Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogansstrain Lai
© Liao et al; licensee BioMed Central Ltd. 2009
Received: 29 March 2009
Accepted: 9 December 2009
Published: 9 December 2009
Pathogenic Leptospira species cause leptospirosis, a zoonotic disease of global importance. The spirochete displays active rotative mobility which may contribute to invasion and diffusion of the pathogen in hosts. FliY is a flagellar motor switch protein that controls flagellar motor direction in other microbes, but its role in Leptospira, and paricularly in pathogenicity remains unknown.
A suicide plasmid for the fliY gene of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai that was disrupted by inserting the ampicillin resistance gene (bla) was constructed, and the inactivation of fliY gene in a mutant (fliY-) was confirmed by PCR and Western Blot analysis. The inactivation resulted in the mRNA absence of fliP and fliQ genes which are located downstream of the fliY gene in the same operon. The mutant displayed visibly weakened rotative motion in liquid medium and its migration on semisolid medium was also markedly attenuated compared to the wild-type strain. Compared to the wild-type strain, the mutant showed much lower levels of adhesion to murine macrophages and apoptosis-inducing ability, and its lethality to guinea pigs was also significantly decreased.
Inactivation of fliY, by the method used in this paper, clearly had polar effects on downstream genes. The phentotypes observed, including lower pathogenicity, could be a consequence of fliY inactivation, but also a consequence of the polar effects.
The genus Leptospira is composed of both saprophytic and pathogenic species . Pathogenic Leptospira spp., such as L. interrogans, L. borgpetersenii, L. weilii and L. kirschner, are the causative agents of leptospirosis, a serious world-wide disease in humans and animals [2, 3]. The disease in humans occurs mostly after contact, often through skin wounds, with soil or water contaminated by urine of infected animals. Its severity varies from mild to rapidly fatal. Severe symptoms are characterized by visible jaundice involving hepatic injury, acute renal failure, carditis and hemorrhage, and case fatality varies from a few percent to 25% [3–6]. However, the mechanisms of disease caused by pathogenic Leptospira spp. remain largely unknown.
Both pathogenic and saprophytic leptospires express two endoflagella (periplasmic flagella). One of the endoflagella is attached at one end of the cell and is located between the protoplasmic cylinder and the outer membrane sheath [7–9]. The endoflagella, rotating within the periplasmic space, are responsible for spirochete motility. In pathogenic Leptospira species, this motility is considered to contribute to invasion into hosts and diffusion within the hosts during infection [9, 10]. In previous studies, we found that pathogenic leptospires can adhere to host cells with one or two termini of the microbial bodies, while non-pathogenic leptospiral strains lacked this ability [11, 12]. The adhering positions were located at the terminal knobs in which flagellar basal bodies are found [1, 7]. At the bottom of the flagellar structure, there is a basal body composed of MS and C rings [13, 14]. In flagellated bacteria, some proteins in the Fli family form the C ring, which functions as the flagellar rotor and contains the directional switching capability of the flagellar motor [15–18]. However, a possible role for the leptospiral endoflagella in pathogenicity has never been explored.
A complete set of flagella-associated genes were found in the genomic sequences of L. interrogans serovar Lai strain Lai and serovar Copenhageni strain Fiocruz L1-130, including four genes that encode flagellar motor switch proteins (FliG, FliM, FliN and FliY) [19, 20]. In bacteria, the flagellar motor switch proteins play a critical role in control of flagellar motor direction [14, 17, 18]. Thus far FliY has been found in some spirochetes and a few bacteria but does not exist in most bacteria [21, 22]. Particularly, FliY of Bacillus subtilis was shown to be a CheY-P-hydrolyzing protein in the chemotactic signaling cascade . In addition, leptospiral FliY carries a carboxy-terminal domain of 60 amino acid residues that is homologous to a domain of YscQ in Yersinia pestis [19, 20]. The YscQ protein was identified as a member of the flagellar associated type III secretion system (T3SS), with multiple functions such as controlling the directional rotation of flagella and the export of virulence factors including Yop proteins [23, 24]. The C ring of Escherichia coli does not have FliY, but its FliN has a high sequence homology with FliY of L. interrogans strain Lai  and FliN is an essential agent for motility and virulence protein export . These data suggest that FliY of pathogenic Leptospira species may have important functions in motility and virulence.
In the present study, we constructed a fliY gene knock-out (fliY-) mutant of L. interrogans serovar Lai strain Lai based on homologous recombination using a suicide plasmid. To examine the possible role of FliY in pathogenesis, the mutant and wild-type strain were compared in assays of motility in liquid medium and migration on semisolid agar, adhesion to macrophages, stimulation of apoptosis in infected host cells, and lethality to guinea pigs.
Products of fliYgene amplification and rFliY expression
Characterization of the fliY-mutant
Persistently lower motility of the fliY-mutant
Altered adhesion of the fliY-mutant
Host-cell apoptosis induced by the wild-type and the fliY-mutant strains
Attenuated lethality of the fliY-mutant strain in guinea pigs
Lethality of the fliY- mutant and the wild-type strain in infected guinea pigs.
(×108 per animal)
Recent reports have shown that flagellin and other flagella-associated proteins from many bacteria participate in adhesion to host cells and colonization of hosts [26–28]. In vitro studies have suggested that the role of flagella could be to increase invasion into host cells and survival within macrophages [29, 30]. However, the correlation between flagella and pathogenicity of pathogenic Leptospira spp. had not been investigated until now. L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai is the most prevalent pathogenic leptospiral strain, which is responsible for over 70% of human leptospirosis cases in China . We therefore inactivated the fliY gene in L. interrogans strain Lai using a suicide plasmid, which is a frequently adopted strategy for determining the function of a target gene. Recently, Croda and his colleagues used plasmid pB2SK to successfully construct a suicide plasmid with spectinomycin resistance for inactivating the ligB gene of L. interrogans serovar Copenhageni strain Fiocruz L1-130 . In the present study we first used another plasmid, p2NIL, with an ampicillin resistance gene (bla) to construct a fliY gene knock out (fliY-) mutant. A fliY- mutant has been constructed, but that fliY inactivation by ampicillin cassette insertion also negatively affected downstream genes; therefore, care has to be taken when interpreting the phenotypes observed for this mutant.
The inactivation of the fliY gene has shown different effects on formation of flagella in different bacteria. In Bacillus subtilis, the deletion of fliY resulted in the loss of flagella . However, the flagella were still produced in the fliY-deleted strain of Bacillus cereus . Although the leptospiral fliY- mutant generated in this study displayed remarkably attenuated motility compared to the wild-type strain, it maintained the typical spiral shape and propeller movement which is caused by the periplasmic endoflagella [1, 7]. As mentioned previously, the major function of flagellar motor switch proteins is to control flagellar motor direction [16, 19–22]. Thus, we infer that the fliY gene inactivation should not affect the formation of the endoflagella.
It is well known that adhesion to host cells is a primary and critical step for bacterial infection [35, 36]. Recently, the importance of cell adhesion for pathogenic Leptospira spp. has been demonstrated [11, 12, 37, 38]. Adhesion to host cells also acts as an essential role for pathogenicity of other spirochetes [39, 40]. Mononuclear macrophages are the most important phagocytes in the human innate and acquired immnune systems. However, many pathogenic bacteria can evade host immunity by inducing apoptosis of macrophages [41–43]. Similarly, pathogenic Leptospira spp. can escape from the host immune system by promoting macrophage apoptosis [11, 44–46]. In the present study, we provide evidence that the ability of the fliY- mutant to adhere to J774A.1 cells, to induce apoptosis in the cells, and to cause death in guinea pigs is much lower than for the wild-type strain. All the phentotypes observed, including lower pathogenicity, could be a consequence of fliY inactivation, or a consequence of the polar effects, or of both.
T3SS is one of protein export systems used by most Gram-negative bacteria . Morphologically, as a transmembrane channel, T3SS is composed of multiple protein complexes called an injectisome, responsible for transporting virulence factors into host cells, some of which cause cell metabolic disorder and death [47–49]. However, the flagellar export apparatus can also function as a bacterial virulence protein secretion system . For example, FliF of Pseudomonas aeruginosa, a flagellar associated protein component in the MS ring, is involved in adhesion by controlling secretion of bacterial adhesins . Although the T3SS and flagellar export apparatus are two relatively separate systems in many pathogenic bacteria , the T3SS and flagellar export apparatus in Yersinia enterocolitica play a common role in secretion of bacterial phospholipases during infection . Taken together, these observations suggest that inactivation of the leptospiral fliY gene (or of the downstream located fliPQ genes) may decrease the export of some unknown adhesion- and cytotoxicity-associated virulence proteins.
Inactivation of fliY clearly had polar effects on downstream genes. The phentotypes observed, including decreasing motility, adhesion to macrophages and host-cell apoptosis, and attenuating lethality in infected guinea pigs, could be a consequence of fliY inactivation, but also a consequence of the polar effects.
Bacterial strains and cell lines
L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was offered by the National Institute for the Control of Pharmaceutical and Biological Products in Beijing, China. The leptospires were cultured in Korthof liquid medium containing 8% heat-inactivated rabbit serum (RS) at 28°C. To maintain virulence, the strain was passaged intraperitoneally in specific pathogen-free Dunkin-Hartley ICO:DH (Poc) guinea pigs (2 weeks old, each weighing about 120 g) before use, according to the description by Merien et al. and Viriyakosol et al. [44, 54]. Animal protocols were approved by the Animal Ethics Review Committee of Zhejiang University.
Cell line and culture
The murine mononuclear-macrophage-like cell line (J774A.1) was obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were cultured in RPMI 1640 medium (GIBCO, USA), supplemented with 10% heat-inactivated fetal calf serum (FCS) (GIBCO), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, USA) at 37°C in an atmosphere of 5% CO2.
PCR and sequencing
Primer information for amplification of the fliY and bla genes.
Primer sequence (5'-3')
F: GCC GGA TCC (BamH I) ATG GGT GAA GGT TCC CTA TCA CAG
R: GCC AAG CTT (Hind III) TCA CTT ACC CTC CGG CTT AAT CCG
F: GCC AGA TCT (Bgl II) TCT AAA TAC ATT CAA ATA TGT
R: GCC AGA TCT (Bgl II) CTT GGT CTG ACA GTT ACC AAT
F: ATG AAA ATG AGA CAT AAA
R: TCA TTT ATA ACT CCT TAC
F: ATG ACG GAA TTA GAC GTT ATG
R: CTA AAA TTT TTC GAT CAT CAA
Expression, purification and immunization of recombinant FliY
pUCm-T fliY and expression vector pET32a (Novagen, USA) were digested with BamH I and Hind III, respectively. The recovered fliY segment was ligated into linearized pET32a using T4 DNA ligase (TaKaRa), and then transformed into E. coli BL21DE3 (Novagen) to form E. coli BL21DE3pET32a-fliY. Recombinant FliY (rFliY) was expressed under inducement of 0.5 mM IPTG for 4 h at 37°C. The expressed rFliY was extracted by Ni-NTA affinity chromatography and the purity of rFliY was determined by SDS-PAGE. New Zealand rabbits, provided by the Laboratory Animal Center of Zhejiang University, were immunized intradermally four times at an interval of once a week with the purified rFliY that was pre-mixed with Freund's adjuvant. On the 15th day after the last immunization, the rabbit serum was collected and the immunodiffusion test was used to examine the titer of antiserum.
Generation and characterization of the fliY-mutant
Confirmation of the fliYgene inactivation in mutants
The fliY- mutant was cultured at 28°C in 8% RS Korthof liquid medium containing 100 μg/ml ampicillin. Genomic DNA of the mutant was extracted using Bacterial Genomic DNA Extraction Kit (BioColor), and the disrupted fliY gene in the mutant was identified by PCR and the Western Blot assay. The product of the fliY-bla gene is larger in the mutant (2019 bp) than the fliY gene in the wild-type strain (1065 bp). By using 1:2500 diluted anti-rFliY serum as the primary antibody and 1:3000 diluted HRP-labeling goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, USA) as the secondary antibody, a Western Blot assay was performed to detect the expression of FliY protein in the mutant. In the genomic sequence of L. interrogans serovar Lai strain Lai, the fliP and fliQ genes are located downstream from the fliY gene. In order to further confirm the inactivation of the fliY gene, two separate RT-PCRs were performed to detect mRNAs of the fliP and fliQ genes, with primers shown in table 2. In addition, an operon predictor tool http://www.microbesonline.org/ was used for analysis of the operon structure.
The motility and shapes of the fliY- mutant and wild-type strain in 8% RS Korthof liquid medium were observed under dark-field microscope after incubation at 28°C for 10 d (the primary generation), 50 d (the 5th generation) and 100 d (the 10th generation). The colony sizes of the mutant and wild-type strain on 8% RS semisolid Korthof plate (0.25% agar) that had been incubated at 28°C for three weeks were measured for three times as described above.
Fontana silver staining
J774A.1 cells (5 × 104 cells/ml) were seeded on coverslips in 12-well tissue culture plates (Corning, USA) and pre-incubated for 24 h at 37°C in an atmosphere of 5% CO2. The freshly cultured leptospires of the fliY- mutant and wild-type strain were harvested by centrifugation (12,000 × g, 15min, 15°C) and washed twice with autoclaved PBS. The pellets were suspended in pre-warmed antibiotics-free 10% FCS RPM1640 to a final concentration of 108 leptospires/ml by dark-field microscopy with a Petroff-Hausser counting chamber (Fisher Scientifics, PA). The cell monolayers were washed three times with autoclaved PBS and then infected with each of the suspensions at an MOI of 100 (100 leptospires per cell) for 10, 20, 30, 40, 50 and 60 min, respectively. After infection, the coverslips were washed three times with PBS to remove non-adherent leptospires, fixed in 5% formaldehyde, stained with silver nitrate, and observed under a light microscope . The adhesion ratio was defined as the number of adhering leptospires per 100 infected host-cells × 100% .
Assessment of cell death by flow cytometry
Apoptosis was measured by flow cytometry using annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) labeling as previously published [11, 60]. The J774A.1 cell monolayers were infected with either the fliY- mutant or wild-type strain with an MOI of 100 at 37°C for 1, 2, or 4 h . After infection, the cells were washed three times with PBS, collected with a cell scratcher, and centrifuged at 1,000 × g for 5 min. The pellets were washed three times with PBS, resuspended in annexin-V binding buffer with FITC-conjugated annexin-V, and incubated for 15 min at room temperature in the dark, following the manufacturer's instructions (Caltag Laboratories, USA). After PI was added, the cell suspension was immediately analyzed by FACSCalibur flow cytometry and CellQuest Pro software (Beckman Coulter, USA). Cells in the early apoptotic phase bind annexin-V but exclude PI, and those in the late apoptotic/necrotic phase stain with both annexin-V and PI, while necrotic cells stain with PI alone .
Animals and challenge infections
The Dunkin-Hartley guinea pigs (150 ± 5 g, 3 weeks old) used in this study were provided by the Laboratory Animal Center of Zhejiang University. The animals were challenged intraperitoneally with different dosages of either wild-type L. interrogans serovar Lai strain Lai or the fliY- mutant, and then observed for 10 d . The animal experiments were approved by the Animal Ethics Review Committee of Zhejiang University.
Data from a minimum of three experiments were averaged and presented as mean ± SD (standard deviation). One-way analysis of variance (ANOVA) followed by the Dunnett's multiple comparisons test were used to determine significant differences. Statistical significance was defined as P value ≤ 0.05.
This work was supported by a Grant (30370072) from the National Natural Science Foundation of China and a grant (2007XZA02) from the Natural Scientific National Foundation of Zhejiang Medical College of China. We are grateful to Dr. Tanya Parish and Dr. Amanda C. Brown (Center for Infectious Disease, Institute for Cell and Molecular Science, Queen Mary's School of Medicine and Dentistry, UK) for having graciously provided the plasmid p2NIL used in this study.
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