Regulation of mimicus metalloprotease (VMP) production by the quorum-sensing master regulatory protein, LuxR

El-Shaymaa Abdel-Sattar 1, 2, Shin-ichi Miyoshi 1 and Abdelaziz Elgaml 1, 3 *

1 Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University,

1-1-1, Tsushima-Naka, Kita-Ku, Okayama 700-8530, Japan

2 Chemical Industries Development Company, Assiut 71511, Egypt

3 Microbiology and Immunology Department, Faculty of Pharmacy, Mansoura University,

Elgomhouria Street, Mansoura 35516, Egypt

* Correspondence:

Corresponding author: Abdelaziz Elgaml

Postal address: Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,

Okayama University, 1-1-1, Tsushima-Naka, Kita-Ku, Okayama 700-8530, Japan

Tel: +81-86-251-7968, Fax: +81-86-251-7926

E-mail: [email protected] ; [email protected]

Running title: Regulation of V. mimicus metalloprotease by LuxR

Key words: Vibrio mimicus; Metalloprotease; Quorum-sensing; LuxR protein

1 Abstract

Vibrio mimicus is an estuarine bacterium, while it can cause severe diarrhea, wound infection and otitis media in humans. This pathogen secretes a relatively important toxin named V. mimicus metalloprotease (VMP). In this study, we clarified regulation of the VMP production according to the quorum-sensing master regulatory protein named LuxR. First, the full length of luxR gene, encoding LuxR, was detected in V. mimicus strain E-37, an environmental isolate. Next, the putative consensus binding sequence of LuxR protein could be detected in the upstream (promoter) region of VMP encoding gene, vmp. Finally, the effect of disruption of luxR gene on the expression of vmp and production of VMP was evaluated. Namely, the expression of vmp was significantly decreased by luxR disruption and the production of VMP was severely altered. Taken together, here we report that VMP production is under the positive regulation of the quorum-sensing master regulatory protein, LuxR.

2 Introduction

Vibrio mimicus is a gram-negative estuarine bacterium, while it is an important enteric human pathogen that can cause severe diarrhea after consumption of raw seafood, and is also associated with other extra-intestinal infections including wound infection and otitis media [1,

2].

Different kinds of virulent or toxic factors are associated with the virulence of V. mimicus including phospholipase, heat stable enterotoxin, toxin, hemolysin, hemagglutinin, and proteolytic enzymes [3]. Among these factors, the extracellular metalloprotease (V. mimicus protease: VMP) is relatively important [1, 4]. VMP is a member of the vibriolysin metalloproteases [4, 5], which exhibits two functions: proteolysis and haemagglutination [6,

7]. It has potential roles in the modulation of other virulence factors by limited proteolysis, enhancement of vascular permeability in the skin as well as in enterotoxigencity as revealed from its role in fluid accumulation in rabbit ileal loops [8, 9].

It is well established that Vibrio species, including V. mimicus, coordinate the expression of virulence genes and other community behaviors according to the quorum-sensing system, which is dependent on the bacterial population cell density [10-12]. Among Vibrio species, the quorum-sensing system of the luminescent bacterium, V. harveyi is considered as a standard model [10]. The quorum-sensing system of V. harveyi is mediated by three signaling molecules named auto-inducer 1 (AI-1), auto-inducer 2 (AI-2) and cholera auto-inducer 1

(CAI-1) [13-15]. V. harveyi possesses AI-1, AI-2 and CAI-1 synthases named LuxM, LuxS and CqsA, respectively. AI-1, AI-2 and CAI-1 membrane bound sensor proteins named LuxN,

LuxPQ and CqsS, respectively, are also present. The system also includes LuxU-LuxO (the central response regulators of the quorum-sensing circuit), and LuxR (the master regulatory protein of target genes controlled by the quorum-sensing system). Moreover, five small RNAs

(sRNAs) regulating LuxR are also present [13-16]. At low population cell density, the

3 signaling molecules are absent, and their sensor proteins act as kinases which add phosphate group to LuxU protein, which in turn transfers the phosphate group to LuxO. Consequently, at low cell density, LuxO presents in the phosphorylated active form of the protein, which acts together with the sigma factor 54 RpoN, sRNAs, sRNA binding protein (Hfq) to repress the production of the master regulatory protein, LuxR. On the other hand, at high population cell density, in the presence of sufficient concentration of the signaling molecules, they interact with their specific sensor proteins and change their function to phosphatases that act to dephosphorylate LuxO. The dephosphorylated form of LuxO is inactive form of the protein; therefore, it cannot inhibit LuxR anymore. Therefore, at high cell density, LuxR can exert its action to change the transcriptional status of the genes controlled by the quorum-sensing regulon [10, 13-16].

Previously our lab proved the presence of V. harveyi-like LuxS/AI-2 dependent quorum-sensing system in V. mimicus and clarified its role in VMP production through construction of disruptants of the genes encoding LuxS and LuxO, respectively [1]. Namely,

VMP production was decreased by luxS disruption, while it was increased by luxO disruption, indicating that VMP production is positively regulated according to the V. mimicus quorum-sensing system. However, it was not confirmed whether VMP production is regulated according to LuxR or not. Here, we report the evidence of the positive regulation of VMP production by the V. mimicus quorum-sensing system master regulatory protein, LuxR.

Materials and methods

Bacterial strains, plasmids and culture conditions:

Bacterial strains and plasmids used in the present study are listed in Table 1. Luria

Bertani (LB) medium containing 0.5% NaCl or 1.0% NaCl was used for cultivation of

4 Escherichia coli and V. mimicus strains, respectively. When required the media was supplemented with an appropriate antibiotic as follows: streptomycin 50 μg ml-1, kanamycin

50 μg ml-1 and chloramphenicol 10 μg ml-1.

Thiosulfate-citrate-bile-salt-sucrose (TCBS) medium supplemented with chloramphenicol 10 μg ml-1 was used to select the luxR mutant strain named LRD-37.

Heart infusion broth (HIB) was used for cultivation of V. mimicus strains during measurement of the vmp gene expression or assaying VMP activity.

Inverse PCR and sequencing for the detection of full length luxR gene

Inverse PCR was carried out as described previously [1]. Briefly, the chromosomal DNA of V. mimicus strain ES-37 was extracted as described previously [20]; then, it was digested with BanII, BglII, KpnI or EcoO1091 separately. Digested fragments were purified by using the gene clean turbo kit (Q-Bio gene, Carlsbad, CA, USA) according to the manufacturer’s manual, and set for self-ligation by using T-4 DNA ligase (TaKaRa Bio, Inc., Kyoto, Japan).

Thereafter, DNA was purified from the ligation mixture by ethanol precipitation, and used as template in the subsequent inverse PCR reaction.

Inverse PCR reactions were carried out using the primer set luxR-Inv (designed from the previously detected partial sequence of luxR gene, accession number AB232378) (Table 2).

The reaction mixtures composed of 2.5 U Ex Taq DNA polymerase (TaKaRa Bio, Inc., Kyoto,

Japan), 10 x Taq buffer with Mg2+, 0.2 mM dNTP, 10 pmols of each of the forward and reverse primers, 150 ng of the template DNA, and the reaction volume was adjusted to 50 μl using nuclease free water. The PCR program consisted of 35 cycles of denaturation at 95°C for 45 sec, annealing at 62°C for 1 min and extension at 72°C for 7 min. After PCR, electrophoresis in 1.0% agarose gel was carried out to detect the products. Then, the amplified products were purified, and their sequences were determined by using Big Dye TM Terminator

5 Cycle Sequencing Ready Reaction Kit (ABI PRISM, Foster City, Calif., U.S.A.) according to the manufacturer’s manual. Samples were analyzed by the ABI PRISMTM 310 Genetic

Analyzer. Two new primer sets, luxR-Full and luxR-ORF (Table 2) designed from the nucleotide sequences of the inverse PCR products, were used for amplification and sequencing of a 812 bp region of DNA that involve the full length open reading frame of luxR gene, as well as a considerable amount of upstream and downstream regions.

BLAST tool of National Center for Biotechnology and Information was used to search for similar sequences, and Genetxy–Mac (version 9.0) soft ware (Genetxy, Tokyo) was used to identify the promoter and terminator.

Sequencing of the promoter region of vmp gene for detection of the LuxR binding consensus sequence

The upstream (promoter) region of vmp gene was sequenced by the method as already described above using the primer vmp-Ri2 (Table 2). Thereafter, the obtained sequence was analyzed, using Genetxy–Mac (version 9.0) soft ware (Genetxy, Tokyo), for the determination of the transcriptional starting site, putative promoter regions (-10 and -35), start codon (ATG), as well as the binding consensus sequence of the LuxR protein as reported previously [21-25].

Construction of the luxR disruptant

The luxR mutant was constructed by the single crossover homologous recombination as described previously [26]. An 529 bp region of luxR was amplified by PCR using the primer set luxR-Mut, the forward and reverse primers containing the recognition sequences for KpnI

(GGTACC) and SacI (GAGCTC), respectively (Table 2). The PCR product was digested with

KpnI and SacI, and inserted into similarly digested suicide vector pKTN701 [19]. The hybrid plasmid, thus obtained, was transformed into E. coli strain SY327λpir, then into E. coli strain

6 SM10λpir. Thereafter, it was transferred to V. mimicus strain ES-37 by conjugation, and the conjugates were cultivated on TCBS agar plates supplemented with chloramphenicol for

24-48 h at 37°C. One suitable luxR mutant named strain LRD-37 was selected, and the gene disruption was confirmed by PCR.

Measurement of bacterial growth

The growth curves of V. mimicus strains were obtained by measuring the optical density of the bacterial cultures, grown at 37°C in HIB medium under aeration with shaking, at 600 nm (OD600) at time intervals.

Reverse transcription (RT-PCR) for measuring the expression of vmp gene

The total RNA of the bacterial cells, cultivated at 37°C, was extracted at early log and early stationary phases of the bacterial growth using RNeasy Mini Kit (Qiagen GmbH, Hilden,

Germany) according to the manufacturer’s manual. Total RNA thus obtained was added to the

Ready-To-Go RTPCR kit (GE Healthcare Bio-science, Buckinghamshire, UK) and incubated at 42°C for 30 min for reverse transcription reaction. Then, the reaction was stopped by heating at 95°C for 5 min to inactivate the reverse transcriptase. Thereafter, PCR amplification with the primer set vmp-Exp (Table 2) was performed. The PCR program consisted of 32 cycles of 30 s denaturation at 95°C, 30 s annealing at 50°C, and 60 s extension at 72°C. The PCR products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide. In these experiments, the house keeping gene mdh was used as an internal control and the RT reaction without the reverse transcriptase was used as a negative control.

Assay of VMP activity

7 Quantitative assay of VMP was performed using azocasein (Sigma-Aldrich, St. Louis,

MO, USA) as described previously [27]. Briefly, the samples were allowed to act on 1.0 mg of azocasein [in 0.6 ml of 50 mM Tris-HCl buffer (pH 8.0)] at 30°C for an appropriate time.

Thereafter, 1.4 ml of 5% trichloroacetic acid was added to stop the reaction and the samples were centrifuged. An aliquot of the supernatant was withdrawn, mixed with the same volume of 0.5 M NaOH, and the absorbance at 440 nm was measured. The amount of VMP capable of hydrolyzing 1 µg of the azocasein in 1 min was defined as one protease unit (PU). In these experiments, sterile HIB medium was used as negative control.

Nucleotide sequence accession numbers

The sequences containing the full length luxR gene and promoter region of vmp gene, obtained in this study, were submitted to DDBJ/EMBL/GenBank database and assigned with the accession numbers AB539839 and LC099949, respectively.

RESULTS

Detection and full length sequencing of luxR gene:

By inverse PCR and sequencing, an 812 bp DNA segment involving the full length open reading frame (ORF) of luxR gene (612 bp) and other regulatory regions could be detected

(Fig. 1). The obtained sequence of luxR ORF had 76% and 86% similarity with sequence of the corresponding gene of V. harveyi and V. cholera, respectively.

Next, in order to clarify whether the LuxR takes part in regulation of vmp expression and

VMP production in V. mimicus, the upstream (promoter) region of vmp gene was sequenced and searched for the LuxR binding consensus sequence.

8 Upstream (promoter) region of vmp gene:

As seen in Fig. 2, by sequencing the promoter region of vmp gene, the binding consensus sequence of LuxR could be detected. Moreover, the transcriptional starting site, putative promoter regions and start codon were assigned.

These results suggest that the master regulatory protein of the quorum-sensing system,

LuxR, take part in the regulation of production of the VMP in V. mimicus through the regulation of the expression of vmp gene. To confirm this hypothesis, we constructed a luxR mutant and studied effect of luxR disruption on transcription of vmp and production of VMP.

Effect of luxR disruption:

To analyze the effect of LuxR on the vmp expression and VMP production, the luxR mutant strain LRD-37 was constructed from its wild type strain ES-37. Then, ES-37 and

LRD-37 were compared for vmp expression and VMP production. The transcription level of vmp gene was severely decreased by the luxR disruption. The vmp mRNA could not be detected in both wild type and mutant strains during the early log phase of bacterial growth

(data not shown), however, at early stationary phase, it could be detected in ES-37 and could not be detected in LRD-37 (Fig. 3A). Moreover, the VMP production was significantly altered by luxR mutation. There was no significant difference in VMP production between ES-37 and

LRD-37 during early stages of the bacterial growth; though, this difference became evident during late stages of the growth. (Fig. 3B).

These results confirm that VMP production in V. mimicus is under positive regulation of the quorum sensing system master regulatory protein, LuxR.

Discussion

9 In pathogenic , including V. mimicus, the coordinated regulation of the virulence gene expression is a critical key factor to successful colonization, invasion, in vivo growth and/or in situ toxin production [11, 28]. One of the most important regulatory cascades involved in this regulation is the cell to cell communication system called quorum-sensing.

The quorum-sensing pathway among Vibrio species controls the expression of the master regulatory protein named LuxR, which in turn regulates majority of the virulence factor production and other community behaviors controlled by this system [10]. However, minority of these factors and behaviors may escape LuxR regulation and controlled through other non-LuxR pathway [25, 26, 29-31].

During V. mimicus infection, the bacterium is known to produce an important vibriolysin metalloprotease termed VMP. The importance of VMP is regarded to its direct toxic effects as well as indirect roles in processing other protein toxins [1, 4-9]. Previously, our lab confirmed the presence of quorum-sensing regulon in V. mimicus and clarified its role in the regulation of production of the VMP, one of the relatively important virulence factors of the pathogen [1].

However, it was not clarified whether VMP production is under the control of LuxR or not. In order to complete this missing ring, in the present study, the regulation of VMP production according to the LuxR was considered. To fulfill with the role of LuxR in the regulation of

VMP production, the full length sequence of luxR gene of V. mimicus strain ES-37, an environmental isolate, was detected. The detected sequence had a high level of identity with the corresponding quorum-sensing master regulatory protein genes of other Vibrio species.

Moreover, the promoter region of vmp gene was found to have a considerable putative consensus binding sequence of LuxR as those previously reported [21-25]. These findings suggest that LuxR regulates the VMP production at the transcriptional level. Disruption of luxR gene resulted in a significant decrease of vmp expression and VMP production, confirming that LuxR is a positive transcriptional regulator of the VMP production. This

10 regulatory pattern of VMP production by V. mimicus is similar to other metalloproteases from the vibriolysin family including those of V. cholerae and V. vulnificus [32, 33].

Taken together, it is concluded that VMP production by V. mimicus is positively regulated by the quorum-sensing system through the transcriptional activation of the master regulatory protein, LuxR. This reflects the significant role of quorum sensing system during V. mimicus infection. Namely quorum sensing system controls the production of VMP, which in turn enhances the vascular permeability, controls the enterotoxigencity as well as modulates other virulence factors by limited proteolysis.

Acknowledgments

This study was supported by a grant from the Program of Japan Initiative for Global Research

Network on Infectious Diseases (J-GRID), the Ministry of Education, Culture, Sports,

Science and Technology in Japan.

Conflict of interest

The authors declare no conflict of interest in the present publication.

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15 Table headings

Table 1. Bacterial strains and plasmids used.

Table 2. Oligonucleotide primers used.

16 Figure legends

Figure 1. Complete sequence of the luxR gene of V. mimicus strain ES-37. The sequence contains the 612 bp luxR open reading frame (letters highlighted in grey color), putative promoter sequences (bold single underlined letters), start and stop codons (bold double underlined letters) and putative terminator (bold dotted underlined letters).

Figure 2. Sequence of the upstream (promoter) region of the vmp gene of V. mimicus strain

ES-37. The transcriptional starting site, +1 (bold dotted underlined letter), putative promoter regions (bold single underlined letters), and start codon (bold double underlined letters) are indicated. The putative consensus binding sequence of LuxR is highlighted in grey color.

Figure 3. Effect of luxR disruption on the expression of vmp (A) and production of VMP (B).

(A) Strain ES-37 and LRD-37 were cultivated in HIB broth at 37°C, total RNA was extracted at early stationary phase, and the level of mRNA was measured by RT-PCR. Thereafter, PCR products were electrophresed on 1.5% agarose gel, visualized by staining with ethidium bromide. Lane M; 1 kb DNA ladder, lane 1; strain ES-37, lane 2; strain LRD-37. In these experiments, the house keeping gene mdh was used as an internal control and the RT reaction without the reverse transcriptase was used as a negative control. (B) Culture supernatants were collected at indicated cultivation times (8 to 32 h) of growth of strains ES-37 (dotted prism) and LRD-37 (black prism) cultivated in HIB broth at 37°C. The proteolytic activity

(PU/ml) of the culture supernatants was measured using azocasein as a substrate. Specific protease activity (PU/OD600) was calculated. In these experiments, the cultivation medium

(HIB) was used as a negative control and showed no activity.

17 Fig. 1. Full length sequence of the luxR gene

-35 -10 1 CACAATTTTCACCCAACAAAGATTGACCTCACTCATATGCACCATTACACTCATAGGGCT 60

61 TTAAATAGCAAATAACAAAATAATCATTAGAGCAAAATGCTCAAACAACTCAATTTGGCA 120

121 AGGATATTTACCTATGGACGCATCAATCGAAAAACGCCCTCGGACTCGGCTATCTCCACA 180

181 AAAACGTAAACTACAACTGATGGAAATCGCATTAGAAGTGTTTGCAACTCGCGGCATTGG 240

241 CCGCGGCGGTCATGCCGACATCGCTGAAATTGCGCAAGTTTCAGTAGCGACAGTATTTAA 300

301 CTACTTCCCAACTCGTGAAGATCTGGTCGACGACGTACTGAACTTTGTAGTTCGTCAGTA 360

361 TTCCAACTTCCTCACTGATCACATCGATCTAGATTTAGAAGCTAAAAGCAACCTACAAAC 420

421 ACTGTGCAAAGAAATGGTGAAACTCGCCATGACTGACTGCCATTGGCTAAAAGTATGGTT 480

481 TGAATGGAGTGCTTCGACTCGTGATGAAGTTTGGCCGTTGTTTGTTTCGACCAACCGCAC 540

541 CAACCAGTTACTGATCAAAAACATGTTCATCAAAGCGATGGAGCGTGGTGAATTGTGTGA 600

601 GAAACACGATGTAGACAATATGGCGAGCCTGTTCCACGGCATCTTCTACTCTATCTTCCT 660

661 ACAAGTAAATCGCTTGGGCGAACAGGAAGCAGTGTATAAGTTAGTCGATAGCTATCTCAA 720

721 CATGCTGTGTATCTACAAGAACTAGTTTCTAAGGCAGTTTTAAGGGCGCATTAAGCGCCC 780

781 TTTTTATTTCCATACTCAAAGAAACTGCACTC 812

1 Fig. 2. Sequence of upstream (promoter) region of the vmp gene

1 TGTGTGGTATGCCAATATTGAAACGACGGCAACCTACAAAGCGGGTGCTGATGCCAAATC 60

61 TACCGATGTGAAAATTAATCCATGGGTATTTATGATCGCTGGTGGTTATAAGTTCTAAAC 120

121 CCGTTTGAATTAAGCTAAGCCGCTCAATGAGCGGCTTTGTTTTCTCTCATTTTTCAATGT 180

181 GATATCCCTCCAATTTCACGGCTGCGCTTTCTTTTCTCTTCATTATTGCCGTAGGGTGTT 240

241 TATTTAAAAAGACGCAAATTTATCAATAGAAAATTATCGACCTGATTTATAAAGCTTTAA 300

301 ATTTTCTGTTCATGTGTGGCACTTGGGCTTAATGAGTAGTTTGTGACACGTAAACTTCAC 360

-35 -10 361 ACCCAGCATTTACTTTTTTTTAACCCAGCTTTGGCGTTTTCAATTTATCTGTACCACCTT 420

+1 421 GAGTGGACACGTAATTTTACGTGCAATCGGCGTTGATAGGAGCGGTTCAACGCTTAATAA 480

481 ACTCAACATCTCTAGGATTGAGAAATG 507

1 Fig. 3. Effect of luxR disruption on the vmp expression (A) and VMP production (B)

A

vmpvmp mRNAmRNA (497 (497 bp) bp ) mdhmdh mRNAmRNA (354 (354 bp) bp )

MM 1 2 1 3 2

B

) 120 600 ES-37 100 LRD-37

80

60

40

20

Specific proteolyticSpecificactivity(PUOD / 0

8 16 24 32 Cultivation time (h)

1 Table 1. Bacterial strains and plasmids used.

Strain or plasmid Relevant features a Reference Vibrio mimicus ES-37 Environmental isolate [7] LRD-37 ES-37 strain, luxR::Cmr This study

Escherichia coli △(lac pro), argE(Am), rif, nalA, recA56, [17] SY327λpir rpoB, λpir, Smr, host for π-requiring plasmids. thi-1, thr, leu, tonA, lacY, supE, SM10λpir recA::RP4-2-Tc::Mu, λpir, oriT of RP4, [18] Kmr; conjugational donor.

Plasmids R6K-ori suicide vector for gene pKTN701 [19] replacement; Cmr. pKVmluxR pKTN701 with luxR; Cmr. This study Cmr: chloramphenicol-resistant, Smr: streptomycin-resistant, Kmr: kanamycin-resistant.

1

Table 2. Oligonucleotide primers used.

Accession Gene Nucleotide sequence 5’~ 3’ Position number

luxR-Inv AB232378 Forward CAACCGCACCAACCAGTTACTG 370--391 Reverse CAAACAACGGCCAAACTTCATC 399--420 a luxR-Full This study Forward TGCACCATTACACTCATAGGG (-76)--(-93) Reverse GAGTGCAGTTTCTTTGAGTATGG 657--679 a luxR-ORF This study Forward TTACCTATGGACGCATCAATC (-6)--15 Reverse GTTCTTGTAGATACACAGCATGTTG 585--609 b luxR-Mut AB539839 Forward GCGGTACCGATGGAAATCGCATTAGAAGTG 66--87 Reverse GCGCGAGCTCTATCGACTAACTTATACACTG 557--578 mdh AY800095 Forward ATATCGCGCCTGTCACTCCTG 17–37 Reverse TCACATCCAGAGYCGYYACACC 349–370 vmp-Ri2 AB435238 Reverse GTTGTTGTGCAAGCGCTTGTTC 109–130 vmp-Exp AB435238 Forward CCTCTAGTGGATATTAACGTG 1009–1029 Reverse TTCACACCGACTGTGTTAAACG 1484–1505 a The primers were designed from the nucleotide sequences of the inverse PCR products of luxR gene obtained in this study. b The artificial restriction enzyme site is indicated by the underline.

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