Small RNA sR158 Participates in Oxidation Stress Tolerance and Pathogenicity of Edwardaiella piscicida by Regulating TA System YefM-YoeB

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To survive in stressful and challenging environments, bacteria have evolved sophisticated mechanisms to sense their environment and alter gene expression patterns by regulators [9].Among various regulators, bacterial small RNAs (sRNAs) have become a research hotspot.Some sRNAs play critical regulatory roles in response to various environmental stresses, bioflm formation, pathogenicity, and other major processes [10][11][12][13][14].
Bacterial sRNAs are typically untranslated transcripts, 50 to 500 nucleotides in length.Regulation of target mRNAs by sRNA is achieved through base matching [15].According to the regulation mode, bacterial sRNAs are divided into transcoded and cis-coded sRNAs.Trans-encoded sRNA is expressed at diferent sites from its target genes and partially complements its target genes by a specifc seed sequence.Te expression site of cis-coded is the same as its unique target site, and they are completely complementary [16,17].Transencoded sRNAs often exhibit their roles in the presence of Hfq, an RNA chaperone protein [9,18].Our previous research has shown that Hfq in E. piscicida plays an indispensable role in response to stress and infection [19].
Te toxin-antitoxin (TA) system is widespread in the genomes of prokaryotes and archaea but was originally discovered as a plasmid-stabilizing molecule [20,21].Te TA operon encodes a stable toxin and an antitoxin that are easily degraded.Te toxin is usually a protein, and the antitoxin can be either a protein or RNA.Up to now, seven diferent types of TA systems have been identifed [22,23], among which type II TA systems are a research hotspot [22].YefM-YoeB is a common type II TA system and is involved in stress resistance in many pathogenic bacteria [24,25].In E. piscicida, YefM-YoeB is crucial to responding to adverse circumstances and pathogenicity [26].
Te type II TA system is often regulated by itself [22], but almost no other regulators have been identifed.In our previous study, an Hfq-dependent sRNA, sR158, was identifed [27].sR158 is located at the downstream of the TA system YefM-YoeB.In this study, the roles of sR158 in stress adaptation and pathogenicity were identifed.Our study is the frst to report that type II TA system expression is regulated by sRNA.

Construction of Missing Mutant Strain.
Te primers are shown in Table 1.Te mutant was constructed, as previously reported [26].To obtain the mutant, ΔsR158, we constructed the deletion of 83 bp fragment of sR158 by using overlapping PCR.After amplifying two PCR fragments using primer pairs sR158KOF1/sR158KOR1 and sR158KOF2/ sR158KOR2, the overlapping PCR fragments were obtained with primer sR158KOF1/sR158KOR2 and cloned into suicide plasmid pDM4, resulting in recombinant plasmid pDMsR158.Te transformants were obtained by converting pDMsR158 to S17-1λpir.E. piscicida were conjugated with transformants.Transconjugants were selected and cultured on LB agar plates containing polymyxin B, chloramphenicol, and 12% sucrose for 48-72 h.PCR was used for screening the sucrose-resistant and chloramphenicol-sensitive colonies with primers sR158KOF3/sR158KOR3.To confrm the inframe deletion, DNA sequencing was performed on the PCR products obtained.

Resistance to Oxidative
Stress.TX01 and ΔsR158 were grown to exponential growth phase, and then bacteria were collected and washed in PBS.About 10 5 bacteria were added per 250 μL hydrogen peroxide (3.2 mM) or PBS (control).After 60 minutes of incubation, mixture was diluted and coated with 50 μL on LB plates.Tese plates were incubated at 28 °C for 36 h, and the number of colonies on it was then recorded.Survival rates were calculated, as described previously [26].

Bioflm and Motility Assay.
Te bioflm formation and motility assay were performed, as described previously [26].

Invading Host Cell
Lines.FG cells and E. piscicida in 96well plates were incubated for 1 h and 2 h at 25 °C at a MOI of 50 :1.After washing, the FG cells were lysed, and bacteria attached with and invaded into hose cells were examined by plate counting.Bacteria were grown in DMEM containing murine monocyte-macrophage cells, as described previously [26].

Pathogenicity Analysis In Vivo.
Te challenge experiment was performed, as described previously [28].Briefy, healthy tilapias (5 groups, 40 per group) were acclimated for 2 weeks, and then were intramuscularly infected with the same dose (1 × 10 6 CFU) of E. piscicida (TX01 and ΔsR158) and PBS (control).Before collecting tissues, these fsh were euthanized with 200 mg/L tricaine methanesulfonate (MS-222) (Sigma, United States).At 24 and 48 hours postinfection (hpi), fve fsh were dissected aseptically, and spleen and head kidney were taken for the examination of viable bacteria.Te rest of the fsh were observed and the number of deaths was recorded.

Construction of sR158 Mutant Strain.
In our previous study, 148 sRNAs of E. piscicida were found and identifed [27].One of them, sR158, a novel sRNA, was found to be located at the downstream of YefM-YoeB (Figure 1), which was important to oxidation pressure, bioflm formation, and virulence in E. piscicida [26].
To examine the function of sR158, sR158 mutant and ΔsR158 was structured by markerless in-frame.Next, we examined the efects of the sR158 deletion on the expression of yefM-yoeB, stress adaptation, and pathogenicity of the bacteria.

Efect of sR158 on Expression of yefM-yoeB.
Since sR158 is located immediately downstream of yefM-yoeB and sRNA belongs to an important regulator [27], we hope to know the efect of sR158 on the expression of yefM-yoeB.Te results of RT-qPCR showed that the expression of yefM-yoeB in ΔsR158 was signifcantly lower than that of TX01, but the expression of ETAE_RS07655 was not afected (Figure 2), which indicates the expression of yefM-yoeB was upregulated by sR158.Since sR158 is an Hfq-associated sRNA and Hfq is an important RNA chaperone protein [19], we want to enquire whether this regulation of sR158 to yefM-yoeB expression depends on Hfq.Te results of RT-qPCR showed that the expression of yefM-yoeB in Δhfq was equivalent to that of yefM-yoeB in ΔsR158.Tese results confrm that sR158 regulates the expression of yefM-yoeB, whose regulatory function is Hfq-dependent.Type II TA expression is transcriptionally autoregulated by itself.For example, YefM and YefM-YoeB regulated the expression of the yefM-yoeB operon [26], and HigA and HigBA regulated the expression of the higBA operon [28].However, as far as we know, there are no reports of sRNA regulating TA systems.

Efect of sR158 on Oxidation Stress Tolerance.
Oxidative stress is an unavoidable environmental threat during the infection of the host by E. piscicida, and ΔyefM-yoeB damages the capability of bacteria to tolerate oxidation pressure [26].Growth analysis was performed to examine the efect of sR158 on the antioxidant stress of bacteria.Te growth of ΔsR158 was similar to that of wild strain TX01, indicating that the absence of sR158 did not afect its growth in normal LB medium and agar plates (Figures 3(a ΔsR158 displayed obviously delayed growth compared to that of TX01 (Figure 3(c)).Consistently, the survival rate of ΔsR158 under oxidative pressure was only 32.5%, which is signifcantly lower than that of TX01 (65.1%) (Figure 3(d)).Tese results illustrate that the deletion of sR158 reduces the antioxidant capacity of E. piscicida.sRNA is widely involved in bacterial stress resistance [9].For example, sRNA MicF in E. coli was closely associated with oxidative stress [30].sRNA RsaC in Staphylococcus aureus modulates the oxidation pressure response during manganese defciency [31].Te deletion of sRNA EsR240 reduced E. tarda′s survival under oxidative stress [32].Consistently, our results showed that sR158 defciency weakened E. piscicida′s tolerance to oxidation stress, probably by regulating the TA system YefM-YoeB.

Efects of sR158 on Bacterial Motility and Bioflm.
As regulators of gene expression, many bacterial sRNAs participate in some important physiologies, such as motility and bioflm formation [33].In the study of bacterial bioformation ability, it was found that the bioflm formation ability of ΔsR158 was signifcantly higher than that of TX01 (Figure 4(a)), indicating deletion of sR158 enhances the bioflm-forming capacity of bacteria in the community.To explore the efect of sR158 on bacterial motility after 24 h, TX01 (31 ± 1.7 mm) and ΔsR158 (32 ± 1.7 mm) showed similar movement zone diameters (Figure 4  ΔyefM-yoeB did not afect the motility of E. piscicida (data not shown), which is consistent with the result of sR158.However, ΔyefM-yoeB enhanced bacterial bioflm formation [26], which is also in accordance with the result of sR158.Tis fnding indicates that sR158 regulates E. piscicida′s bioflm formation by promoting yefM-yoeB expression.It has been reported that sRNA RsmZ and RsmY of Pseudomonas aeruginosa also regulate bioflm [34,35].In Staphylococcus epidermidis, the role of RsaE/RoxS in bioflm matrix production was also reported [36].Tese reports, along with our results, manifest that sRNAs participate in bacterial bioflm formation.

Efects of sR158 on Cell Invasion and Intracellular Survival.
To detect the involvement of sR158 in pathogenicity to host cells, Japanese founder gill cells were cultured and incubated with TX01 or ΔsR158 for 2 h to detect adhesion and invasion of host cells.Te results showed that the recovery amount of ΔsR158 was obviously higher than that of TX01 at 1 hpi and 2 hpi (Figure 5(a)), which indicates sR158 defciency enhances the infection of E. piscicida in host cells.No signifcant diference was observed between ΔsR158 survival in RAW264.7 cells and TX01 at four detection time points (Figure 5(b)), suggesting sR158 is not associated with E. piscicida survival within host phagocytes.Consistently, the ΔyefM-yoeB recovered from FG cells was signifcantly higher than TX01.Te amount of ΔyefM-yoeB in RAW264.7 cells was equivalent to that of TX01 [26], which further indicates the relevance of sR158 and YefM-YoeB.

Efect on General Virulence of Bacteria in Fish.
In vitro experiments have shown that sR158 was involved in E. piscicida invasion of host cells.We hope to elucidate the function of sR158 in host infection.Te results showed that  the number of bacteria in ΔsR158 were signifcantly higher than that in TX01 (Figure 6(a)).Tilapias were infected with TX01 and ΔsR158, and the mortality of fsh was monitored.Te results showed that all ΔsR158-infected fsh died at 16 days, while TX01-infected fsh still had a 20% survival rate at 20 days (Figure 6(b)).Te above results show that deleting sR158 increases the pathogenicity of E. piscicida.
Many bacterial sRNAs have been found to play a role in virulence in recent years.For example, in Streptococcus pneumoniae, multiple sRNAs were involved in niche-specifc roles in virulence [37].In Staphylococcus aureus, sarA transcript-derived sRNA teg49 regulated virulence genes independent of SarA [37].In Salmonella typhimurium, sRNA IsrJ promoted bacterial invasion and enhanced the translocation efciency of the T3SS-1 efector protein SptP into eukaryotic cells [38].sRNA STnc150 downregulated the protein expression of FimA, and deletion of STnc150 enhanced the bacterial adhesion ability of S. typhimurium to host cells and reduced LD50 in mice [39].A great number of virulence-related sRNAs were reported in a variety of human pathogens, such as Vibrio cholerae and V. vulnifcus [40].However, few sRNAs from aquatic pathogens were reported.Lately, in E. piscicida, several sRNAs have been speculated to be involved in virulence [41].EsR240 was confrmed to participate in E. piscicida′s virulence [32].In our previous study, fve Hfq-dependent sRNAs (sR012, sR043, sR082, sR084, and sR145) were involved in E. piscicida′s pathogenicity.Te deletion of sR012, sR043, and sR082 abated bacterial virulence, but the deletion of sR084 and sR145 boosted bacterial pathogenicity [27].In this study, the deletion of Hfq-dependent sR158 also increased E. piscicida's pathogenicity, including adhesion to host cells, tissue colonization, and general virulence, which were consistent with phenotypes of the TA system YefM-YoeB deletion [26].However, within 14 days after infection, ΔsR158 exhibited less virulence than the wild strain.We speculate the mutation of sR158 perhaps leads E. piscicida to be trapped in host cells and unable to spread swiftly to the whole body, causing the survival of ΔsR158 to be higher than that of the wild strain.After 14 days, since the number of ΔsR158 in tissues increased, the survival of ΔsR158 was lower than that of the wild strain, indicating the virulence of ΔsR158 was stronger than that of the wild strain.
In conclusion, our results confrm that the novel sRNA sR158 of E. piscicida positively regulates YefM-YoeB expression, which is Hfq-dependent.Te phenotypes of sR158 defciency are consistent with those of yefM-yoeB, including reduced resistance against oxidation stress, enhanced bioflm formation, increased invasion of host cells and tissues, and boosted general virulence.Tese fndings indicate that sR158 participates in the stress resistance and pathogenicity of E. piscicida, probably by regulating YefM-YoeB.Our result is the frst report that the type II TA system is regulated by sRNA, which provides new insights into the regulatory role of bacterial sRNA [42,43].

Figure 3 :
Figure 3: Resistance to oxidation stress.TX01 and ΔsR158 grew to the logarithmic phase were diluted at diferent concentrations, then dripped onto the normal LB solid plate (a) or LB solid plate containing 250 μM hydrogen peroxide (c), and incubated for 36 h.(b) Bacterial growth curves for TX01 and ΔsR158.(d) Logarithmic TX01 and ΔsR158 were challenged with hydrogen peroxide or PBS.Next, living bacteria were determined by plate counting.Data are expressed as mean ± SEM (N � 3), where N represents the number of experiments performed.

Figure 4 :Figure 5 :
Figure 4: Bioflm and motility assay.(a) Bioflm assay.Te two strains identifed bioflm formation by crystal violet staining and measured A 570 in the fnal eluent.(b) Te motility of Edwardsiella piscicida.1.5 μL of bacteria was dripped on the fresh swimming plate (0.3% agar).Te plate was incubated to observe the bacterial movement.Data are expressed as mean ± SEM (N � 3), where N represents the number of experiments performed.

Table 1 :
Oligonucleotide primers used in this study.