Streptococcus iniae Capsule Impairs Phagocytic Clearance and Contributes to Virulence in Fish

Bacterial strains, culture, transformation, and DNA techniques.WT S. iniae strain K288 was isolated from the brain of a diseased HSB at the Kent SeaTech aquaculture facility in Mecca, CA (8). S. iniae strain 94-426 was originally isolated from a diseased tilapia. All S. iniae strains were grown at 30°C (unless otherwise stated) in Todd-Hewitt broth (THB) (Hardy Diagnostics) or Todd-Hewitt agar (THA). Enumeration of CFU for in vitro assays and in vivo infections was performed by plating dilutions on THA. Beta-hemolytic activity of S. iniae was assessed on sheep blood agar plates (tryptic soy agar with 5% sheep red blood cells added). In all assays, overnight cultures of S. iniae were diluted 1:10 in fresh THB and grown to mid-log phase (optical density at 600 nm of 0.40). S. iniae strains were rendered electrocompetent for transformation through growth in THB containing 0.6% glycine according to procedures described for GBS (18); transformants were propagated at 30°C in THB with 0.25 M sucrose. Antibiotic selection was achieved with chloramphenicol (Cm) at 4 μg/ml, erythromycin (Erm) at 5 μg/ml, or spectinomycin at 100 μg/ml. Escherichia coli used in cloning was grown at 37°C (unless otherwise stated), with shaking, under aerobic conditions in Luria-Bertani broth (Hardy Diagnostics) or statically on Luria agar. When necessary, E. coli was grown in antibiotics, i.e., ampicillin at 100 μg/ml, spectinomycin at 100 μg/ml, Erm at 500 μg/ml, or Cm at 20 μg/ml. Mach1 chemically competent E. coli (Invitrogen) and MC1061 electrocompetent E. coli used in transformations were recovered through growth at 30°C in SOC medium (Invitrogen). A PureLink Quick plasmid miniprep kit (Invitrogen) was used to isolate plasmids propagated in E. coli. S. iniae genomic DNA was isolated using the UltraClean DNA isolation kit (MoBio).

Cell lines and culture conditions.The adherent carp monocytic/macrophage cell (CLC) line (European Collection of Cell Cultures 95070628) and the WBE27 white bass embryonic epithelial cell line (ATCC CRL-2773) (48) were grown at 28°C with 5% CO₂. Cells were passaged fewer than 10 times and maintained in 125-ml tissue culture flasks in Dulbecco modified Eagle medium (DMEM)(Gibco) containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco).

Statistical analyses.Data analyses were performed using the statistical tools included with GraphPad Prism (GraphPad Software, Inc.). In vitro assay data were analyzed using unpaired two-tailed t tests. Fish infection survival data were analyzed using a log rank test. A P value of <0.05 was considered statistically significant. In vitro assays were repeated three times, in quadruplicate, and the data presented (means ± standard errors of the means [SEM]) are from a single representative assay.

Allelic exchange mutagenesis.Allelic exchange mutagenesis of S. iniae strains K288 and 94-426 was carried out as previously described (7), with the only significant modification being the use of the Gateway cloning system (Invitrogen). For PCR, all primers were designed based on the cpsD gene region of the S. iniae capsule operon deposited under GenBank sequence accession number AY904444. PCR was used to amplify ∼400 bp of S. iniae chromosomal DNA fragments directly upstream and downstream of cpsD, with primers adjacent to cpsD constructed to possess 25-bp 5′ extensions corresponding to the 5′ and 3′ ends of the chloramphenicol acetyltransferase (cat) gene from pACYC (34), respectively. The upstream and downstream PCR products were then combined with a 660-bp amplicon of the complete cat gene by using fusion PCR (51). The resultant PCR amplicon containing an in-frame substitution of cpsD with cat was subcloned into the Gateway entry vector pCR 8/GW/TOPO and transformed into chemically competent Mach1 E. coli (Invitrogen). Plasmid DNA was extracted, and a Gateway LR recombination reaction was performed to transfer the fusion PCR amplicon into the corresponding Gateway entry site of a temperature-sensitive knockout vector, pKODestErm (created for Gateway cloning from pHY304 (9), to generate the knockout plasmid pKOcpsD. Following propagation in MC1061 E. coli, the pKOcpsD construct was introduced into WT S. iniae by electroporation. Transformants were identified at 30°C by Erm selection and shifted to the nonpermissive temperature for plasmid replication (37°C). Differential antibiotic selection (Cm^r and Erm^s) was used to identify candidate allelic exchange mutants. Targeted in-frame replacement of cpsD was confirmed unambiguously by PCRs documenting the desired insertion of cat and absence of cpsD sequence in chromosomal DNA isolated from both of the final Δ cpsD mutants and by phenotype, with the observation of rapid sinking in liquid culture.

Transmission electron microscopy.Capsular polysaccharide of mid-log-phase WT K288 and K288 ΔcpsD was visualized via transmission electron microscopy using a lysine acetate fixation protocol as previously described (23). The only notable deviation in this protocol was the use of an overnight room temperature incubation in the second fixation step. Samples were embedded in LR White (Fluka), sectioned, and counterstained with uranyl acetate. Grids were viewed and photographed using a JEOL 1200EX II transmission electron microscope (JEOL, Peabody, MA) at a magnification of ×15,500 and an acceleration voltage of 80 kV.

Cytochrome c assay.Anionic cell surface charge was measured through a cytochrome c binding assay as previously described (8). An overnight culture of each S. iniae strain was diluted 1:10 and grown to mid-log phase. Five milliliters of the bacteria was pelleted at 13,000 × g for 5 min and resuspended in 1 ml of MOPS (morpholinepropanesulfonic acid) (pH 7.0). The bacteria were pelleted and then resuspended in 450 μl of MOPS and 50 μl of 10-mg/ml cytochrome c (Sigma). The solution was vortexed and incubated at room temperature for 15 min. The bacteria were pelleted, and 200 μl of the supernatant was added to a flat-bottom 96-well plate. The amount of unbound cytochrome c was determined by absorbance of the supernatant at 530 nm.

Growth rate analysis and hemolytic activity.Mid-log-phase cultures of WT S. iniae and the ΔcpsD mutant were diluted 1:10 in a 96-well plate. Growth was monitored via optical density readings at 600 nm, in quadruplicate, every 30 min for 8 h. Hemolytic activity against sheep red blood cells was measured as described previously (19).

Invasion and adherence assays.Invasion and adherence assays were performed in collagenized 96-well tissue culture plates (Costar). White bass epithelial cells (WBE27) were seeded at a density of 1 × 10⁵ cells per well and allowed to grow overnight. The medium was replaced with 100 μl DMEM containing 2% FBS. Bacteria from a mid-log-phase culture were diluted in DMEM with 2% heat-inactivated FBS, and 100 μl was added to achieve a multiplicity of infection (MOI) of 10 (bacteria to cells). Following centrifugation at 350 × g for 5 min, the plate was incubated for 1 h at 28°C with 5% CO₂. The cells were washed three times with DMEM and incubated in fresh DMEM with 20 μg/ml penicillin (Invitrogen) and 200 μg/ml of gentamicin (Invitrogen) for 2 h to kill extracellular bacteria. Cells were then washed three times with phosphate-buffered saline (PBS) and lysed by trituration in 100 μl of 0.01% Triton X-100 (Sigma). Surviving intracellular bacteria were quantified by plating serial dilutions of lysed cell supernatant on THA. Adherence assays were carried out in a similar manner except that no antibiotics were used and the bacteria were incubated with the cells for 30 min and washed five times with PBS to remove nonadherent bacteria prior to enumeration of CFU.

Capsular polysaccharide isolation and purification.Capsular polysaccharide was extracted from 2 liters of 94-426 S. iniae culture by using methods described for other encapsulated species (20). Briefly, overnight cultures were treated with a final concentration of 1% Cetavlon, a polycationic detergent that precipitates polyanionic polysaccharides. The precipitate was collected by centrifugation and resuspended in water, and CaCl₂ added to a final concentration of 1 mM to separate polysaccharide from detergent. Nucleic acids were precipitated from solution by adding 25% (vol/vol) ethanol, followed by centrifugation. Capsule in the supernatant was subsequently precipitated by ethanol at a final concentration of 80% (vol/vol). Contaminating protein, traces of Cetavlon, and other low-molecular-mass contaminants were removed with proteinase K digestion and extensive dialysis against a buffer composed of 10% ethanol, 50 mM NaCl, and 5 mM Tris. Capsule was further purified with a Sephacryl 200 gel filtration column using 50 mM ammonium formate elutions. Column fractions were tested for neutral sugar estimation by phenol sulfuric acid assay (14). Void-volume fractions were pooled and concentrated by speed vacuuming and analyzed by deoxycholate-polyacrylamide gel electrophoresis (42) and Alcian blue staining (42).

Glycosyl composition analysis.The glycosyl composition of capsular polysaccharide was determined by the preparation and analysis of trimethylsilyl methylglycosides (40). Briefly, samples were methanolyzed with 1 M methanolic HCl at 80°C for 18 h, followed by re-N-acetylation of methylglycosides by use of pyridine-acetic anhydride in the presence of methanol at 100°C for 1 h. The free hydroxyl groups of re-N-acetylated methylglycosides were trimethylsilylated using Tri-Sil reagent (Pierce) at 80°C for 20 min. The volatile trimethylsilyl methylglycosides were then analyzed by combined gas-liquid chromatography-mass spectrometry (GLC-MS) using a DB-1 capillary column (J&W Scientific) (30 m by 0.25 mm), and detection was done with a mass selective detector (Hewlett-Packard HP 5890 series II GC interfaced to a 5971A mass selective detector).

In vivo fish challenges.Groups of 20 (∼40-g) HSB (Morone chrysops × Morone saxatilis) were used for in vivo infection studies. Fish were maintained at 25°C in ∼75-liter flowthrough tanks. An overnight culture of each S. iniae strain was diluted 1:10 and grown to mid-log phase. The bacteria were pelleted, resuspended in PBS, and diluted appropriately to deliver the desired dose in a 100-μl intraperitoneal (i.p.) injection. Survival was monitored for 7 days. Fish challenges were carried out in an ALAAC-certified facility following IACUC-approved protocols.

Survival in whole blood.Blood was extracted via syringe from the caudal vein of three HSB and collected in a heparinized tube. Three hundred microliters of each blood sample was immediately added to two 2-ml siliconized microcentrifuge tubes with approximately 300 CFU of mid-log-phase bacteria. The tubes were incubated with shaking at 30°C for 1 h. Two 100-μl aliquots from each blood sample were spread onto THA to enumerate surviving bacteria. Survival was calculated as a percentage of remaining bacteria relative to the starting inoculum.

Total cell killing and intracellular survival.Total phagocytic survival assays were carried similarly to invasion and adherence assays. Bacteria were incubated with CLCs at an MOI of 0.1. Cells were lysed and plated as described above for invasion and adherence assays. Survival is expressed as CFU per ml of lysed cell supernatant at each time point. Intracellular growth assays were carried out in a manner similar to that for entry assays. Bacteria were incubated with the CLCs at 28°C at an MOI of 10. After 1 h, the medium was replaced with fresh DMEM with 20 μg/ml penicillin (Invitrogen) and 200 μg/ml of gentamicin (Invitrogen) to kill extracellular bacteria. After 4 h in antibiotics, the medium was replaced with fresh DMEM containing 2% FBS. The cells were lysed and plated to determine surviving CFU as described above. Survival is expressed as CFU per ml of lysed cell supernatant at each time point. For a visual comparison of phagocytosis in CLCs, bacteria were labeled by being grown to an optical density at 600 nm of 0.40 in THB plus 50 μg/ml fluorescein isothiocyanate (FITC) (Molecular Probes). Bacteria were washed twice in PBS and added at an MOI of 10 to CLC monolayers as described above. After incubation with antibiotics as described above to kill extracellular bacteria, monolayers were washed two times with PBS, and SYTOX Orange (Molecular Probes) was added to a final concentration of 0.5 μM to each well. Bacteria were visualized with a Zeiss Axiovert 100 inverted microscope with appropriate fluorescent filters. FITC-labeled intracellular bacteria appeared green, and remaining extracellular bacteria killed by antibiotics were labeled with SYTOX Orange and appeared red. Images were captured with a charge-coupled device camera using the Axiovision software package (Zeiss).

Resistance to AMPs.Mid-log-phase cultures of S. iniae were diluted in fresh THB to ∼3 × 10⁴ CFU/ml, and 180 μl of this bacterial suspension was added to wells of a 96-well plate. Dilutions of the antimicrobial peptides moronecidin (28) (1.5 μM final concentration in the well) and polymyxin B (Sigma) (60 μM final concentration in the well) were prepared in distilled water and added to wells in 20-μl volumes; distilled water alone was used as a control. To measure antimicrobial killing kinetics, 20-μl aliquots from each well were serially diluted in PBS and plated at specified time points after addition of the antimicrobial peptide for CFU determination. Each treatment was performed in four replicate wells. Kinetic killing data were calculated for each time point by dividing the treatment group CFU by the control CFU.

Oxidant susceptibility assay.Bacterial strains were grown to mid-log phase and diluted 1:10 in PBS, and 100 μl was added to a 96-well plate, resulting in ∼3 × 10⁶ CFU/well. Hydrogen peroxide (H₂O₂) (Fisher Scientific) was added to a 0.035% final concentration. Bacteria were incubated at 30°C, and the reaction was quenched at time end points by adding 1,000 U of catalase (Sigma). Dilutions were plated on THA to determine the number of surviving CFU.

Mutagenesis of S. iniae cpsD reduces surface capsular polysaccharide.Precise, in-frame allelic replacement of the cpsD gene was achieved in S. iniae strains K288 and 94-426 to create K288 ΔcpsD and 94-426 ΔcpsD (Fig. 1A). Each S. iniae ΔcpsD allelic replacement mutant exhibited reduced buoyancy in liquid culture (Fig. 1B). Loss of capsule was corroborated by loss of anionic charge on the surface of ΔcpsD mutants, as determined by decreased cytochrome c binding (36) (Fig. 1C). As reported for capsule mutants of other streptococci (15, 31) and also observed in our observations of increased chain length in GBS strain COH1 ΔcpsE isogenic capsule mutants (44) (data not shown), the S. iniae ΔcpsD mutants formed chains of greater length than the WT parent strains (Fig. 2A). Compared to the wild type, a clear reduction in surface capsular polysaccharide in the K288 ΔcpsD mutant was visualized through transmission electron microscopy (Fig. 2B). The ΔcpsD mutants had identical growth rates and similar hemolytic activity to the WT S. iniae strains (data not shown). It should be noted that complementation of the K288 ΔcpsD mutant was attempted by cloning the S. iniae cpsD gene into the multiple cloning sites of the constitutive high-expression plasmid pDCerm (25) and the tetracycline-inducible expression plasmid pLR16T (41). Expression of cpsD in pDCerm did not restore a wild-type phenotype; however, complementation with pLR16T (over a tight range of tetracycline levels) resulted in partial restoration of WT liquid culture buoyancy and coccus chain length phenotypes to the K288 ΔcpsD mutant (data not shown).

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FIG. 1.

Allelic exchange mutagenesis of S. iniae cpsD results in a capsule-deficient phenotype. (A) A knockout plasmid, pKOcpsD, was created, containing erythromycin resistance (Erm^R), a temperature-sensitive origin of replication (t.s. rep), and a chloramphenicol resistance gene (cat) flanked by homologous regions of DNA upstream (Up) and downstream (Dwn) of cpsD. The knockout plasmid was used for precise in-frame replacement of the S. iniae cpsD gene with cat. (B and C) Allelic replacement of cpsD resulted in reduced capsule production as seen by reduced buoyancy in liquid culture (B) and a reduction in negative cell surface charge measured indirectly through amount of positively charged cytochrome c remaining unbound to the bacteria (mean ± SEM) (C). OD, optical density.

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FIG. 2.

Deletion of cpsD increases coccus chain length and reduces extracellular capsular polysaccharide. (A) Bright-field microscopy (magnification, ×400) reveals increased coccus chain length of the ΔcpsD mutants compared to WT S. iniae. (B) Transmission electron microscopy (magnification, ×15,500) shows a decrease in capsule in the K288 ΔcpsD mutant compared to WT K288.

S. iniae capsule mutants show increased epithelial cell adherence and invasion.A frequent observation in capsule-deficient streptococci is an enhancement of cellular adherence and invasion compared to those of WT strains (23, 35). Consistent with this pattern, the ΔcpsD mutants displayed a ∼10-fold increase in adherence and a ∼100-fold increase in intracellular invasion of cultured white bass epithelial cells compared to the parent strains (P < 0.0001) (Fig. 3).

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FIG. 3.

Reduction in capsule increases adherent and invasive properties of S. iniae. S. iniae strain K288 was less adherent and invasive than capsule-deficient strain K288 ΔcpsD after incubation with WBE27 white bass epithelial cells. Adherent bacteria were enumerated after 30 min, and invasive intracellular bacteria were enumerated at 2 h (mean ± SEM). Similar results were observed for WT strain 94-426 and it ΔcpsD mutant.

S. iniae capsular sugars are reduced in the ΔcpsD mutant.GLC-MS composition analysis of a capsular monosaccharide preparation revealed that the capsule of WT S. iniae strain 94-426 potentially contains l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine (Table 1). It is possible that some of these sugars exist in noncapsular polysaccharides. We found a significant reduction in putative capsular monosaccharides, with the exception of d-mannose, in the capsule-deficient ΔcpsD S. iniae isogenic mutant (Fig. 4). Given its relative abundance in both WT and ΔcpsD S. iniae, it is possible that d-mannose exists as a noncapsular, cell surface polysaccharide component.

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FIG. 4.

GLC-MS analysis of S. iniae extracellular polysaccharides shows a reduction in 94-426 ΔcpsD monosaccharides compared to wild type. GLC-MS elution spectra after capsular preparation from on overnight culture of S. iniae are shown. The spectra define the potential capsular monosaccharides of S. iniae and indicate a significantly decreased abundance of capsular monosaccharides in the 94-426 ΔcpsD capsule-deficient mutant compared to the wild type. Sugar abbreviations: Fuc, l-fucose; Man, d-mannose; Gal, d-galactose; Glc, d-glucose; GlcA, d-glucuronic acid; GalNAc, N-acetyl-d-galactosamine; and GlcNAc, N-acetyl-d-glucosamine.

Reduction of capsule attenuates S. iniae infection in hybrid striped bass.The effect of the ΔcpsD mutation on S. iniae virulence was assessed through an i.p. infection challenge in HSB (Fig. 5A and B). Both WT S. iniae strains resulted in 100% HSB mortality within 1 week at an inoculum of 3 × 10⁶ CFU. In contrast, injections of up to 100-fold-greater inocula of the respective ΔcpsD mutants caused no mortality or visual signs of infection in the fish.

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FIG. 5.

Capsule contributes to S. iniae virulence in vivo as indicated in Kaplan-Meier survival plots showing attenuation of the ΔcpsD mutants. (A) Hybrid striped bass (groups of 20) were injected intraperitoneally with 3 × 10⁶ CFU of WT K288; with 3 × 10⁶, 10⁷, or 10⁸ CFU of the K288 ΔcpsD mutant; or with PBS. (B) Hybrid striped bass (groups of 20) were injected intraperitoneally with 3 × 10⁶ CFU of WT 94-426 or with 3 × 10⁶ CFU of the 94-426 ΔcpsD mutant. Mortality (100%) was observed only in the WT-injected fish for each strain.

S. iniae capsule promotes resistance to whole-blood and macrophage killing.To elucidate potential mechanisms for the observed in vivo attenuation of the capsule mutants, S. iniae survival in fresh whole HSB blood was measured. Both of the S. iniae ΔcpsD mutants were significantly more susceptible to blood killing than the WT strains (Fig. 6A), indicating increased clearance by innate immune defenses. To further assess the role of the capsule in promoting S. iniae survival, WT S. iniae and the isogenic ΔcpsD mutant strains were incubated with a cultured fish macrophage cell line. The capsule-deficient mutant was over 20-fold more susceptible to killing by the macrophages in the in vitro assay (P < 0.0001) (Fig. 6B).

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FIG. 6.

S. iniae capsule decreases susceptibility to killing by whole blood and macrophages. (A) Survival of capsule-deficient ΔcpsD mutants is significantly decreased compared to that of wild-type S. iniae following 1 h of incubation in fresh whole fish blood. (B) K288 ΔcpsD is more sensitive than wild-type S. iniae to total phagocytic killing after a 6-h incubation with CLCs. Data are presented as mean ± SEM.

An S. iniae capsule mutant is less susceptible to cationic AMPs.One evolutionarily conserved mechanism for innate immune defense against bacterial infection is the production of cationic AMPs by phagocytes and other host cell types. We compared the susceptibilities of both WT S. iniae strains and the ΔcpsD isogenic mutants to the HSB AMP moronecidin and found the mutant strain to exhibit significantly (P < 0.0001) delayed killing kinetics (i.e., increased resistance) (Fig. 7A). Similar differences (P < 0.001) were observed in parallel assays performed with the bacterially derived cationic AMP polymyxin B (Fig. 7B). These studies indicate that the susceptibility to whole-blood and macrophage killing of the capsule-deficient strains does not derive from enhanced sensitivity to AMPs.

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FIG. 7.

Encapsulated S. iniae is more vulnerable to cationic antimicrobial peptides the ΔcpsD capsule mutants but equally susceptible to hydrogen peroxide killing. (A and B) Antimicrobial peptide kinetic killing profiles for 1.5 μM moronecidin (A) and 60 μM polymyxin B (B) indicate decreased sensitivity to AMPs for the K288 ΔcpsD mutant compared to wild-type K288 S. iniae. Similar results were observed for the 94-426 ΔcpsD mutant. (C) No biologically significant difference in survival of WT K288 and K288 ΔcpsD is observed following 1-, 2-, and 3-h incubations with hydrogen peroxide. Similar results were observed for the 94-426 ΔcpsD mutant. Data are presented as mean ± SEM.

Loss of S. iniae capsule expression does not affect hydrogen peroxide sensitivity.An additional mechanism for phagocyte control of bacterial pathogens is reactive oxygen species generated through the oxidative burst. We compared the sensitivities of WT S. iniae strains and the isogenic mutants to killing by hydrogen peroxide and observed no biologically significant differences (Fig. 7C), suggesting that avoidance of oxidant killing mechanisms does not explain the contribution of capsule to S. iniae survival in the whole-blood and macrophage killing assays.

Capsular polysaccharide expression by S. iniae impedes phagocytotic uptake.Further investigations were performed to determine the step at which S. iniae capsule expression interfered with phagocyte killing, using a cultured fish macrophage cell line. The macrophages bound (Fig. 8A) and internalized (Fig. 8B) the capsule-deficient mutants much more efficiently than WT S. iniae. Upon phagocytosis by the macrophages, both WT and ΔcpsD mutant strains were rapidly and effectively killed intracellularly (Fig. 8C). Thus, the contribution of the S. iniae capsule to resisting phagocytic clearance was through impeding phagocytosis, not enhancing intracellular survival.

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FIG. 8.

Capsule hinders phagocytosis of S. iniae but does not confer intracellular protection against phagocytic killing. (A) The capsule-deficient K288 ΔcpsD mutant has increased binding affinity to the surface of CLC compared to WT K288 (mean ± SEM). (B) The K288 ΔcpsD mutant also is phagocytosed by CLCs more rapidly than WT K288. Fluorescence imaging (magnification, ×400) shows phagocytosed intracellular bacteria labeled with FITC (green), and adherent extracellular bacteria are labeled with SYTOX Orange (red). (C) Once phagocytosed, the K288 ΔcpsD mutant and WT K288 are both effectively killed over time as seen through enumeration of viable intracellular S. iniae in CLCs at 2, 4, 6, and 20 h postincubation (mean ± SEM).