Flagellar motility contributes to the invasion and survival of Aeromonas hydrophila in Anguilla japonica macrophages

Accepted Manuscript Flagellar motility contributes to the invasion and survival of Aeromonas hydrophila in Anguilla japonica macrophages Yingxue Qin , Guifang Lin , Wenbo Chen , Bei Huang , Wenshu Huang , Qingpi Yan PII:

S1050-4648(14)00165-X

DOI:

10.1016/j.fsi.2014.05.016

Reference:

YFSIM 2988

To appear in:

Fish and Shellfish Immunology

Received Date: 31 December 2013 Revised Date:

3 May 2014

Accepted Date: 13 May 2014

Please cite this article as: Qin Y, Lin G, Chen W, Huang B, Huang W, Yan Q, Flagellar motility contributes to the invasion and survival of Aeromonas hydrophila in Anguilla japonica macrophages, Fish and Shellfish Immunology (2014), doi: 10.1016/j.fsi.2014.05.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Flagellar motility contributes to the invasion and survival of Aeromonas hydrophila in Anguilla japonica macrophages

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Yingxue Qin, Guifang Lin, Wenbo Chen, Bei Huang, Wenshu Huang, Qingpi Yan∗ Fisheries College, Key Laboratory of Science and Technology for Aquaculture and Food Safety, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, P.R.China, Jimei University, Xiamen, Fujian 361021, PR China

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Abstract: The interaction between pathogenic bacteria and the host phagocytes is complicated. It is generally believed that only obligate intracellular pathogens can invade and survive in host phagocytes. In this study, we revealed that the pathogenic Aeromonas hydrophila

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B11 can also invade and survive in the macrophages of its host Anguilla japonica i n vitro. To further investigate the mechanisms of A. hydrophila invasion and survival in host macrophages, a mini-Tn10 transposon mutagenesis system was used to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOFKm) and the recipient Aeromonas hydrophila B11. Out of 465 individual colonies, 13 mutants impaired in survival within macrophages were selected, and the mutant BM116 was the most seriously impaired strain. Molecular analysis showed that an ORF of approximately 1335 bp GenBank accession numbers JQ974982 of the mutant BM116 was inserted by mini-Tn10. This ORF putatively encodes a deduced 445 amino acids protein that displays the highest identity (99.6%) with the flagellar hook protein FlgE of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type B11, the mutant B116 and the complemented strain were investigated. The results reveal that the flagella of the mutant BM116 was absent and that these mutant bacteria exhibited defective motility, adhesion, and invasion and survival in host macrophages when compared with the wild type and the complemented strain. These findings indicate that flgE is required for flagellum biogenesis in A. hydrophila and that flagellar motility is required for A. hydrophila invasion and survival in the macrophages of its host. Our findings provide an important new understanding of the nonintracellular pathogenic bacteria invasion and survival in host phagocytes and the interactions between the pathogens and their host. Key words: Aeromonas hydrophila; survival; phagocytes; flagella 1 Introduction Many microbes have developed not only invasion strategies to enter the host but also strategies to evade host immunity. Studies have suggested that one of the ideal strategies to escape from the host immune system is to choose the professional phagocytes as habitats [1,2,3]. Certain bacterial factors of Helicobacter pylori, such as catalase, ClpP ATP-dependent caseinolytic protease and its chaperone CloA, and RuvC Holliday junction resolvase, have been implicated in H. pylori avoidance and the destructive effects of reactive oxygen species (ROS), which aid in H. pylori survival and its long-term ∗ Corresponding author, Tel.:+86 592 6183028; Fax: +86 592 6181476. E-mail address: [email protected]( Q.-P. Yan).

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persistence in host macrophages and also lead to chronic inflammation, gastric or duodenal ulcers and even gastric cancer [4]. Legionella pneumophila, Coxiella burnetii and Leishmania spp. employ the strategy of differentiating from a transmissive form that inhibits the phagosome-lysosome fusion for several hours to a cell type fit for intracellular replication to exploit macrophages as a replication niche [5]. Staphylococcus aureus, a bacteria that has not been traditionally considered among the bona fide intracellular pathogens, has also been convincingly shown to invade and persist for varying lengths of time in many cell types and even survive in professional phagocytes [1]. It could be deduced that the functions required to interact with the host cell and to enhance survival or replication in professional phagocytes are important virulence factors of pathogens. The survival mechanisms of many clinical pathogens have been illuminated, but only a few fish pathogens have been reported to resist killing by phagocytes, and fewer have been demonstrated to survive and persist in phagocytes [6,7,8]. Piscirickettsia salmonis had been confirmed to be capable of surviving and replicating in rainbow trout macrophages [7]. Yersinia ruckeri was also found to be able to survive in vitro inside trout macrophages for at least 24 h, and transmission electron microscopy further demonstrated that Y. ruckeri bacteria were sequestered in autophagocytic compartments without fusion with primary lysosomes [8]. However, hardly any fish pathogens have been studied to uncover the mechanisms of their survival in phagocytes. Aeromonas hydrophila is a widespread representative of Aeromonas found in water, water habitants, domestic animals and foods (fish, shellfish, poultry, and raw meat) [9]. It is not only a common pathogen involved in heavy mortalities in farmed and feral fish [10,11,12] but also one of the causative agents of gastrointestinal and extraintestinal infections in humans [13,14]. Though many virulence determinants (such as haemolysins, proteases, enterotoxins and so on) have been known to bestow upon A. hydrophila the ability to cause disease, little is known about whether A. hydrophila can survive in phagocytes and what is the mechanism for its survival in phagocytes. The aim of this study is to investigate whether A. hydrophila can invade and survive in the macrophages of Anguilla japonica and, if so, to investigate possible mechanisms by which the bacteria avoid destruction.

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2 Materials and methods 2.1 Bacterial strains and growth conditions The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli was grown at 37°C in LB. A. hydrophila grown at 28°C in trypticase soy agar (TSA). The bacteria were harvested and resuspended in phosphate-buffered saline (PBS, pH 7.4) after overnight incubation. The density of bacterial suspension was adjusted according to the OD550. The medium was supplemented with the appropriate antibiotics at the following concentrations: 100 µg/ml kanamycin (Km); 50 µg/ml ampicillin (Ap) and 50 µg/ml streptomycin (Sm); 25 µg/mL chloromycetin (Cm). Table 1 Strains and plasmids used in this study. Strain or plasmid Strains

Characteristic(s)

Source or reference

ACCEPTED MANUSCRIPT A. hydrophila B11 BM01~BM459 BM116 BM116(flgE+)

wild-type strain (SmR)

[15] R

R

mini-Tn10Km insertion mutant (Sm Km ) R

This study

R

flgE :: mini - Tn10Km(Sm Km )

This study R

R

r

BM116 complemented with pACYC184-flgE(Sm Km Cm )

This study

Escherichia coli thi thr leu tonA lacY supE recA RP4-2-Tc::Mu::Km (λpir)

[16]

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SM10

-

F , φ 80dlacZ ∆M15, ∆(lacZYA-argF )U169, deoR , recA1

endA1 , hsdR17 (rK-, mK+), phoA, supE44 , λ-, thi -1, gyrA96 , relA1

Plasmid pMD18-T pLOF/Km pACYC184 pACYC184-flgE

Cloning vector(ApR)

Takara

R

R

Tnl0-based delivery plasmid with(Km Ap ); R

Takara

R

(Cm Tc )

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E.coli DH5α

[16]

provided by Prof. Nie

pACYC184 derivative containing 1461 bp fragment of flgE R

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putative promoter and ORF(Cm )

This study

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2.2 Preparation of A. japonica macrophage suspension Healthy A. japonica (376.2±31.4 g) individuals were obtained from an aquaculture farm. A. japonica macrophages were prepared as previously described [7,17]. Briefly, fish were anaesthetised with 4-ethyl-amino-benzocaine, and the head-kidneys were removed and pooled under aseptic conditions. The tissues were then pushed through a 100 µm nylon mesh and suspended in L-15 medium (Biological Industries, Israel) supplemented with 10 IU/ml heparin and 100 IU streptomycin/penicillin (S/P)/ml and 2% foetal calf serum (FCS). The cell suspension was layered onto a 34%/51% discontinuous Percoll (Amersham Pharmacia Biotech, UK) density gradient with a syringe and centrifuged at 400×g for 30 min at 4°C. The band of cells in the layer above the 34%/51% interface was collected, washed twice and resuspended with L-15 medium with 10% FCS, 100IU S/P/ml and 10 IU heparin/ml. Then, the cells were incubated at 28°C. After 4 h, non-adherent cells were removed by washing with L-15 and monolayers were collected. Next, the cell suspension was adjusted to ~2×107 cells/ml in L-15 medium with 10% FCS, 100IU S/P/ml and 10 IU heparin/ml and transferred to 6-well plates at 1 ml/well. 2.3 A. hydrophila invasion and survival in macrophages in vitro In vitro bacterial invasion was performed as described by Larsen et al. [18] with some modifications. Briefly, this assay was performed according to the following steps: (I) 1ml of the macrophage suspension was added to each well of 6-well culture plates and incubated for 2 h. After that, 1 ml of the bacterial suspension [multiplicity of infection (MOI) = 100 (100 bacteria per macrophage added) was added to each well and incubated at 28°C for 1 h. (II) After bacteria invasion, the macrophages were pooled in sterile tubes and centrifuged at 100×g for 5 min at 28°C, and the supernatant was carefully removed without disturbing the packed cells. After washing the packed cells twice with cold PBS, the cells were resuspended in 2 ml PBS. (III) The cell suspensions were treated with 250 µg/mL gentamycin for 20 min at 4°C to eliminate extracellular bacteria and were then washed twice. The supernatant fluid was withdrawn and tested for sterility by the plate counting method. The packed cells were resuspended in fresh L-15 medium with 10

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IU heparin/ml, 10% FCS and 100 units S/P. (IV). The cell suspension was incubated at 28°C in 5% CO2 for 0 h, 1 h, 2 h, 4 h, 12 h, and 24 h. After incubation, the cells were centrifuged for 5 min at 100×g at 28°C, the supernatant was aspirated, and 1 ml of sterile distilled water was added for 30 min to lyse the cells. The CFU value for the cell lysate was determined by plate counting [7,8]. 2.4 Mutagenesis of A. hydrophila strain B11 and isolation of mutants defective in survival within macrophages The mini-Tn10Km transposon was introduced into A. hydrophila strain B11 on the suicide vector pLOFKm carried by E. coli Sm10 through a filter mating technique developed by Herrero et al. [16] with minor modifications. Briefly, 0.22-µm filters with a 1:4 mixture of the donor strain E. coli Sm10 (pLOFKm) carrying the mini-Tn10Km and the recipient strain A. hydrophila were incubated for 4 h at 28°C on TSA plates supplemented with 3 mM IPTG. The filters were transferred into Eppendorf tubes containing 1 ml TSB and vortexed. Then, 100 µl of the suspension was spread on TSA plates supplemented with Km100 and Sm50 to select for A. hydrophila strains carrying the mini-Tn10Km transposon. Following incubation at 28°C for 24 h, single colonies were selected for analysis of the intracellular survival of the A. hydrophila mutants according to the assay described above except that the incubation time in the last step was 1 h. 2.5 Southern blot Southern blots were performed according to the procedure described by Rock and Nelson [19] with some modifications. Briefly, total genomic DNA was extracted from A. hydrophila B11 and the mutants using a bacterial genomic DNA extraction kit (Takara, Japan). Genomic DNA was digested with the SacI (Takara) restriction endonuclease which does not cut within the transposon, and electrophoresed on a 0.8% agarose gel in Tris-acetate-EDTA (TAE) buffer. The DNA was then transferred to a nylon membrane as described [20]. Blots were probed with a digoxigenin (DIG)-dUTP-labeled probe (Roche) for the mini-Tn10Km transposon. The KmR gene probe was created by PCR amplification of a 176-bp region of the KmR gene using the primers Km3 (5’-CGGGGATCGCAGTGG-3’) and Km4 (5’-TGGG AAGCCCGATGC-3’) and a DIG-PCR probe synthesis kit (Roche). After hybridisation at 42°C for 16 h, the membrane was washed and immunological detection was observed using a DIG Detection Kit (Roche). We expected to detect single bands for mutants with a single transposon insertion.

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2.6 Molecular analysis of the mutants The DNA sequence flanking the transposon mutants was determined using TAIL-PCR [21]. The arbitrary primer was supplied by the Genomic Walking Kit (Takara, Japan), the nested primers specific to the transposon are listed in Table 2. Among the nested primers, LSP4, LSP5 and LSP6 were used to amplify upstream sequences flanking the transposon, and RSP4, RSP5 and RSP6 were used to amplify the downstream sequences flanking transposon. TAIL-PCR products were purified using a Gel Extraction Kit (Omega, USA), after which they were cloned into pMD18-T (Takara, Japan) and sequenced. DNA sequences were analysed by BLASTN and other softwares such as ClustalW and MegAlign (DNAStar).

Table 2 Primer LSP4 LSP5 LSP6 RSP4

The specific primers used in TAIL-PCR

Sequence 5' ATGCTTGATGGTCGGAAGAGGC 3' 5' CATCGGGCTTCCCATACAATCG 3' 5' ATTATCGCGAGCCCATTTATACCC 3' 5' CCTGTTGAACAAGTCTGGAAAGAAATG 3'

ACCEPTED MANUSCRIPT RSP5 RSP6

5' GATCTTGCCATCCTATGGAACTG 3' 5' TTACGCTGACTTGACGGGACGG 3'

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2.7 Complementation and biological characteristics analysis The flgE gene of B11 was amplified by the primers flgE-EcoRV-for: GATATC TGGAAGG TGACCAGAAGGGCAACG and flgE-2HA-SphI-rev: GCATGC TTACTAGAGGCTAGCATAA TCAGGAACATCATACGGATAACGGATCTGCAGTATGGTCTGC (enzyme restriction sites are underlined, and the HA-tag sequence is italic). The PCR product was digested with EcoR V and Sph I (Takara). The fragment was ligated into pACYC184 (and also digested with EcoR V and Sph I) using T4 DNA ligase (Takara) to yield pACYC184-flgE. pACYC184-flgE was introduced into the mutant BM116 by electroporation to complement the flgE mutation. The complemented strain BM116 (flgE+) was selected on chloromycetin plates and FlgE protein expression was detected by Western blot with an anti-HA tag antibody according to the method described by Sambrook et al. [20]. Briefly, the bacterial cells were suspended in electrophoresis sample buffer and heated at 100 for 5min. 20 µl lysates of each sample were separated in a 12% gel. The gel was transferred to PDVF membrane (GE Healthcare). The membrane was blocked with 1% skim milk in PBS, incubated with a 1:3000 dilution of anti-HA antibody (Sigma). After washing, the membrane was incubated with a 1:5000 dilution of goat anti-rabbit horseradish peroxidase conjugated secondary antibody (Sigma) and then revealed using the enhanced chemiluminescence system (LAS-4000mini, Fuji, Japan). Formvar-coated grids were floated on 20 µl drops of bacterial cell suspensions. Excess sample was withdrawn by touching the edge of the grid to a cut edge of Whatman filter paper. The grids were negatively stained with a 2% solution of uranyl acetate and observed with a Tecnai F20 (Philips) transmission electron microscope. The motility of wild-type strain B11 and mutant BM116 and complementation were tested by the following assay. Single colonies of bacteria were stabbed into test tubes filled with semi-soft medium and incubated at 28°C for ~10 h to observe their motility. Bacterial adhesion assays were performed as described previously by Balebona et al. [22] and Vesterulund et al. [23] with some modification. In brief, bacteria suspendsions were adjusted to an OD550 = 0.30 (≈108 cfu/mL) in PBS and 50 µl of the bacteria suspendsions was added to microscope slides covered with mucus. Microscope slides were incubated in a humidified chamber at 28°C for 1 h. Nonadherent bacteria were washed away by dipping the slides into PBS. The slides were air-dried at 28°C for 20 min and fixed with absolute methyl alcohol at 28°C for 20 min and then stained with crystal violet for 2 min, washed and air-dried. The slides were examined by light microscope. The average number of bacteria adhering to 1 mm2 of the glass surface was then determined. For each assay, 10 such 1- mm2 areas were counted and the average value was calculated. Bacterial invasion and survival in macrophages in vitro was performed as described above except that in the III step the cell suspension was incubated at 28°C in 5% CO2 for 0 h and 1 h. The results at 0 h represent the number of invasive bacteria in macrophages, and the results at 1 h indicate the number of surviving bacteria at 1 h after entry into the macrophages. 2.8 Statistical analysis All data were statistically analysed with SPSS16.0. The mean ± standard deviation was

ACCEPTED MANUSCRIPT calculated for each sample. Unless otherwise stated, all experiments were performed at least three times in triplicate assays. Significant differences between the means were determined by analysis of variance (ANOVA). 3 Results 3.1 A. hydrophila B11 invade and survive in macrophages The results in Fig. 1 show that ~2.0×10 CFU/ml bacteria invaded into the macrophages 5 after 1 h co-incubation and that ~1.5×10 CFU/ml bacteria of A. hydrophila B11 survived in the macrophages for 1 h. A total of ~28% and ~2.1% of the invading bacteria survived in the macrophages at 12 h and 24 h after entering the cells, respectively. These results indicated that A. hydrophila B11 can invade into macrophages and that some of the invading bacteria can persist in the cells for at least 24 h.

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Fig. 1 A. hydrophila B11 invade and survive in macrophages after phagocytosis. Values denoted by different letters are significantly different when compared by ANOVA (P<0.05) 3.2 Isolation of mutants defective in survival within macrophages To elucidate the mechanisms of A. hydrophila survival in macrophages, the mini-Tn10Km transposon was introduced on a suicide plasmid pLOFKm into A. hydrophila B11 to construct a mutagenesis library. Transposants were selected on TSA containing 100 µg/ml Km and 50 µg/ml Sm. Out of 465 individual colonies screened, 13 mutants were identified to be defective in survival within macrophages when compared with the wild-type strain (P<0.01) (Fig. 2). Only 12000 CFU/ml of the mutant BM116 bacteria survived, which was the smallest number among the 13 mutants; this mutant was selected for further study.

Number of survival bacteria CFU/ml

160000

a

140000 120000 100000 80000 60000

b 40000

bc

b

c d de

de f g

20000

d

de f

f

0 B11

BM09

BM10

BM11 BM116 BM128 BM262 BM271 BM373

BM378

BM383 BM385 BM409 BM459

Strain

Fig. 2 Isolation of mutants defective in survival in macrophages. Values denoted by different letters are significantly different when compared by ANOVA (P 0.01)

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3.3 Southern blot analysis of the wild-type strain B11 and the mutants The Southern blot analysis of the wild-type strain and the mutants showed that only a single band was present in the mutants and the plasmid positive control pLOFKm; no signal was detected in the negative control wild-type strain B11 (Fig. 3). This result demonstrated that the mutation was caused by the insertion of transposon mini-Tn10Km and that only a single copy of the transposon was present in the chromosome of mutants.

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Figure 3. Detection of mini-Tn10 insertion in wild-type strain B11 and the mutants by Southern blot.

B11 ATCC 7966

TGAVAQQFHQ GALQFTNNAL DLSIQGNGFF VTSDGLTNLD RTFTRAGAFK LNENSYMVNN ********** ********** ********** ********** ********** ********** TGAVAQQFHQ GALQFTNNAL DLSIQGNGFF VTSDGLTNLD RTFTRAGAFK LNENSYMVNN

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B11

MSFNNALSGV NAAQKDLNVT ANNIANVNTT GFKESRAEFA DVYANSIFVN AKTQVGNGVA ********** ********** ********** ********** ********** ********** MSFNNALSGV NAAQKDLNVT ANNIANVNTT GFKESRAEFA DVYANSIFVN AKTQVGNGVA

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ATCC 7966

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3.4 Molecular analysis of the BM116 mutant The DNA sequence flanking the mini-Tn10 transposon inserted in the mutant BM116 was amplified by TAIL-PCR (Liu et al., 1995). TAIL-PCR products of ~1500 bp and ~2000 bp were obtained when the upstream and downstream sequences of the inserted transposon, respectively, were amplified. These products were purified using a Gel Extraction Kit (Omega, USA), cloned into pMD18-T (Takara, Japan) and sequenced. Sequence analysis showed that a 1461 bp sequence of the mutant BM116 containing mini-Tn10 was obtained, which included a 1335 bp ORF (GenBank accession numbers JQ974982) and its putative promoter. This ORF was found to putatively encode a deduced 445 amino acids protein that displays the highest identity (99.6%) with the flagellar hook protein FlgE of A. hydrophila subsp. hydrophila ATCC 7966 (Fig. 4).

ATCC 7966 B11

QGNYLQGYEI NTDGTPKAVS INATKPIQIP DRAGEPKMTE LVEASFNLSI ESKTKPTSPT ********** ********** ********** ********** ********** ********** QGNYLQGYEI NTDGTPKAVS INATKPIQIP DRAGEPKMTE LVEASFNLSI ESKTKPTSPT

ATCC 7966

AFDPTNSATF AHSTSVTIYD SLGAPHVITK YFVRHEDPAA PGTPLTPGTW SMYMYEGNKP ********** ********** ********** ********** ********** ********** B11 AFDPTNSATF AHSTSVTIYD SLGAPHVITK YFVRHEDPAA PGTPLTPGTW SMYMYEGNKP

ATCC 7966

IDIAGGSPSP ATGVPTGVKM EFTSGGKLDP TKTVPADPIK TVALGTTAGI ITNGADPAQT ********** ********** ********** ********** **.***.*** ********** B11 IDIAGGSPSP ATGVPTGVKM EFTSGGKLDP TKTVPADPIK TVTLGTAAGI ITNGADPAQT

ATCC 7966

LEIRLGDVTQ YSSPFNVTKL TQDGATVGNL TKVEITPDGI VSATYSNATT LKVAMVALAK ********** ********** ********** ********** ********** ********** B11 LEIRLGDVTQ YSSPFNVTKL TQDGATVGNL TKVEITPDGI VSATYSNATT LKVAMVALAK

ATCC 7966

FANSQGLTQV GDTSWRQSLL SGDALPGTPN SGTLGSIKSS ALEQSNVDLT SQLVNLITAQ ********** ********** ********** ********** ********** ********** B11 FANSQGLTQV GDTSWRQSLL SGDALPGTPN SGTLGSIKSS ALEQSNVDLT SQLVNLITAQ

ATCC 7966

RNFQANSRSL EVNSSLQQTI LQIR ********** ********** **** B11 RNFQANSRSL EVNSSLQQTI LQIR

ACCEPTED MANUSCRIPT Fig.4. Comparison of the amino acid sequence of A. hydrophila B11 FlgE with the homologous FlgE of A. hydrophila subsp. hydrophila ATCC 7966. The deduced amino acid of the Tn10 insertion is indicated with a bold, enlarged letter.

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3.5 Complementation After the recombinant expression plasmid pACYC184-flgE was introduced into mutant BM116, the expression of protein FlgE in the complementation strain was detected by Western blot. The result confirmed that FlgE is expressed in the complementation strain, but not in mutant BM116 (Fig.5).

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Fig.5 Detection of FlgE expression in the mutant BM116 and the complementation strain by Western blot.

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3.6 Transmission electron micrographs Molecular analysis revealed that the flgE gene of the BM116 mutant was inactivated by transposon Tn10. flgE is known to encode the hook protein of bacterial flagellum. The flagella of A. hydrophila strains were examined by transmission electron microscope (Fig. 6). These micrographs show that flagella were presented on the cells surface of wild-type B11 and the complementation strain, while no flagella were observed on the cell surface of the mutant BM116, indicating that flgE is required for flagellum biogenesis in A. hydrophila.

Figure 6. Transmission electron micrographs of A. hydrophila strains. (A) The wild-type strain B11; (B) The mutant BM116; (C) The BM116 complementation strain (flgE+) 3.7 Bacterial motility The results in Fig. 7 show that the wild-type strain exhibits motility in semi-soft medium, mutant BM116 displays defective motility, and the complementation strain exhibits restored motility.

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Figure 7. Motility of wild-type strain B11, mutant BM116 and the complementation strain in semi-soft medium.

a

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3.8 Bacteria adhesion The adhesion ability of wild-type B11, mutant BM116 and the complementation strain was compared. The results in Fig. 7 show that approximately 1.7×105 cell/cm2 wild-type bacteria and 1.2×105 cell/cm2 complementation bacteria adhered to the slides, while the number of adherent bacteria of the mutant BM116 strain was only approximately 6.2×104 cell/cm2, which demonstrated that the mutant BM116 was significantly impaired in its adhesion ability. These results suggested that mutagenesis of the flgE gene significantly affects bacterial adhesion.

B11

BM116 Strain

BM116(flgE +)

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Fig. 8. Bacterial adhesion. Values denoted by different letters are significantly different when compared by ANOVA (P<0.05)

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3.9 Bacterial invasion and survival in macrophages in vitro In view of the relationship between motility and viulence of A. hydrophila , the number of invasive and surviving bacteria of the wild-type B11, mutant BM116 and complementation strains in macrophages was determined in vitro. The results at 0 h represent the number of invasive bacteria in macrophages, and the results at 1 h indicate the number of surviving bacteria at 1 h after entry into the macrophages. The data in Fig. 9 show that there was no difference between the number of invasive wild-type bacteria and that of the complemented strain, while the number of invasive BM116 was significantly lower than those of wild-type B11 and the complementation strain (Fig. 9). Furthermore, there were only ~24.6% invasive mutant bacteria that survived in macrophages 1 h after entry into the cells, while the survival rates of wild-type B11 and the complementation strain were ~73.6% and ~65.1%, respectively, which suggested that the invasive ability and survival of the mutant bacteria BM116 in host macrophages were seriously impaired.

ACCEPTED MANUSCRIPT B11

BM116

BM116(flgE+)

250000 Number of survival bacteria CFU/ml

a a 200000 c 150000

c b

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0 0 h 1h Time after bacteria invasion

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Fig.9. Bacterial invasion and survival in macrophages. Values denoted by different letters are significantly different when compared by ANOVA (P<0.01)

Discussion Previous studies confirmed that a variety of human pathogens, for example, Listeria monocytogenes, Shigella flexneri and Legionella pneumophila are obligate intracellular pathogens [24]. The mechanisms for the survival of these pathogens in phagocytes have been fully explored [3,24,25]. It has been elucidated that one major advantage of the intracellular lifestyle is a rich source of nutrients inside a cell and the low risk of mixed intracellular infection by different pathogens on a single-cell level. Hence, in the intracellular milieu, competition between microbes seems to be rare [2]. Another advantage offered by the intracellular habitat is that the microbial invader can be shielded from attack by antibodies and bactericides [1,2]. It is notable that some studies have even found that pathogens such as V. vulnificus, Vibrio anguillarum, and Staphylococcus aureus, which have not been traditionally regarded as obligate intracellular pathogens, can also invade and survive in haemocytes or professional phagocytes for varying lengths of time [1,18,26]. In this study, we revealed that the pathogenic A. hydrophila B11 can also invade the macrophages of its host in vitro and some bacteria survive in the macrophages for at least 24 h. Bacteria have to withstand the disruption of lysosomal enzymes in phagocytes once they gain entry into these cells, but they can avoid other potential threats from outside of the cells at the same time. In this case, gaining an intracellular niche, even briefly, might afford a window of opportunity for A. hydrophila and other bacteria to survive and even promote diseases. To some extent, this result supports a hypothesis that intracellular survival is a bacterial strategy by which to subvert immunological defence mechanisms and extracellular bactericidal concentrations of antibiotics, which might lead to chronic or relapsing infections [1]. However, the interaction between the host and pathogen is complex. Studies examining the factors that contribute to the intracellular survival of the non-obligate intracellular bacteria are poorly documented. In this study, we revealed that when the flagellar hook protein gene flgE of A. hydrophila B11 was disrupted by transposon insertion, the flagella of the bacterium was disappeared. Furthermore, the ability of bacteria motility, adhesion, and invasion and intracellular survival in host macrophages were also significantly impaired. It has been confirmed that A. hydrophila has two distinct flagellar systems, the polar flagellum needed for swimming in liquid was expressed in all culture conditions and multiple

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lateral flagella for swarming over surfaces, which were expressed on solid or semisolid media [27,28]. Lateral flagella are distinct from the polar flagellum, but their synthesis is dependent upon the presence of a polar flagellum[29]. Further studies revealed that the lateral flagellar gene region of A. hydrophila contains 38 genes [27] and the polar flagellum region of A. hydrophila contains 55 genes [28], more than 20 genes are shared between these two flagellar systems, including the flgE gene which encodes the hook protein. A study of the role of flagella and motility in the pathogenesis of Vibrio vulnificus revealed that the flgE knockout mutant completely lost its flagellum, but that it could be restored by the complemented strain by providing the intact flgE gene [30]. Li et al. [31] also reported that when they constructed a specific flgE mutant of the oral spirochete Treponema denticola, the mutant displayed no visible motility and lacks periplasmic flagella. Similar results were obtained in this study. When the flgE gene of A. hydrophila B11 was disrupted by the mini-Tn10transposon, the mutant BM116 was unable to form flagella. These results all support the view that flgE is necessary for flagella formation. In this study, we also found that the motility and adhesion ability of the mutant BM116 was significantly defective in the absence of flagella. Studies on A. hydrophila AH-3 also displayed that complete loss of motility caused ~85% reduction in adherence[29]. It has previously been described that adhesion of V. alginolyticus to glass surfaces is dependent on swimming speed and that higher speed results in higher adhesion [32]. Larsen and Boesen’s [18] also reported that high swimming speeds increase the likelihood that the mutant collides with the macrophages with a higher force, which may increase its uptake by the macrophages. Therefore we concluded that one possible explanation of our results is that motility defects lead to a reduced bacterial moving speed and reduced collisions between the mutant bacteria and the host cells, eventually leading to the reduced invasion by BM116. It is not known with certainty how A. hydrophila survives inside the macrophages after invasion. However, many studies have shown that obligate intracellular pathogenic bacteria have evolved a number of very effective strategies that permit intracellular survival within phagocytes. These strategies mainly include: (a) the inhibition of phagosome-lysosome fusion; (b) inhibition of phagosome acidification; (c) recruitment and retention of tryptophan-aspartatecontaining coat protein on phagosomes to prevent their delivery to lysosomes; (d) expression of members of the host-induced repetitive glycine-rich protein family of proteins; (e) lysis of the phagocytes and escape to enter the cytoplasm [25, 33, 34]. In this study, the data suggest that the survival ability of the mutant in host macrophages was also seriously impaired with the absence of bacterial flagella. It can be deduced that A. hydrophila uses the strategy of escape from the phagosomes or lysosomes to the cytoplasm to avoid the damage of the bactericidal materials and survival in the host phagocytes. Although no direct evidence in this study has proven the hypothesis, some previous reports have revealed that bacterial flagella or motility contribute to bacterial survival in host phagocytes. Sano et al. [35] found that Salmonella ∆fliA mutants lacking flagella showed significantly reduced motility outside and inside host cells, and highly motile Salmonella bacilli can escape from host cells while flagellum-less ∆fliA mutants did not. Stamm et al. [36] demonstrated that Mycobacterium marinum can escape from phagosomes into the cytoplasm of infected macrophages by actin-based motility.

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Although some specific mechanisms of A. hydrophila invasion and survival in host phagocytes remain uncertain, our findings provide an important new understanding of this process. Our future research efforts will focus on the strategies that permit A. hydrophila intracellular survival within phagocytes. In summary, the present study revealed that the pathogenic A. hydrophila B11 can invade the macrophages of its host A. japonica in vitro and some bacteria can survive in the macrophages for at least 24 h. flgE is required for flagellum biogenesis in A. hydrophila, and flagellar motility contributes to the invasion and survival of A. hydrophila in host macrophages. Acknowledgments This work was supported by grants from The National Natural Science Foundation of China under contract No. 31272699 and 41176115 and the Natural Science Foundation of Fujian Province under contract No. 2013J01137. We gratefully acknowledge Prof. Nie and Dr. Y. Huang for providing some of the strains and plasmids used in this study.

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Highlights •Aeromonas hydrophila B11 can invade and survive in the macrophages of its host. •flgE is required for flagellum biogenesis in A. hydrophila. •Flagellar motility contributes to bacterial invasion and survival in the macrophages.