three fragments were PCR assembled together using the 20 bp homology regions in primers FA226Fw and FA227Rv, and introduced into pGPI-SceI by restriction cloning with XbaI and EcoRI (Thermo Fisher Scientific).The point mutation lexA(S119A) was introduced using the Q5-Site-directed mutagenesis kit (New England Biolabs) with primers FA229Fw and FA230Rv.The resulting mutant allele was verified by Sanger sequencing with primers FA236Fw and FA237Rv.Plasmid carrying the lexA(S119A)::Tet r cassette was named pFA118 and was delivered to K56-2 by triparental mating as described before.Tetracycline-resistant and trimethoprim sensitive exconjugants were selected and the presence of the mutant allele lexA(S119A) was confirmed by PCR and DNA sequencing.The lexA(Ind-) phenotype was confirmed by its higher susceptibility to UV compared to the wild type strain K56-2.
The FA115 and AM031 strains were generated by introducing pFA158 and pAM028 into K56-2 by triparental mating as described before and selecting for trimethoprim-gentamicin and trimethoprim-chloramphenicol resistant exconjugants, respectively.pFA158 was generated by cloning the dsRed2 coding sequence into NdeI/XbaI sites of plasmid pSCrhaB2plus.dsRed2 coding sequence was PCR amplified from plasmid pUC18T-mini-Tn7T-Tp-dsRedExpress [3] with primers FA265Fw and FA266Rv.pAM028 was constructed by replacing the I-SceI gene in pAH-25 [4] by egfp, which was previously PCR amplified with primers FA218Fw and FA219Rv and digested with NdeI and XbaI.
The oligonucleotides used in this study are listed in Table S3.

Determination of MIC
The MIC was measured by performing the microdilution test and following CLSI reference methods [6].Bacterial growth was estimated by measuring OD 600 at 24 h with a TECAN Infinite MPlex plate.We determined that a given antibiotic concentration prevented bacterial growth when it reduced bacterial growth to an OD 600 comparable to that of the negative control well (LB medium with no bacteria) after 20 h of incubation in static conditions.

UV stress assays
Log-phase cultures (OD 600 =0.3) of wild type and mutant strains were washed and suspended in fresh LB medium.Serial 10-fold dilutions were prepared in fresh LB medium and spotted on the surface of LB agar plates.The plates were then irradiated with 222 nm UVC light at a dose of 100 W/cm 2 for different times using a UVO-Ccrosslinker (EQUILAB, SL, Madrid).
After irradiation, plates were incubated at 37 ºC for 24 h.The survival percentage of the population was calculated as the number of CFUs divided by the total number of CFUs in the non-irradiated population.Table S1     T. elliotti EFVs (24 h post-feeding).Ciliates were incubated with bacteria (ratio 1:200 protist:bacteria) in buffer.At 0 and 24 h ciliates and EFVs were lysed with 1% Triton-X100 and RNA was isolated from collected bacteria.Bacteria incubated in Tris-HCl for 24 h in the absence of ciliates served as control.The stars indicate significant differences (** p < 0.01) when compared to the control group (bacteria incubated in buffer for 24 h) according the unpaired t student's test.performing the microdilution test and following CLSI reference methods [6].Bacterial growth was estimated by measuring OD 600 at 24 h.MIC was determined as 4 µg/mL for K56-2, 2 µg/mL for lexA(Ind-), <2 µg/mL for ΔsurA and 1 µg/mL for ΔrecA.

Figure S2 .
Figure S1.Representative microscopy images of EFVs purified from B. cenocepacia K56-2 and T. elliotti co-cultures.(A) Purified EFVs before washing steps.(B) Aggregate of purified EFVs after washing and centrifugation steps as described in the Methods section.

Figure S3 .Figure S4 .Figure S5 .Figure S6 .
Figure S3.Biphasic killing curve obtained in follow-up persister assays.Bacteria that 116 survived 20 h exposure to 40 µg/ml ciprofloxacin in persister assays were grown in fresh LB 117 medium overnight, harvested and inoculated in LB supplemented with 40 µg/ml ciprofloxacin 118 for 20 h for a follow-up persister assay.The number of surviving bacteria was estimated by 119 CFU counting on LB plates.Data shown correspond to the avearge of two independent 120 experiments.En each experiment three independent cultures were grown and exposed to 121 the antibiotic.122 123

Figure S7 .
Figure S7.Upregulation of SOS response genes in B. cepacia ATCC25416 recovered from

Figure S8 .Figure S9 .Figure S10 .
Figure S8.Paraquat (PQ) treatment in LB. (A) MIC assay for B. cenocepacia K56-2 167 exposed to different concentrations of PQ in LB for 24 h.Bacterial growth was estimated by 168 measuring OD 600 after 24 h in static conditions in a 96 well plate.(B) As expected, exposure 169 to 1 mM PQ in LB for 30 min did not result in bacterial death.The number of surviving K56-2 170 bacteria was quantified by CFU counts on LB agar plates at 0 and 30 min after exposure to 171 PQ. 172 173

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Bacterial strains and protists used in this study

Table S2 .
Plasmids used in this study rThis study

Table S3 .
Oligonucleotides used in this study.Oligonucleotides were synthesized by Integrated DNA Technologies.