# The Influence of Outer Membrane Protein on Ampicillin Resistance ofVibrio parahaemolyticus

Authors: Xiangyu Meng; Danyang Huang; Qing Zhou; Fan Ji; Xin Tan; Jianli Wang; Xiaoyuan Wang
Journal: Canadian Journal of Infectious Diseases and Medical Microbiology
 (2023)

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## Abstract

The antibiotic resistance of the food-borne pathogenVibrio parahaemolyticus has attracted researchers’ attention in recent years, but its molecular mechanism remains poorly understood. In this study, 7 genes encoding outer membrane proteins (OMPs) were individually deleted in V. parahaemolyticus ATCC33846, and the resistance of these 7 mutants to 14 antibiotics was investigated. The results revealed that the resistance of the 7 mutants to ampicillin was significantly increased. Further exploration of 20-gene transcription changes by real time-qPCR (RT-qPCR) demonstrated that the higher ampicillin resistance might be attributed to the expression of β-lactamase and reduced peptidoglycan (PG) synthesis activity through reduced transcription of penicillin-binding proteins (PBPs), increased transcription of l,d-transpeptidases, downregulated d,d-carboxypeptidase, and alanine deficiency. This study provides a new perspective on ampicillin resistance in OMP mutants with respect to PG synthesis.


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## Body

## 1. Introduction

Vibrio parahaemolyticus is a Gram-negative bacterium causing intestinal infections. It is commonly found in seafood, such as blood clams and shrimp [1], causing significant economic losses as an aquatic pathogen [2]. Due to the overuse of antibiotics, multidrug-resistant V. parahaemolyticus has been isolated in recent years. The strains have demonstrated resistance to ampicillin, cefazolin, penicillin, and so on [3].Studies investigating the mechanism of ampicillin resistance mainly focus on three points: drug permeation, peptidoglycan (PG) synthesis, andβ-lactamase. In Neisseria meningitidis, a single point mutation in the porin PorB can strongly affect the binding and permeation of β-lactam antibiotics [4]. Affinity binding studies of four transformants revealed decreased affinity of PBP4 for ampicillin [5]. In Enterococcus faecium, l,d-transpeptidase-mediated resistance may emerge in various pathogens [6]. In V. parahaemolyticus, a novel class A carbenicillin-hydrolyzing β-lactamase, bla (CARB-17), was responsible for the intrinsic penicillin resistance [7].The role of outer membrane protein (OMP) in antibiotic resistance is usually related to permeation [8] as antibiotic susceptibility is related to OMP channel size [9]. Ampicillin enters E. coli through OmpF [10]. In carbapenem-resistantEnterobacter aerogenes, the expression of OMPs is deficient due to overexpressed sRNA or decreased due to single-point mutation [11]. In addition, performing gene knockout in V. parahaemolyticus is challenging, so few studies have reported antibiotic resistance in OMP mutants.In this work,VP_RS22195, VP_RS23020, VP_RS16800, VP_RS16465, VP_RS20840, VP_RS03765, and VP_RS11205 were knocked out by ATCC33846. Subsequently, the growth, outer membrane (OM) permeability, and minimum inhibitory concentration (MIC) of OMP deletion mutants were evaluated. In total, 14 antibiotics targeting different sites in cells were selected, including β-lactams (ampicillin), aminoglycosides (streptomycin, kanamycin, gentamicin, tobramycin), quinolones (ciprofloxacin, nalidixic acid), furans (nitrofurantoin), cationic antimicrobial peptides (polymyxin B), rifampicin, clarithromycin, tetracycline, chloramphenicol, and novobiocin. After ampicillin treatment, the transcriptional changes of 20 genes in each mutant were further detected. This study gives a better understanding of the role of OMPs in V. parahaemolyticus OM and the cell wall.


## 2. Materials and Methods

### 2.1. Strain and Growth Condition

Table1 lists all strains and plasmids used in this study. V. parahaemolyticus ATCC33846 was used to construct OMP deletion strains. The bacteria were grown in Luria-Bertani (LB) broth medium containing 10 g/L NaCl, 10 g/L trypsin (OXOID), and 5 g/L yeast extract (OXOID) at 37°C and 200 rpm for liquid culture.Table 1
Bacteria and plasmids are used in this study.
Strains or plasmidsDescriptionsSourcesStrainsATCC33846Wild-typeVibrio parahaemolyticusATCCCC118(λpir)λ-pir lysogen of CC118 (D(ara-leu) araD DlacX 74 galE galK phoA20 thi-1rpsE rpoB argE(Am) recA1)[12]S17-1(λpir)λ-pir lysogen of S17-1 (thi pro hsdR–hsdM + recA RP4 2-Tc::Mu-Km::Tn7 (Cmr))[13]ATCC33846ΔVP_RS22195Derived from ATCC33846 by removingVP_RS22195This studyATCC33846ΔVP_RS23020Derived from ATCC33846 by removingVP_RS23020This studyATCC33846ΔVP_RS16800Derived from ATCC33846 by removingVP_RS16800This studyATCC33846ΔVP_RS16465Derived from ATCC33846 by removingVP_RS16465This studyATCC33846ΔVP_RS20840Derived from ATCC33846 by removingVP_RS20840This studyATCC33846ΔVP_RS03765Derived from ATCC33846 by removingVP_RS03765This studyATCC33846ΔVP_RS11205Derived from ATCC33846 by removingVP_RS11205This studyPlasmidspDS132Suicide plasmid, Cmr[14]pACYC184Plasmid containingp15A origin, Cmr, TcrATCCpWJW101Plasmid containingloxL-Gm-loxR, Gmr[15]pDTW109Plasmid containingcre gene, Gmr[16]pΔVP_RS22195Derived from pDS132 by addingVP_RS22195 homologous armsThis studypΔVP_RS23020Derived from pDS132 by addingVP_RS23020 homologous armsThis studypΔVP_RS16800Derived from pDS132 by addingVP_RS16800 homologous armsThis studypΔVP_RS16465Derived from pDS132 by addingVP_RS16465 homologous armsThis studypΔVP_RS20840Derived from pDS132 by addingVP_RS20840 homologous armsThis studypΔVP_RS03765Derived from pDS132 by addingVP_RS03765 homologous armsThis studypΔVP_RS11205Derived from pDS132 by addingVP_RS11205 homologous armsThis study


### 2.2. Construction of Deletion Plasmid pOTC-SB

The deletion plasmid pOTC-SB was composed of genesCmR, p15A, traJ, oriT, and cre. The CmR and p15A gene fragment (1) was amplified from template pACYC184 with primers Cm-p15A-F and Cm-p15A-R, and then digested with FastDigest enzymes SpeI and PstI (Thermo Scientific). The traJ and oriT gene fragment (2) was amplified from template pDS132 with primers traJ-oriT-F and traJ-oriT-R and then digested with FastDigest enzymes SalI and SpeI (Thermo Scientific). The cre gene fragment (3) was amplified from template pDTW109 by primers Ptac-cre-F and Ptac-cre-R. The sacB gene fragment was amplified from template pDS132 by primers sacB-F and sacB-R. Finally, the ligation product of (1) and (2) was connected to (3) by using one-step cloning (ClonExpress II One Step Cloning Kit, Vazyme). The resulting plasmid was named pOTC as it contained oriT and cre. Linearized pOTC by BstZ17I and sacB were combined by one-step cloning to construct pOTC-SB.


### 2.3. Construction of OMP Deletion Mutants

Upstream and downstream homology arms were amplified from the chromosomal DNA ofV. parahaemolyticus ATCC33846 by the corresponding U/D-(target gene)-F/R primers, as displayed in Table 2. The loxL-Gm-loxR fragment was amplified from pWJW101 [15] by primers Gm-R and Gm-F, and the homology arms and loxL-Gm-loxR were integrated by fusion PCR (4). Then, the pDS132 plasmid and (4) were separately digested with the FastDigest enzymes XbaI or SalI and ligated by T4 ligase (VP_RS23020 was connected to pDS132 by one-step cloning). E. coli CC118 was transformed with products and cultured on LB plates containing 30 μg·ml−1 gentamicin, and the plasmid extracted from E. coli CC118 was electrotransformed into E. coliS17-l (λpir).Table 2
Primers used in this study.
NamesSequence (5′–3′)Ptac-cre-FTTG​GAT​ACA​CCA​AGG​AAA​GTG​GTA​CCT​GAA​CGA​CCC​CGA​ATA​TTG​GPtac-cre-RACA​GAT​GTA​GGT​GTT​CCA​CAC​TGC​AGC​TAA​TCG​CCA​TCT​TCC​AGC​AtraJ-oriT-FCACT​AGTTCG​GGT​CGG​GTG​AAT​CTTtraJ-oriT-RACTTTCCTTGGTGTATCCCmR-p15A-FTGT​GGA​ACA​CCT​ACA​TCT​GCmR-p15A-RCACT​AGTATC​GTA​TGG​GGC​TGA​CTTsacB-FCAG​CGC​TAG​CGG​AGT​GTA​TAC​CAC​CTT​TAT​GTT​GAT​AAG​AAA​TAA​AAG​AAAsacB-RCAA​CAT​AGT​AAG​CCA​GTA​TAC​GGA​TCG​ATC​CTT​TTT​AAC​CCA​TCGmR-RCGT​AAT​ACG​ACT​CAC​TAT​AGG​GCGmR-FGCG​CAA​TTA​ACC​CTC​ACT​AAA​GVP_RS22195-U-FAATCT​AGACAG​AAT​CAC​AGC​ACG​AGGVP_RS22195-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGC​TCC​CAC​AAC​AGT​CCA​CAT​AGCVP_RS22195-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCT​AAG​TAA​GAT​GTT​GTT​CCG​CGVP_RS22195-D-RAATCT​AGATGG​CAG​CGA​ACA​AAG​GTGVP_RS23020-U-FGGG​TTA​AAA​AGG​ATC​GAT​CCT​GTC​GTT​TTT​CGC​AAC​AAT​CVP_RS23020-U-DGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGA​AAA​TGG​CTT​GCT​CCC​GTVP_RS23020-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CAG​CAC​GCA​TGT​AAC​CGT​GVP_RS23020-D-RAAT​TTG​TGG​AAT​TCC​CGG​GAG​TAT​GAC​GTA​TAC​CCG​CAT​AVP_RS16800-U-FAATCT​AGACCT​CTT​TTG​GCT​CGG​GTTVP_RS16800-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGA​AAC​GGG​CTA​ACT​ACT​GTVP_RS16800-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCG​TTA​CGG​ATG​TAT​GTG​TVP_RS16800-D-RAATCT​AGAGAC​ACT​GAA​CTG​CCT​GTCVP_RS16465-U-FAATCT​AGAAAG​ACG​AAC​CAG​GCA​AGGVP_RS16465-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGA​GTG​TCA​GCA​AGT​GCA​ACVP_RS16465-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCG​ATG​CTC​CGT​TTA​GTA​GVP_RS16465-D-RAATCT​AGAGAA​TGG​CTA​GTA​AGG​AAT​GTVP_RS20840-U-FAAAGTC​GACGAA​GAC​CGT​CAT​TTA​GCTVP_RS20840-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGG​CAA​GTC​TAT​GGG​GAA​GCVP_RS20840-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCA​TTC​TCA​TCA​AAA​GGG​GVP_RS20840-D-RAAAGTC​GACGCC​ATA​CGG​AGG​ATT​CATVP_RS03765-U-FAATCT​AGAAAA​TCG​GTA​TGC​CAC​TTCVP_RS03765-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGG​CCG​CCT​AAT​AAC​ACT​GACVP_RS03765-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CGA​CCG​TAG​AAT​AGC​AGC​CVP_RS03765-D-RAATCT​AGACAT​CGC​TCA​GTG​GGT​TTVP_RS11205-U-FAATCT​AGACGC​CTC​TAT​CGT​TCG​CATVP_RS11205-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGG​ACA​ATT​ACC​GGG​GGT​GAGVP_RS11205-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCC​TCT​TTA​GAA​AGT​TCT​ACC​GVP_RS11205-D-RAATCT​AGATTC​AAC​ATC​TCT​TGG​ACT​CRT-VP_RS22195-FCTA​AGC​AAC​CAA​GTT​AGC​CAA​CRT-VP_RS22195-RTTC​TTC​TTG​AGC​AGC​CAT​TGRT-VP_RS23020-FAGT​TGA​ATC​TCG​CCG​TAA​ATRT-VP_RS23020-RCTGCTGGTTCTGCTTTCGRT-VP_RS16800-FTGCTACATCGCCATCACGRT-VP_RS16800-RGCA​TCA​ACT​ACC​GCT​TCT​TGRT-VP_RS16465-FGCT​GGT​GAC​GAG​AAA​GAC​TRT-VP_RS16465-RTTGAAGCCGATACCGAAGRT-VP_RS20840-FCAA​CAG​GCT​CAC​TAG​GTG​CTRT-VP_RS20840-RTTTGCGTCCATATCGTCCRT-VP_RS03765-FCTC​AAA​CTA​TCG​GCA​CTG​GTRT-VP_RS03765-RTTCGCTTGTGGGTGCTCRT-VP_RS11205-FCGT​AAG​CGA​TTT​TCT​GTG​CRT-VP_RS11205-RAAAGCGGCTGGGATTGGRT-VP_RS17515-FGCT​TGT​CCG​TTT​GTG​TAT​CCCRT-VP_RS17515-RTGC​TCA​ACT​GTT​AGT​TAC​GCC​TCRT-VP_RS13510-FAAT​CAT​TGC​TCG​TTA​CCA​CAGRT-VP_RS13510-RCCG​ACG​TAT​AGG​CTT​TCT​CTT​CRT-mrcB-FGCGACAGAAGACCGAGATRT-mrcB-RCGT​TAA​GGT​ACT​GCC​ACC​TRT-VP_RS02165-FTCGCTTACCGTGCCATCRT-VP_RS02165-RTTT​TAC​ATC​CAG​CAT​CAC​CACRT-mrdA-FGTTTTGATGGGCTTGCTGRT-mrd-RCCACTTTGATGCGGTTGTRT-VP_RS21250-FTTGACGGGATGAAAACGGRT-VP_RS21250-RAACAACTGCGATAAGGCGRT-VP_RS22785-FTGGCATCCAAACCTCACCRT-VP_RS22785-RCTT​GAA​GAC​TTT​CTC​GCT​GTRT-dacB-FAGA​CCA​ACT​ATT​TCC​ACC​TGRT-dacB-RAGTAGATAACGGCATCGGRT-VP_RS22200-FACGCCACCTGCGTCTATTRT-VP_RS22200-RCATTCCGATGCCGAAATCRT-VP_RS09310-FTAA​TGA​CGG​CAC​TTA​TGG​TRT-VP_RS09310-RACA​GAC​AAC​TGC​TCT​ACG​GRT-VP_RS15980-FAGAAGAAGGCGACAACCGRT-VP_RS15980-RGCACCGAGCGATAAAACGRT-uhpA-FAAG​GCT​ATC​TAA​GCA​AAC​GCRT-uhpA-RGCA​ATG​TCG​GAG​GTA​AGG​TRT-VP_RS14060-FTAGAGCTCGGCGCAGATGRT-VP_RS14060-RTTGCTTGCCCGGATACAGNote. The restriction enzyme sites are in bold.After conjugation ofV. parahaemolyticus ATCC33846 and E. coliS17-l (λpir) with the relevant plasmid, 5 μg·ml−1 polymyxin B and 10 μg·ml−1 chloramphenicol were used to select transformants of V. parahaemolyticus. Then, 10% (W/V) sucrose and gentamicin (10 μg·ml−1) were used to select deletion mutants. The mutants were verified by colony PCR and genome verification.Following knockout,Gm was removed by Cre in pOTC-SB. The deletion mutants were conjugated with S17-1 containing pOTC-SB and then spread-plated with 30 μg·ml−1 chloramphenicol. Agarose gel electrophoresis revealed three results. The first result indicated a single band with the length of homology arms plus Gm; the second result showed a single band with the length of homology arms plus 100 bp; the third result displayed a combination of the above two types of bands. To ensure thorough Cre, the strains of the first and third colonies were cultured again in LB with chloramphenicol until the second result appeared. Then, negative selection was performed on an LB plate with gentamicin to ensure the removal of Gm from the genome by Cre. The pOTC-SB was removed by culturing in LB with 10% sucrose without antibiotics. The negative selection was performed on an LB plate with chloramphenicol to verify the removal of pOTC-SB, which has chloramphenicol resistance. The verification of OMP deletion mutants by agarose gel electrophoresis is shown in Figure 1.Figure 1
The verification of OMP deletion mutants was performed using primers of (gene)-U-F and (gene)-D-R by DNA agarose gel electrophoresis. M marker; WT, wild type, representing ATCC33846; lanes A-H showed ΔVP_RS22195, ΔVP_RS23020, ΔVP_RS16800, ΔVP_RS16465, ΔVP_RS20840, ΔVP_RS03765, and ΔVP_RS11205 respectively.After OMP deletion mutants were constructed, mutants and wild-type strains were streaked and purified on the LB plate 5 times, then cultured in LB liquid overnight and stored at −70°C. Before each assay, strains were streaked and activated on the LB plate for 25 h, and then a single colony was cultured in LB liquid for 14 h. A bacterial solution was then used in each assay.


### 2.4. Growth Curve

The overnight cultured strains were added to 50 ml of LB broth medium with an initial OD600 of 0.02. The mixture was cultured at 37°C and 200 rpm. All assays were performed in triplicate.


### 2.5. Permeation Assay

The OD600 of overnight-cultured strains was adjusted to 0.5. The sediments were washed twice and suspended in 10 mmol·L−1 pH 7.4 PBS. Subsequently, 48 μl NPN solution was added to 1152 μl of the bacteria solution before being observed with the fluorescence spectrophotometer. The detection was carried out under excitation light at 350 nm, emission light at 428 nm, and a 2.5 mm slit width. All assays were carried out in triplicates, and three parallels were performed in each group.


### 2.6. The Minimum Inhibitory Concentration (MIC) Assay

All antibiotics except ampicillin were diluted by a two-fold dilution method to adjust the concentration to 0.0078125–256μg·ml−1 in 96-well plates. As the 2-fold dilution of high-concentration solutions would result in large intervals, another concentration setting was used for ampicillin. For solutions with a concentration under 100 μg·ml−1, a 2-fold dilution method was used to achieve a concentration range of 0.98–125 μg·ml−1. For solutions with a concentration range of 100–1000 μg·ml−1, the resulting concentration was set to 100, 200, 300, …, 1000 μg·ml−1. In the concentration range between 1000 and 2000 μg·ml−1, the concentration points were set as 1000, 1250, 1500, and 1750 μg·ml−1. In addition, for concentrations above 2000 μg·ml−1, final concentrations of 2000, 2500, and 3000 μg·ml−1 were set up. The strains cultured overnight were then transferred to 5 ml LB test tubes with an initial OD600 of 0.02 for 4-5 h. Then, the OD600 of freshly cultured strains was adjusted to 0.001. Then, 100 μl of diluted bacteria solution was added to each well, and the culture was incubated at 37°C for 18 h. OD600 was measured by BioTek Cytation 5. All assays were carried out in duplicate and performed three times in parallel in each group.


### 2.7. Real-Time Polymerase Chain Reaction (RT-qPCR)

The overnight cultured strains were transferred into 5 ml LB test tubes with an initial OD600 of 0.02 for 4–5 h. Half of the bacterial solution was taken out into a sterilized empty test tube. Ampicillin was added to the experimental groups at the final concentration of 16 μg·ml−1, and sterilized water with the same volume of ampicillin was added to tubes as the control. After incubating at 37°C and 200 rpm for 0.5 h, the sediment was used for RNA extraction. The methods used for RNA extraction, reverse transcription, and RT-qPCR were the same as those described in [17]. All assays were performed in triplicates.


## 2.1. Strain and Growth Condition

Table1 lists all strains and plasmids used in this study. V. parahaemolyticus ATCC33846 was used to construct OMP deletion strains. The bacteria were grown in Luria-Bertani (LB) broth medium containing 10 g/L NaCl, 10 g/L trypsin (OXOID), and 5 g/L yeast extract (OXOID) at 37°C and 200 rpm for liquid culture.Table 1
Bacteria and plasmids are used in this study.
Strains or plasmidsDescriptionsSourcesStrainsATCC33846Wild-typeVibrio parahaemolyticusATCCCC118(λpir)λ-pir lysogen of CC118 (D(ara-leu) araD DlacX 74 galE galK phoA20 thi-1rpsE rpoB argE(Am) recA1)[12]S17-1(λpir)λ-pir lysogen of S17-1 (thi pro hsdR–hsdM + recA RP4 2-Tc::Mu-Km::Tn7 (Cmr))[13]ATCC33846ΔVP_RS22195Derived from ATCC33846 by removingVP_RS22195This studyATCC33846ΔVP_RS23020Derived from ATCC33846 by removingVP_RS23020This studyATCC33846ΔVP_RS16800Derived from ATCC33846 by removingVP_RS16800This studyATCC33846ΔVP_RS16465Derived from ATCC33846 by removingVP_RS16465This studyATCC33846ΔVP_RS20840Derived from ATCC33846 by removingVP_RS20840This studyATCC33846ΔVP_RS03765Derived from ATCC33846 by removingVP_RS03765This studyATCC33846ΔVP_RS11205Derived from ATCC33846 by removingVP_RS11205This studyPlasmidspDS132Suicide plasmid, Cmr[14]pACYC184Plasmid containingp15A origin, Cmr, TcrATCCpWJW101Plasmid containingloxL-Gm-loxR, Gmr[15]pDTW109Plasmid containingcre gene, Gmr[16]pΔVP_RS22195Derived from pDS132 by addingVP_RS22195 homologous armsThis studypΔVP_RS23020Derived from pDS132 by addingVP_RS23020 homologous armsThis studypΔVP_RS16800Derived from pDS132 by addingVP_RS16800 homologous armsThis studypΔVP_RS16465Derived from pDS132 by addingVP_RS16465 homologous armsThis studypΔVP_RS20840Derived from pDS132 by addingVP_RS20840 homologous armsThis studypΔVP_RS03765Derived from pDS132 by addingVP_RS03765 homologous armsThis studypΔVP_RS11205Derived from pDS132 by addingVP_RS11205 homologous armsThis study


## 2.2. Construction of Deletion Plasmid pOTC-SB

The deletion plasmid pOTC-SB was composed of genesCmR, p15A, traJ, oriT, and cre. The CmR and p15A gene fragment (1) was amplified from template pACYC184 with primers Cm-p15A-F and Cm-p15A-R, and then digested with FastDigest enzymes SpeI and PstI (Thermo Scientific). The traJ and oriT gene fragment (2) was amplified from template pDS132 with primers traJ-oriT-F and traJ-oriT-R and then digested with FastDigest enzymes SalI and SpeI (Thermo Scientific). The cre gene fragment (3) was amplified from template pDTW109 by primers Ptac-cre-F and Ptac-cre-R. The sacB gene fragment was amplified from template pDS132 by primers sacB-F and sacB-R. Finally, the ligation product of (1) and (2) was connected to (3) by using one-step cloning (ClonExpress II One Step Cloning Kit, Vazyme). The resulting plasmid was named pOTC as it contained oriT and cre. Linearized pOTC by BstZ17I and sacB were combined by one-step cloning to construct pOTC-SB.


## 2.3. Construction of OMP Deletion Mutants

Upstream and downstream homology arms were amplified from the chromosomal DNA ofV. parahaemolyticus ATCC33846 by the corresponding U/D-(target gene)-F/R primers, as displayed in Table 2. The loxL-Gm-loxR fragment was amplified from pWJW101 [15] by primers Gm-R and Gm-F, and the homology arms and loxL-Gm-loxR were integrated by fusion PCR (4). Then, the pDS132 plasmid and (4) were separately digested with the FastDigest enzymes XbaI or SalI and ligated by T4 ligase (VP_RS23020 was connected to pDS132 by one-step cloning). E. coli CC118 was transformed with products and cultured on LB plates containing 30 μg·ml−1 gentamicin, and the plasmid extracted from E. coli CC118 was electrotransformed into E. coliS17-l (λpir).Table 2
Primers used in this study.
NamesSequence (5′–3′)Ptac-cre-FTTG​GAT​ACA​CCA​AGG​AAA​GTG​GTA​CCT​GAA​CGA​CCC​CGA​ATA​TTG​GPtac-cre-RACA​GAT​GTA​GGT​GTT​CCA​CAC​TGC​AGC​TAA​TCG​CCA​TCT​TCC​AGC​AtraJ-oriT-FCACT​AGTTCG​GGT​CGG​GTG​AAT​CTTtraJ-oriT-RACTTTCCTTGGTGTATCCCmR-p15A-FTGT​GGA​ACA​CCT​ACA​TCT​GCmR-p15A-RCACT​AGTATC​GTA​TGG​GGC​TGA​CTTsacB-FCAG​CGC​TAG​CGG​AGT​GTA​TAC​CAC​CTT​TAT​GTT​GAT​AAG​AAA​TAA​AAG​AAAsacB-RCAA​CAT​AGT​AAG​CCA​GTA​TAC​GGA​TCG​ATC​CTT​TTT​AAC​CCA​TCGmR-RCGT​AAT​ACG​ACT​CAC​TAT​AGG​GCGmR-FGCG​CAA​TTA​ACC​CTC​ACT​AAA​GVP_RS22195-U-FAATCT​AGACAG​AAT​CAC​AGC​ACG​AGGVP_RS22195-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGC​TCC​CAC​AAC​AGT​CCA​CAT​AGCVP_RS22195-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCT​AAG​TAA​GAT​GTT​GTT​CCG​CGVP_RS22195-D-RAATCT​AGATGG​CAG​CGA​ACA​AAG​GTGVP_RS23020-U-FGGG​TTA​AAA​AGG​ATC​GAT​CCT​GTC​GTT​TTT​CGC​AAC​AAT​CVP_RS23020-U-DGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGA​AAA​TGG​CTT​GCT​CCC​GTVP_RS23020-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CAG​CAC​GCA​TGT​AAC​CGT​GVP_RS23020-D-RAAT​TTG​TGG​AAT​TCC​CGG​GAG​TAT​GAC​GTA​TAC​CCG​CAT​AVP_RS16800-U-FAATCT​AGACCT​CTT​TTG​GCT​CGG​GTTVP_RS16800-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGA​AAC​GGG​CTA​ACT​ACT​GTVP_RS16800-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCG​TTA​CGG​ATG​TAT​GTG​TVP_RS16800-D-RAATCT​AGAGAC​ACT​GAA​CTG​CCT​GTCVP_RS16465-U-FAATCT​AGAAAG​ACG​AAC​CAG​GCA​AGGVP_RS16465-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGA​GTG​TCA​GCA​AGT​GCA​ACVP_RS16465-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCG​ATG​CTC​CGT​TTA​GTA​GVP_RS16465-D-RAATCT​AGAGAA​TGG​CTA​GTA​AGG​AAT​GTVP_RS20840-U-FAAAGTC​GACGAA​GAC​CGT​CAT​TTA​GCTVP_RS20840-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGG​CAA​GTC​TAT​GGG​GAA​GCVP_RS20840-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCA​TTC​TCA​TCA​AAA​GGG​GVP_RS20840-D-RAAAGTC​GACGCC​ATA​CGG​AGG​ATT​CATVP_RS03765-U-FAATCT​AGAAAA​TCG​GTA​TGC​CAC​TTCVP_RS03765-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGG​CCG​CCT​AAT​AAC​ACT​GACVP_RS03765-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CGA​CCG​TAG​AAT​AGC​AGC​CVP_RS03765-D-RAATCT​AGACAT​CGC​TCA​GTG​GGT​TTVP_RS11205-U-FAATCT​AGACGC​CTC​TAT​CGT​TCG​CATVP_RS11205-U-RGCC​CTA​TAG​TGA​GTC​GTA​TTA​CGG​ACA​ATT​ACC​GGG​GGT​GAGVP_RS11205-D-FCTT​TAG​TGA​GGG​TTA​ATT​GCG​CCC​TCT​TTA​GAA​AGT​TCT​ACC​GVP_RS11205-D-RAATCT​AGATTC​AAC​ATC​TCT​TGG​ACT​CRT-VP_RS22195-FCTA​AGC​AAC​CAA​GTT​AGC​CAA​CRT-VP_RS22195-RTTC​TTC​TTG​AGC​AGC​CAT​TGRT-VP_RS23020-FAGT​TGA​ATC​TCG​CCG​TAA​ATRT-VP_RS23020-RCTGCTGGTTCTGCTTTCGRT-VP_RS16800-FTGCTACATCGCCATCACGRT-VP_RS16800-RGCA​TCA​ACT​ACC​GCT​TCT​TGRT-VP_RS16465-FGCT​GGT​GAC​GAG​AAA​GAC​TRT-VP_RS16465-RTTGAAGCCGATACCGAAGRT-VP_RS20840-FCAA​CAG​GCT​CAC​TAG​GTG​CTRT-VP_RS20840-RTTTGCGTCCATATCGTCCRT-VP_RS03765-FCTC​AAA​CTA​TCG​GCA​CTG​GTRT-VP_RS03765-RTTCGCTTGTGGGTGCTCRT-VP_RS11205-FCGT​AAG​CGA​TTT​TCT​GTG​CRT-VP_RS11205-RAAAGCGGCTGGGATTGGRT-VP_RS17515-FGCT​TGT​CCG​TTT​GTG​TAT​CCCRT-VP_RS17515-RTGC​TCA​ACT​GTT​AGT​TAC​GCC​TCRT-VP_RS13510-FAAT​CAT​TGC​TCG​TTA​CCA​CAGRT-VP_RS13510-RCCG​ACG​TAT​AGG​CTT​TCT​CTT​CRT-mrcB-FGCGACAGAAGACCGAGATRT-mrcB-RCGT​TAA​GGT​ACT​GCC​ACC​TRT-VP_RS02165-FTCGCTTACCGTGCCATCRT-VP_RS02165-RTTT​TAC​ATC​CAG​CAT​CAC​CACRT-mrdA-FGTTTTGATGGGCTTGCTGRT-mrd-RCCACTTTGATGCGGTTGTRT-VP_RS21250-FTTGACGGGATGAAAACGGRT-VP_RS21250-RAACAACTGCGATAAGGCGRT-VP_RS22785-FTGGCATCCAAACCTCACCRT-VP_RS22785-RCTT​GAA​GAC​TTT​CTC​GCT​GTRT-dacB-FAGA​CCA​ACT​ATT​TCC​ACC​TGRT-dacB-RAGTAGATAACGGCATCGGRT-VP_RS22200-FACGCCACCTGCGTCTATTRT-VP_RS22200-RCATTCCGATGCCGAAATCRT-VP_RS09310-FTAA​TGA​CGG​CAC​TTA​TGG​TRT-VP_RS09310-RACA​GAC​AAC​TGC​TCT​ACG​GRT-VP_RS15980-FAGAAGAAGGCGACAACCGRT-VP_RS15980-RGCACCGAGCGATAAAACGRT-uhpA-FAAG​GCT​ATC​TAA​GCA​AAC​GCRT-uhpA-RGCA​ATG​TCG​GAG​GTA​AGG​TRT-VP_RS14060-FTAGAGCTCGGCGCAGATGRT-VP_RS14060-RTTGCTTGCCCGGATACAGNote. The restriction enzyme sites are in bold.After conjugation ofV. parahaemolyticus ATCC33846 and E. coliS17-l (λpir) with the relevant plasmid, 5 μg·ml−1 polymyxin B and 10 μg·ml−1 chloramphenicol were used to select transformants of V. parahaemolyticus. Then, 10% (W/V) sucrose and gentamicin (10 μg·ml−1) were used to select deletion mutants. The mutants were verified by colony PCR and genome verification.Following knockout,Gm was removed by Cre in pOTC-SB. The deletion mutants were conjugated with S17-1 containing pOTC-SB and then spread-plated with 30 μg·ml−1 chloramphenicol. Agarose gel electrophoresis revealed three results. The first result indicated a single band with the length of homology arms plus Gm; the second result showed a single band with the length of homology arms plus 100 bp; the third result displayed a combination of the above two types of bands. To ensure thorough Cre, the strains of the first and third colonies were cultured again in LB with chloramphenicol until the second result appeared. Then, negative selection was performed on an LB plate with gentamicin to ensure the removal of Gm from the genome by Cre. The pOTC-SB was removed by culturing in LB with 10% sucrose without antibiotics. The negative selection was performed on an LB plate with chloramphenicol to verify the removal of pOTC-SB, which has chloramphenicol resistance. The verification of OMP deletion mutants by agarose gel electrophoresis is shown in Figure 1.Figure 1
The verification of OMP deletion mutants was performed using primers of (gene)-U-F and (gene)-D-R by DNA agarose gel electrophoresis. M marker; WT, wild type, representing ATCC33846; lanes A-H showed ΔVP_RS22195, ΔVP_RS23020, ΔVP_RS16800, ΔVP_RS16465, ΔVP_RS20840, ΔVP_RS03765, and ΔVP_RS11205 respectively.After OMP deletion mutants were constructed, mutants and wild-type strains were streaked and purified on the LB plate 5 times, then cultured in LB liquid overnight and stored at −70°C. Before each assay, strains were streaked and activated on the LB plate for 25 h, and then a single colony was cultured in LB liquid for 14 h. A bacterial solution was then used in each assay.


## 2.4. Growth Curve

The overnight cultured strains were added to 50 ml of LB broth medium with an initial OD600 of 0.02. The mixture was cultured at 37°C and 200 rpm. All assays were performed in triplicate.


## 2.5. Permeation Assay

The OD600 of overnight-cultured strains was adjusted to 0.5. The sediments were washed twice and suspended in 10 mmol·L−1 pH 7.4 PBS. Subsequently, 48 μl NPN solution was added to 1152 μl of the bacteria solution before being observed with the fluorescence spectrophotometer. The detection was carried out under excitation light at 350 nm, emission light at 428 nm, and a 2.5 mm slit width. All assays were carried out in triplicates, and three parallels were performed in each group.


## 2.6. The Minimum Inhibitory Concentration (MIC) Assay

All antibiotics except ampicillin were diluted by a two-fold dilution method to adjust the concentration to 0.0078125–256μg·ml−1 in 96-well plates. As the 2-fold dilution of high-concentration solutions would result in large intervals, another concentration setting was used for ampicillin. For solutions with a concentration under 100 μg·ml−1, a 2-fold dilution method was used to achieve a concentration range of 0.98–125 μg·ml−1. For solutions with a concentration range of 100–1000 μg·ml−1, the resulting concentration was set to 100, 200, 300, …, 1000 μg·ml−1. In the concentration range between 1000 and 2000 μg·ml−1, the concentration points were set as 1000, 1250, 1500, and 1750 μg·ml−1. In addition, for concentrations above 2000 μg·ml−1, final concentrations of 2000, 2500, and 3000 μg·ml−1 were set up. The strains cultured overnight were then transferred to 5 ml LB test tubes with an initial OD600 of 0.02 for 4-5 h. Then, the OD600 of freshly cultured strains was adjusted to 0.001. Then, 100 μl of diluted bacteria solution was added to each well, and the culture was incubated at 37°C for 18 h. OD600 was measured by BioTek Cytation 5. All assays were carried out in duplicate and performed three times in parallel in each group.


## 2.7. Real-Time Polymerase Chain Reaction (RT-qPCR)

The overnight cultured strains were transferred into 5 ml LB test tubes with an initial OD600 of 0.02 for 4–5 h. Half of the bacterial solution was taken out into a sterilized empty test tube. Ampicillin was added to the experimental groups at the final concentration of 16 μg·ml−1, and sterilized water with the same volume of ampicillin was added to tubes as the control. After incubating at 37°C and 200 rpm for 0.5 h, the sediment was used for RNA extraction. The methods used for RNA extraction, reverse transcription, and RT-qPCR were the same as those described in [17]. All assays were performed in triplicates.


## 3. Results

### 3.1. Deletion of OMP Affects Cell Growth, OM Permeation, and Antibiotic Resistance

Bacterial growth has four phases, including the lag phase, the exponential phase, the stationary phase, and the decline phase. In the lag phase, bacteria produce new enzymes to digest, build biomass, and prepare for cell division [18]. ATCC33846 started to grow after a lag phase of 2 hours following the inoculum. Compared to ATCC33846, some OMP mutants demonstrated a shorter lag phase, including ΔVP_RS22195, ΔVP_RS16800, ΔVP_RS16465, and ΔVP_RS20840 (Figure 2). The shorter lag phase indicated that the OMPs were the proteins prepared in the lag phase. Deletion of OMPs lightened the burden of preparation for cells in the lagged phase. In the exponential phase, ΔVP_RS16800, ΔVP_RS16465, ΔVP_RS20840, and ΔVP_RS03765 (Figures 2(c)–2(f)) had a higher growth rate than ATCC33846. Furthermore, ΔVP_RS22195, ΔVP_RS16800, ΔVP_RS16465, ΔVP_RS20840, and ΔVP_RS03765 entered the decline phase directly, without an obvious stationary phase. In contrast, ΔVP_RS23020 and ΔVP_RS11205 showed poor growth (Figures 2(b) and 2(g)).Figure 2
Growth curves of the 7V. parahaemolyticus OMP deletion mutants. Wild-typeV. parahaemolyticus ATCC33846 was used as a control. Each point represents the mean value of three biological replicates, and error bars represent standard deviations calculated from three biological replicates.
(a)(b)(c)(d)(e)(f)(g)The N-Phenyl-1-naphthylamine (NPN) probe was used to test the OM permeability of OMP deletion mutants. NPN can be excited to form green fluorescence in the bacterial inner membrane, a hydrophobic environment [19]. A decreased OM permeability was observed in ΔVP_RS16465 (Figure 3).Figure 3
Comparison of OM permeability of the 7V. parahaemolyticus OMP mutants, using wild-type ATCC33846 as a control. The fluorescence intensity was determined with excitation at 350 nm and emission at 428 nm. Each bar represents the mean value of three biological replicates, and error bars represent standard deviations calculated from three biological replicates. A statistically significant difference was determined using independent samples t-test analysis (∗P<0.05).Fourteen antibiotics were selected for the MIC assay (Figure4). In ΔVP_RS23020, increased resistance to antibiotics (ciprofloxacin, nalidixic acid, and novobiocin) inhibiting DNA synthesis was observed. Rifampicin inhibits RNA synthesis, and a 4-fold increase in the rifampicin MIC was observed in ΔVP_RS22195. Resistance to antibiotics (clarithromycin, chloramphenicol, tetracycline, streptomycin, kanamycin, gentamicin, and tobramycin) inhibiting protein synthesis was all increased in ΔVP_RS22195 and ΔVP_RS20840. Among protein synthesis-inhibiting antibiotics, resistance to 3 aminoglycosides (streptomycin, kanamycin, and gentamicin) was increased, whereas the fold change of tobramycin MIC was not obvious. Notably, ΔVP_RS11205 was more resistant to aminoglycosides than other mutants, and the tetracycline susceptibility of ΔVP_RS23020 was increased 2-fold. Nitrofurantoin inhibits carbohydrate metabolism enzymes and interferes with cell wall synthesis [20]. Resistance to nitrofurantoin was not decreased after OMP deletion. In addition, resistance to β-lactams, which act on peptidoglycan (PG) synthesis, showed a general increase. Polymyxin B damages Gram-negative bacterial OM and resistance to polymyxin B was increased 2.8-fold in ΔVP_RS20840. All OMP mutants showed at least a 64-fold increase in ampicillin resistance.Figure 4
Comparison of resistance to 14 antibiotics among the 7V. parahaemolyticus OMP deletion mutants, using wild-type ATCC33846 as a control. The fold change is calculated by log2 (the MIC of mutants/MIC of ATCC33846). Each color block represents the mean value of three biological replicates.


### 3.2. Changes in the Transcription of 20 Genes in OMP Deletion Mutants under the Absence and Stimulation of Ampicillin

RT-qPCR was performed to detect the transcriptional changes of 20 genes to further study the causes of increased ampicillin resistance in OMP deletion mutants (Table3). These genes can be divided into five types: OMP genes, β-lactamase genes, PG synthesis-related genes, stress-regulation-related genes, and lipid A synthesis genes. VP_RS17515 expresses β-lactamase. VP_RS13510, mrcB, mrdA, VP_RS02165, VP_RS03450, VP_RS22785, dacB, VP_RS22200, VP_RS09310, VP_RS15980 were selected as PG synthesis-related genes through BLASTp in NCBI, and the proteins were homologous with PBP1A, PBP1B, PBP2, PBP3, PBP5, PBP4, LdtA, LdtD, and LdtF, respectively, in E. coli. In order to directly observe gene regulation under membrane stress, uhpA and VP_RS14060, which are homologous to the response regulator proteins rcsB and cpxR in E. coli, respectively, were selected. lpxA is a lipid A synthesis gene. β-lactams inhibit bacterial growth by binding to penicillin-binding proteins (PBPs) and interfering with PG synthesis [27]. According to the growth curve, bacteria in the initial exponential phase were used as the experiment sample. The ampicillin MIC value of ATCC33846 was 3.90625–15.625 μg·mL−1, while the MIC values of OMP mutants were above 600 μg·mL−1. Therefore, 16 μg·mL−1 ampicillin, which would not kill ATCC33846 and have a better effect on mutants, was set as the pretreatment.Table 3
Introduction of genes related to this work.
Gene namesDescriptionsVP_RS22195Hypothetical protein, 438 bp, chromosome 240.45% similar to murein lipoprotein Lpp inEscherichia coliVP_RS23020Maltoporin, 1311 bp, chromosome 272.36% similar to maltoporin inEscherichia coliVP_RS16800MipA/OmpV family protein, 781 bp, chromosome 2100% similar to MipA/OmpV family protein inEscherichia coliVP_RS16465OmpA family protein, 1080 bp, chromosome 228.77% similar to porin OmpA inEscherichia coliVP_RS20840OmpA family protein, 1318 bp, chromosome 229.62% similar to porin OmpA inEscherichia coliVP_RS03765OmpA family protein, 1019 bp, chromosome 132.5% similar to murein porin OmpA inEscherichia coliVP_RS11205OmpH family outer membrane protein, 465 bp, chromosome 136.42% similar to periplasmic chaperone Skp inE. coliVP_RS17515Carbenicillin-hydrolyzing class A beta-lactamase CARB-22Hydrolyzingβ-lactams [21]VP_RS13510PBP1A family penicillin-binding proteinPeptidoglycan synthase which is essential for cell elongation [22]mrcBPenicillin-binding protein 1BPeptidoglycan synthase which is essential for cell division [22]mrdAPenicillin-binding protein 2Transpeptidase essential for cell elongation [23]VP_RS02165Penicillin-binding protein 3Transpeptidase essential for cell division [23]VP_RS22785D-alanyl-D-alanine carboxypeptidaseD,D-carboxypeptidase which cleaves terminal D-ala in the peptidoglycan [24]dacBSerine-typeD-Ala-D-Ala carboxypeptidaseD,D-carboxypeptidase which cleaves terminal D-ala in the peptidoglycan [23]VP_RS22200l,D-transpeptidase family proteinAttaching Lpp to mDAP3 of peptidoglycan [23]VP_RS09310, VP_RS15980l,D-transpeptidase family proteinSynthesizing mDAP3–mDAP3cross-links in the peptidoglycan [23]uhpATranscriptional regulator, 29.06% similar to RcsB inE. coliRcsB is response regulator of Rcs sensing outer membrane stress [25]VP_RS14060Response regulator, 62.45% similar to CpxR inE. coliCpxR is response regulator of Cpx sensing inner membrane stress [26]lpxAAcyl-ACP-UDP-N-acetylglucosamine O-acyltransferaseEssential for the biosynthesis of lipid AFigure5 shows the transcription changes of 20 genes in mutants compared to ATCC33846. In ΔVP_RS23020 (Figure 5(b)), all genes exhibited transcriptional upregulation except for VP_RS11205. In ΔVP_RS16465 and ΔVP_RS20840 (Figures 5(d) and 5(e)), VP_RS23020 was upregulated over 9-fold. The transcription of VP_RS17515 was also upregulated over 9-fold in ΔVP_RS23020 but downregulated in ΔVP_RS22195, ΔVP_RS16800, ΔVP_RS20840, ΔVP_RS03765,and ΔVP_RS11205 (Figures 5(a), 5(c), 5(e)–5(g)). Most PG synthesis genes were downregulated in mutants except for ΔVP_RS23020. However, in ΔVP_RS23020, the transcription of VP_RS22200, VP_RS09310, and VP_RS15980 (Ldts) were all upregulated. Among PG synthesis genes, the transcriptional changes of VP_RS22785 and VP_RS09310 showed an interesting phenomenon. These two genes were downregulated over 2-fold in ΔVP_RS22195, ΔVP_RS16800, ΔVP_RS16465, and ΔVP_RS20840. In contrast, they were upregulated over 2-fold in ΔVP_RS23030 and ΔVP_RS11205, VP_RS22785, and VP_RS09310. In ΔVP_RS03765, these two genes showed no obvious downregulation compared to other genes. Moreover, VP_RS20840 showed the same trend as VP_RS22785 in all mutants. Moreover, the transcription of lpxA was downregulated in all mutants except for ΔVP_RS23020.Figure 5
Comparison of transcription of 20 genes in the 7V. parahaemolyticus OMP deletion mutants, using wild-type ATCC33846 as a control. Five kinds of genes are divided by dotted line: OMP genes, β-lactamase genes, PG synthesis-related genes, stress-regulation-related genes, and lipid A synthesis genes. VP_RS17515 expresses β-lactamase. The transcription change fold is calculated by 2−ΔΔCt. Each bar represents the mean value of three biological replicates, and error bars represent standard deviations calculated from three biological replicates.Figure6 displays the transcription changes of OMP genes of ATCC33846 and mutants under ampicillin stimulation compared to the untreated group. All OMP genes were downregulated in ΔVP_RS22195, ΔVP_RS23020, and ΔVP_RS20840 (Figures 6(b)6(c), and 6(f)). Furthermore, VP_RS16800 was downregulated over 2-fold in ATCC33846, ΔVP_RS23020, ΔVP_RS20840, and ΔVP_RS03765 (Figures 6(a), 6(c), 6(f), and 6(g)), and over 4-fold in ΔVP_RS22195 and ΔVP_RS11205 (Figures 6(b) and 6(h)). VP_RS17515 demonstrated over 2-fold upregulation in ATCC33846, ΔVP_RS16800, and ΔVP_RS16465 (Figures 6(a), 6(d), and 6(e)). In addition, transcription of PG synthesis genes showed significant changes in OMP deletion mutants but not after ampicillin stimulation. These findings implied that OMP deletion caused internal resistance to ampicillin instead of inducing a stress response with stimulation of ampicillin.Figure 6
Comparison of transcription of 20 genes in the 7V. parahaemolyticus OMP deletion mutants under stimulation of ampicillin (AMP), using wild-type ATCC33846 as a control. Five kinds of genes are divided by dotted line: OMP genes, β-lactamase genes, PG synthesis-related genes, stress-regulation-related genes, and lipid A synthesis genes. The transcription change fold is calculated by 2−ΔΔCt. Each bar represents the mean value of three biological replicates, and the error bars represent standard deviations calculated from three biological replicates.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.1. Deletion of OMP Affects Cell Growth, OM Permeation, and Antibiotic Resistance

Bacterial growth has four phases, including the lag phase, the exponential phase, the stationary phase, and the decline phase. In the lag phase, bacteria produce new enzymes to digest, build biomass, and prepare for cell division [18]. ATCC33846 started to grow after a lag phase of 2 hours following the inoculum. Compared to ATCC33846, some OMP mutants demonstrated a shorter lag phase, including ΔVP_RS22195, ΔVP_RS16800, ΔVP_RS16465, and ΔVP_RS20840 (Figure 2). The shorter lag phase indicated that the OMPs were the proteins prepared in the lag phase. Deletion of OMPs lightened the burden of preparation for cells in the lagged phase. In the exponential phase, ΔVP_RS16800, ΔVP_RS16465, ΔVP_RS20840, and ΔVP_RS03765 (Figures 2(c)–2(f)) had a higher growth rate than ATCC33846. Furthermore, ΔVP_RS22195, ΔVP_RS16800, ΔVP_RS16465, ΔVP_RS20840, and ΔVP_RS03765 entered the decline phase directly, without an obvious stationary phase. In contrast, ΔVP_RS23020 and ΔVP_RS11205 showed poor growth (Figures 2(b) and 2(g)).Figure 2
Growth curves of the 7V. parahaemolyticus OMP deletion mutants. Wild-typeV. parahaemolyticus ATCC33846 was used as a control. Each point represents the mean value of three biological replicates, and error bars represent standard deviations calculated from three biological replicates.
(a)(b)(c)(d)(e)(f)(g)The N-Phenyl-1-naphthylamine (NPN) probe was used to test the OM permeability of OMP deletion mutants. NPN can be excited to form green fluorescence in the bacterial inner membrane, a hydrophobic environment [19]. A decreased OM permeability was observed in ΔVP_RS16465 (Figure 3).Figure 3
Comparison of OM permeability of the 7V. parahaemolyticus OMP mutants, using wild-type ATCC33846 as a control. The fluorescence intensity was determined with excitation at 350 nm and emission at 428 nm. Each bar represents the mean value of three biological replicates, and error bars represent standard deviations calculated from three biological replicates. A statistically significant difference was determined using independent samples t-test analysis (∗P<0.05).Fourteen antibiotics were selected for the MIC assay (Figure4). In ΔVP_RS23020, increased resistance to antibiotics (ciprofloxacin, nalidixic acid, and novobiocin) inhibiting DNA synthesis was observed. Rifampicin inhibits RNA synthesis, and a 4-fold increase in the rifampicin MIC was observed in ΔVP_RS22195. Resistance to antibiotics (clarithromycin, chloramphenicol, tetracycline, streptomycin, kanamycin, gentamicin, and tobramycin) inhibiting protein synthesis was all increased in ΔVP_RS22195 and ΔVP_RS20840. Among protein synthesis-inhibiting antibiotics, resistance to 3 aminoglycosides (streptomycin, kanamycin, and gentamicin) was increased, whereas the fold change of tobramycin MIC was not obvious. Notably, ΔVP_RS11205 was more resistant to aminoglycosides than other mutants, and the tetracycline susceptibility of ΔVP_RS23020 was increased 2-fold. Nitrofurantoin inhibits carbohydrate metabolism enzymes and interferes with cell wall synthesis [20]. Resistance to nitrofurantoin was not decreased after OMP deletion. In addition, resistance to β-lactams, which act on peptidoglycan (PG) synthesis, showed a general increase. Polymyxin B damages Gram-negative bacterial OM and resistance to polymyxin B was increased 2.8-fold in ΔVP_RS20840. All OMP mutants showed at least a 64-fold increase in ampicillin resistance.Figure 4
Comparison of resistance to 14 antibiotics among the 7V. parahaemolyticus OMP deletion mutants, using wild-type ATCC33846 as a control. The fold change is calculated by log2 (the MIC of mutants/MIC of ATCC33846). Each color block represents the mean value of three biological replicates.


## 3.2. Changes in the Transcription of 20 Genes in OMP Deletion Mutants under the Absence and Stimulation of Ampicillin

RT-qPCR was performed to detect the transcriptional changes of 20 genes to further study the causes of increased ampicillin resistance in OMP deletion mutants (Table3). These genes can be divided into five types: OMP genes, β-lactamase genes, PG synthesis-related genes, stress-regulation-related genes, and lipid A synthesis genes. VP_RS17515 expresses β-lactamase. VP_RS13510, mrcB, mrdA, VP_RS02165, VP_RS03450, VP_RS22785, dacB, VP_RS22200, VP_RS09310, VP_RS15980 were selected as PG synthesis-related genes through BLASTp in NCBI, and the proteins were homologous with PBP1A, PBP1B, PBP2, PBP3, PBP5, PBP4, LdtA, LdtD, and LdtF, respectively, in E. coli. In order to directly observe gene regulation under membrane stress, uhpA and VP_RS14060, which are homologous to the response regulator proteins rcsB and cpxR in E. coli, respectively, were selected. lpxA is a lipid A synthesis gene. β-lactams inhibit bacterial growth by binding to penicillin-binding proteins (PBPs) and interfering with PG synthesis [27]. According to the growth curve, bacteria in the initial exponential phase were used as the experiment sample. The ampicillin MIC value of ATCC33846 was 3.90625–15.625 μg·mL−1, while the MIC values of OMP mutants were above 600 μg·mL−1. Therefore, 16 μg·mL−1 ampicillin, which would not kill ATCC33846 and have a better effect on mutants, was set as the pretreatment.Table 3
Introduction of genes related to this work.
Gene namesDescriptionsVP_RS22195Hypothetical protein, 438 bp, chromosome 240.45% similar to murein lipoprotein Lpp inEscherichia coliVP_RS23020Maltoporin, 1311 bp, chromosome 272.36% similar to maltoporin inEscherichia coliVP_RS16800MipA/OmpV family protein, 781 bp, chromosome 2100% similar to MipA/OmpV family protein inEscherichia coliVP_RS16465OmpA family protein, 1080 bp, chromosome 228.77% similar to porin OmpA inEscherichia coliVP_RS20840OmpA family protein, 1318 bp, chromosome 229.62% similar to porin OmpA inEscherichia coliVP_RS03765OmpA family protein, 1019 bp, chromosome 132.5% similar to murein porin OmpA inEscherichia coliVP_RS11205OmpH family outer membrane protein, 465 bp, chromosome 136.42% similar to periplasmic chaperone Skp inE. coliVP_RS17515Carbenicillin-hydrolyzing class A beta-lactamase CARB-22Hydrolyzingβ-lactams [21]VP_RS13510PBP1A family penicillin-binding proteinPeptidoglycan synthase which is essential for cell elongation [22]mrcBPenicillin-binding protein 1BPeptidoglycan synthase which is essential for cell division [22]mrdAPenicillin-binding protein 2Transpeptidase essential for cell elongation [23]VP_RS02165Penicillin-binding protein 3Transpeptidase essential for cell division [23]VP_RS22785D-alanyl-D-alanine carboxypeptidaseD,D-carboxypeptidase which cleaves terminal D-ala in the peptidoglycan [24]dacBSerine-typeD-Ala-D-Ala carboxypeptidaseD,D-carboxypeptidase which cleaves terminal D-ala in the peptidoglycan [23]VP_RS22200l,D-transpeptidase family proteinAttaching Lpp to mDAP3 of peptidoglycan [23]VP_RS09310, VP_RS15980l,D-transpeptidase family proteinSynthesizing mDAP3–mDAP3cross-links in the peptidoglycan [23]uhpATranscriptional regulator, 29.06% similar to RcsB inE. coliRcsB is response regulator of Rcs sensing outer membrane stress [25]VP_RS14060Response regulator, 62.45% similar to CpxR inE. coliCpxR is response regulator of Cpx sensing inner membrane stress [26]lpxAAcyl-ACP-UDP-N-acetylglucosamine O-acyltransferaseEssential for the biosynthesis of lipid AFigure5 shows the transcription changes of 20 genes in mutants compared to ATCC33846. In ΔVP_RS23020 (Figure 5(b)), all genes exhibited transcriptional upregulation except for VP_RS11205. In ΔVP_RS16465 and ΔVP_RS20840 (Figures 5(d) and 5(e)), VP_RS23020 was upregulated over 9-fold. The transcription of VP_RS17515 was also upregulated over 9-fold in ΔVP_RS23020 but downregulated in ΔVP_RS22195, ΔVP_RS16800, ΔVP_RS20840, ΔVP_RS03765,and ΔVP_RS11205 (Figures 5(a), 5(c), 5(e)–5(g)). Most PG synthesis genes were downregulated in mutants except for ΔVP_RS23020. However, in ΔVP_RS23020, the transcription of VP_RS22200, VP_RS09310, and VP_RS15980 (Ldts) were all upregulated. Among PG synthesis genes, the transcriptional changes of VP_RS22785 and VP_RS09310 showed an interesting phenomenon. These two genes were downregulated over 2-fold in ΔVP_RS22195, ΔVP_RS16800, ΔVP_RS16465, and ΔVP_RS20840. In contrast, they were upregulated over 2-fold in ΔVP_RS23030 and ΔVP_RS11205, VP_RS22785, and VP_RS09310. In ΔVP_RS03765, these two genes showed no obvious downregulation compared to other genes. Moreover, VP_RS20840 showed the same trend as VP_RS22785 in all mutants. Moreover, the transcription of lpxA was downregulated in all mutants except for ΔVP_RS23020.Figure 5
Comparison of transcription of 20 genes in the 7V. parahaemolyticus OMP deletion mutants, using wild-type ATCC33846 as a control. Five kinds of genes are divided by dotted line: OMP genes, β-lactamase genes, PG synthesis-related genes, stress-regulation-related genes, and lipid A synthesis genes. VP_RS17515 expresses β-lactamase. The transcription change fold is calculated by 2−ΔΔCt. Each bar represents the mean value of three biological replicates, and error bars represent standard deviations calculated from three biological replicates.Figure6 displays the transcription changes of OMP genes of ATCC33846 and mutants under ampicillin stimulation compared to the untreated group. All OMP genes were downregulated in ΔVP_RS22195, ΔVP_RS23020, and ΔVP_RS20840 (Figures 6(b)6(c), and 6(f)). Furthermore, VP_RS16800 was downregulated over 2-fold in ATCC33846, ΔVP_RS23020, ΔVP_RS20840, and ΔVP_RS03765 (Figures 6(a), 6(c), 6(f), and 6(g)), and over 4-fold in ΔVP_RS22195 and ΔVP_RS11205 (Figures 6(b) and 6(h)). VP_RS17515 demonstrated over 2-fold upregulation in ATCC33846, ΔVP_RS16800, and ΔVP_RS16465 (Figures 6(a), 6(d), and 6(e)). In addition, transcription of PG synthesis genes showed significant changes in OMP deletion mutants but not after ampicillin stimulation. These findings implied that OMP deletion caused internal resistance to ampicillin instead of inducing a stress response with stimulation of ampicillin.Figure 6
Comparison of transcription of 20 genes in the 7V. parahaemolyticus OMP deletion mutants under stimulation of ampicillin (AMP), using wild-type ATCC33846 as a control. Five kinds of genes are divided by dotted line: OMP genes, β-lactamase genes, PG synthesis-related genes, stress-regulation-related genes, and lipid A synthesis genes. The transcription change fold is calculated by 2−ΔΔCt. Each bar represents the mean value of three biological replicates, and the error bars represent standard deviations calculated from three biological replicates.
(a)(b)(c)(d)(e)(f)(g)(h)


## 4. Discussion

The MIC change folds showed two major increases in resistance to aminoglycosides and ampicillin (Figure4), and the MIC value is shown in Table S1. The MIC values of 3 kinds of aminoglycosides (streptomycin, kanamycin, and gentamicin) increased in all 7 OMP mutant strains, indicating that the mechanism of aminoglycoside resistance could be related to OM. Aminoglycosides act on bacterial ribosomes and inhibit translation [28]. Moreover, aminoglycosides are polycationic at physiological pH and can replace divalent cations on lipopolysaccharide, thereby increasing membrane permeability [29]. However, the results of the MIC fold change of polymyxin B, which could also damage OM by replacing divalent cations on lipopolysaccharides, showed that not all the OM of mutants was more resistant to cationic antibiotics. In addition, after the deletion of OMP genes, the fold change of tobramycin’s MIC was lower than that of other aminoglycosides, and ΔVP_RS03765 was sensitive to tobramycin. Therefore, the aminoglycoside resistance mechanism remains unclear in OMP deletion mutants.A significant increase in ampicillin resistance was observed, with ATCC33846 showing a small MIC value (fluctuating 3.90625–15.625μg·mL−1). However, 10.6% of V. parahaemolyticus isolates from the coast of Korea were sensitive to ampicillin, while 87.2% were resistant [30]. It is speculated that the low ampicillin MIC of ATCC33846 was caused by repeated subculturing through different generations without antibiotics in the laboratory and an ATCC33846 sample at the early exponential phase. In addition, different CARB β-lactamases in various V. parahaemolyticus strains exhibit different ampicillin hydrolysis rates [31].Ampicillin resistance in different OMP mutants may result from reduced PG synthesis activity and expression ofβ-lactamase. PG synthesis can be affected by reduced transcription of PBPs, increased transcription of Ldts, downregulated D,d-carboxypeptidase, and alanine deficiency.Reduced transcription of PBPs was inferred to be one cause of ampicillin resistance. In ΔVP_RS22195, ΔVP_RS03765,and ΔVP_RS11205, PG synthesis-related genes were downregulated (Figures 5(a), 5(f), and 5(g)). VP_RS22195 is located adjacent to VP_RS22200 in the genome and has 40.45% protein similarity to the murein lipoprotein Lpp in E. coli (Table 3). Furthermore, the N-terminus of lipoprotein LpoB is required for the activation of PBP1B in E. coli [32]. Lipoprotein NlpI is a part of PG biosynthetic multienzyme complexes and acts as an adaptor [33]. In addition, transcription of uhpA in ΔVP_RS22195 was downregulated over 15-fold (Figure 5(a)). The lack of lipoprotein increases the periplasmic distance, and OM stress signals cannot be transmitted by the Rcs system [34]. PG synthesis was affected after the deletion of VP_RS22195, resulting in a slower division rate in the exponential phase than ATCC33846 (Figure 2(a)). Another cause might be the upregulated Ldts in ΔVP_RS23020. In a β-lactam-resistant mutant of Enterococcus faecium, Ldtfm was found to account for β-lactam resistance by using a different substrate from d,d-transpeptidase [6].ΔVP_RS16465 and ΔVP_RS20840 might exhibit ampicillin resistance due to the downregulated transcription of VP_RS22785 (Figures 5(d) and 5(e)), which encodes a D,d-carboxypeptidase cleaving pentapeptides into tetrapeptides [23]. The V. choleraed,d-endopeptidase ShyA could recognize but not cleave dimers containing pentapeptides [35]. Therefore, PG was protected from being cleaved by d,d-endopeptidases, and existed PG was maintained, reducing the activity of PG synthesis to resist ampicillin. In addition, with lower tetrapeptides, the transcription of VP_RS09310 was downregulated, which is homologous to l,D-transpeptidase LdtD cleaving tetrapeptide to form mDAP3–mDAP3cross-links (Table 3). However, the deletion of PBP5 in E. coli increased the sensitivity to β-lactams [36]. It is speculated that 3 homologs of PBP5 in V. parahaemolyticus and one of the downregulated homologs do not exert a significant impact on maintaining the normal cell shape. In addition, the OMP gene VP_RS20840 showed the same trend in transcriptional change as VP_RS22785 in all OMP mutants (Figure 5). This finding implies an unknown relationship between VP_RS22785 and VP_RS20840. In addition, alanine deficiency may be involved in increased ampicillin resistance. Previous research reported lysis in an E. coli strain lacking alanine racemase in the absence of D-ala, which is mainly caused by defects in PG synthesis [37]. OmpA was upregulated after the addition of alanine through analysis of proteomics and RT-qPCR [38]. It is speculated that lower levels of alanine entered the cell following OmpA deletion, which might further affect the transcription of PBP genes. VP_RS16465, VP_RS20840, and VP_RS03765 belong to the OmpA protein family (Table 3), while the different transcription trends of VP_RS22785 inferred different functions of OmpA.The existence and expression ofβ-lactamase were one of the causes of ampicillin resistance in V. parahaemolyticus. After ampicillin treatment, VP_RS17515 (β-lactamase) was upregulated in other strains except for ΔVP_RS23020 (Figure 6(c)). The same conclusion was obtained in V. parahaemolyticus V110 [7]. Although the transcription of VP_RS17515 was slightly downregulated in ΔVP_RS23020 under ampicillin stimulation (Figure 6(c)), it was upregulated more than 4-fold in ΔVP_RS23020 (Figure 5(b)). VP_RS23020 encodes maltoporin. In addition, the maltose metabolism pathway was potentially involved in the resistance to antibiotics that target cell wall biosynthesis. In a Lactococcus lactis strain resistant to lactococcin 972, which is a bacteriocin that inhibits cell wall biosynthesis by binding to lipid II, maltose metabolic genes were deleted. However, this strain showed no lactococcin 972 sensitivity in the maltose medium [39].Lipoprotein potentially affected OM biosynthesis through phospholipids. Transcription oflpxA was downregulated over 3.7-fold in ΔVP_RS22195 (Figure 5(a)), indicating that the lack of VP_RS22195 had an effect on OM synthesis. The maturity of lipoprotein is associated with phosphatidylglycerol [40]. Moreover, crosstalk between phospholipids and lipopolysaccharide synthesis was observed. LpxK catalyzes the synthesis of lipid IV A from lipid A disaccharide, which depends on the concentration of unsaturated fatty acids [41]. Furthermore, transcription of lpxA was downregulated over 2-fold in ΔVP_RS16800, ΔVP_RS20840, ΔVP_RS03765, and ΔVP_RS11205. Nevertheless, the relationship between these OMPs and OM synthesis remains unknown.


## 5. Conclusions

Deletion of OMP affects growth and OM permeation, and MIC and OMP mutants demonstrated significantly increased ampicillin resistance. Further RT-qPCR analysis showed several possible causes of ampicillin resistance in OMP mutants, including the expression ofβ-lactamase, the reduction of PG synthesis activity due to reduced transcription of PBPs, increased transcription of Ldts, downregulated D,D-carboxypeptidase, and alanine deficiency. This study provides a new perspective on ampicillin resistance in OMP mutants with respect to PG synthesis. Future work will focus on the role of OMPs in the synthesis of OM and PG.



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*Source: 8079091-2023-01-13.xml*