Viral encephalopathy and retinopathy cause up to 100% mortalities in juveniles of more than 40 finfish species including those most important to the European marine aquaculture industry such as sea bass (Dicentrarchus labrax
) and sea bream (Sparus aurata
]. All these diseases are caused by viral nervous necrosis viruses (VNNVs) which belong to the Nodaviridae
family within the betanodavirus
]. VNNVs are non-enveloped particles of icosahedral symmetry enclosing two single-stranded, positive sense RNAs. One of the RNAs encodes an RNA-dependent RNA polymerase, while the other encodes their coat protein (C protein). According to C gene-derived protein sequences, betanodavirus
isolates from Europe, Asia and Japan could be classified into 4 genotypes, but displaying only 19–23% differences among them [5
]. Most C proteins of geographically-related betanodaviruses share up to 98–99% of their amino acid sequence.
Different types of VNNV killed vaccines have been described [7
], including those made with inactivated virus [8
], VLP virus-like particles [10
], recombinant C proteins [13
], or synthetic peptides derived from the C protein [15
]. Most of those have to be delivered by fish-to-fish injection such as intraperitoneal injection of formalin-inactivated betanodaviruses [16
]. Thus, an oil-adjuvanted intraperitoneal injectable vaccine that protects 12 g sea bass against the RGNNV genotype for one year has been available for emergencies since 2014 and received market authorizations in 2018 in Spain, Italy, Croatia and Greece (https://www.pharmaq.no/updates/pharmaq-has-rec/
). Alternative innovative vaccination immersion protocols have been described for sea bass [17
], and specific antibodies were induced in grouper eggs by vertical transmission from broodfish injected with inactivated VNNV [18
]. Vaccination methods against nodaviruses and their corresponding immune responses in European sea bass have been recently reviewed [19
] including oral delivery alternatives such as those using inactivated bacteria encapsulating dsRNA from VNNV, and chitosan conjugated VNNV DNA [7
]. Most recently, protection has been reported by using alive recombinant bacteria expressing the C protein sequence mixed with the feed [20
]. Although the use of recombinant bacteria will be most appreciated for large scale oral vaccination by avoiding stressful, labour intensive and costly delivery, the release of alive genetically modified organisms (GMOs) will have practical problems. Thus, the presence of recombinant DNA and antibiotic resistance genes in alive or even in inactivated GMOs will raise safety concerns for sustainable aquaculture.
To investigate alternatives to live or dead recombinant bacteria, we have explored here a previously reported platform consisting of formaldehyde-inactivated recombinant bacteria displaying downsized viral antigens in their surface (called spinycterins) [21
]. Such spinycterins were obtained by genetic fusion of selected prokaryotic anchor-motifs to the N-terminal part of small linear immunodominant viral fragments. Despite the high reduction of antigenicity caused by formaldehyde crosslinking, successful production of anti-viral antibodies were demonstrated by immersion of ultrasound-treated zebrafish and/or carps in spinycterins displaying downsized CyHV-3 herpesvirus [21
]. Among the safety advantages, the spinycterin inactivated condition may allow also for lyophilization and/or addition into feeds, contributing also to bypass the low temperature-dependence of fish vaccines. However, several fine-tuning details need improvement to favor further development of spinycterins for small fish vaccines. First, there is no previous evidence that shows that any spinycterin displaying downsized VNNV antigens will induce protection against VNNV challenge [21
]. Second, crosslinking by formaldehyde inactivation caused a ~80% antigenicity loss [21
]. Third, the yields of expression of some the anchor fusions were low or inhibited bacterial growth [21
]. Fourth, safety concerns may still remain when handling and releasing to the environment large amounts of recombinant bacteria and those need to be minimized even when using GMOs which may have some of their DNA intact despite inactivation. Therefore, improvements in the above-mentioned concepts were explored in spinycterins made with downsized VNNV antigens.
Because the fish host VNNV antigenicity is focused on its coat (C) protein, downsizing of the C protein was performed as a means to increase its expression levels in recombinant E. coli
while maintaining the immunogenic potential of the antigen [22
]. To provide for bacterial surface display, several prokaryotic membrane anchor-motifs were fused to the downsized C protein. The anchor-motifs employed in this work, included those used before [21
] and the P9 anchor-motif identified in the envelope of phage ϕ6 [24
]. Because of the importance of nodaviruses in the aquaculture of commercially important fish species such as sea bass and sea bream, we chose one of them (sea bass) to validate protection of spinycterins against the VNNV challenge.
To preserve the initial immunogenicity of recombinant bacteria in the resulting spinycterins, several alternative methods to formaldehyde inactivation were explored. Among the many alternatives described before, bactericidal drugs appeared to be an attractive possibility since they allow for 100% of preservation of antigenicity, while maintaining intact the recombinant bacterial morphology and their inherent adjuvanticity. Among the described bactericidal drugs, those that target double-stranded DNA, introducing stable breaks into its strands by covalent binding and causing dead by oxidative ROS (i.e., bacterial DNA gyrase inhibitors), were preferred in contrast to those targeted to bacterial DNA-dependent RNA synthesis (i.e., rifampicins), cell-wall envelopes (wall synthesis inhibitors), and/or bacterial protein translation (synthesis inhibitors) [25
]. Therefore, we explored the possibilities of some of these drugs to irreversibly inactivate surface displaying bacteria in a cost-efficient manner preferably by damaging their double stranded DNA by introducing stable breaks.
In addition, to increase safety we explored the DNA-repair deficient BLR(DE3) strain. In contrast to the BL21(DE3) E. coli
, the derived BLR(DE3) strain cannot repair double DNA strand breaks, nor revert antibiotic-dependent ROS oxidation damage, thus making their derived recombinants more susceptible to DNA inactivation methods [26
]. Additionally, the BLR(DE3) strain is resistant to tetracycline (TetR) which is more convenient for large-scale manufacturing because it makes possible to reduce any possible contaminant bacterial growth. Furthermore, BLR(DE3) requires Isoleucine (Ileu-
) in the culture media to grow [27
], opening the possibility to develop antibiotic-independent recombinant selective methods to reduce the possibilities to spread resistant genes. All these characteristics make BLR(DE3) highly advantageous for large-scale production, but it is not yet known whether BLR(DE3) can be used to produce spinycterins.
The results obtained in this work showed that a new bacterial culture media containing soy-bean rather than casein hydrolysates made anchor-antigen expression more reproducible by delaying autoinduction and eliminating the IPTG requirement. In addition, Ciprofloxacin inactivation irreversibly damaged the DNA of the recombinant bacteria by covalent binding to the DNA strands and killed the bacteria by oxidative ROS mechanisms while preserving all antigen immunogenicity, therefore increasing safety during both manipulations and delivery of the resulting spinycterins. Finally, BLR(DE3) was a good substitute for BL21(DE3), adding another safety level. Because of all these properties, irreversibly DNA-damaged recombinant BLR(DE3) displaying downsized viral antigens may be used to further develop new adjuvant-less spinycterin vehicles for VNNV antigens in an environmental safer way. These spinycterins may not only contribute to move ahead the state-of-the art of small fish viral vaccinology but also other veterinary vaccination procedures.
This work describes 100% protection of sea bass juveniles against VNNV challenge by spinycterin vehicles and a direct correlation between bacterial surface exposure and fish protection levels. The high level of protection was obtained in the absence of adjuvants with irreversibly DNA-damaged DNA-repair-less spinycterins. Furthermore, to our knowledge, this is the first time that inactivated bacteria displaying a recombinant cystein-free downsized C VNNVN-terminal antigen (frgC91–220
) containing most of the epitopes targeted by fish neutralizing antibodies (B-cell epitopes) have been described for inducing protection against VNNV challenge. The frgC91–220
was fused to several prokaryotic membrane anchors to select the ones with higher membrane expression in E. coli
. A correlation between the levels of bacterial surface expression and fish protection was demonstrated by comparing the corresponding data obtained with YBEL + frgC91–220
spinycterins (higher frgC91–220
surface display and full protection) with those of Nmistic + frgC91–220
surface display and partial protection) and frgC91–220
surface display and lowest protection) spinycterins. These relatively high protection levels were obtained despite the selected frgC91–220
being located outside of the most important shell protrusion C-terminal domain (P domain, amino acids 214–338) in both VLP [43
] and whole virus [31
], which could had been expected to be more antigenic based only on predicted structural criteria (Figure 1
On the other hand, to improve spinycterin manufacturing, yields, reproducibility and safety, the following strategies were combined: a novel and scalable autoinduction soy-bean based media for E. coli expressing recombinant proteins under the control of the T7/lac promoter, inactivation through an irreversible DNA-damage alternative to traditional crosslinking inactivation, and a DNA repair-less E. coli strain as chassis.
A new auto-induction medium for BL21(DE3) E. coli
culture was developed based on previous reports to reduce overexpression toxicity of some recombinant proteins [40
]. Thus, we had previously found that the expression of some of the anchor-fusions to immunogenic proteins in E. coli
under the T7/lac promoter control grown in bacterial culture media based on casein hydrolysates, such as LB or TB, were partially or totally inhibited, apparently due to toxicity during the growth phase [21
]. Since such toxicity may be caused by early autoinduction of E. coli
due to the presence of lactose in the casein hydrolysates during their fast growth rate [40
], we undertook a series of experiments to reduce the residual lactose content of the culture media. Those experiments lead us to develop the so-called SB medium, a bacterial culture medium based on vegetable soy-bean rather than casein hydrolysates of animal origin. To further reduce autoinduction, glucose was also added to the media as previously recommended [40
]. Using the SB media, the highest expression levels by PAGE/Western blotting were obtained for the YBEL + frgC91–220
construct among 6 other anchor-motif alternatives. Similar higher expression results were previously reported for YBEL + frgIICyHV3
when compared to other 6 anchor-motifs in spinycterins grown in TB media [21
]. Although their growth rate was slower in SB medium, detectable levels of recombinant protein expression could be obtained for the 6 anchor-motifs studied (E. coli
, B. subtilis
, phage), in contrast to the problems with some of their yields previously obtained when using the TB medium [21
]. The YBEL + frgC91–220
construct remained with the highest level of expression among the anchor-motifs studied when cultured in SB or TB media.
After the studies on the bacterial culture media, experiments were focused in finding inhibitors of DNA replication by searching for an alternative to crosslinking for bacterial inactivation which destroyed ~80% of the bacterial surface displayed frgIICyHV3
] or most of the anti-polyH binding of YBEL + frgC91–220
spinycterins (this work). Among the possible anti-bacterial drugs s screened for inactivation, those targeting DNA replication focused in their supercoiling steps, such as those belonging to the quinolone family, appeared to be the best alternative, thus quinolones target unwinding DNA gyrase or topoisomerase II (Gram-negative bacteria) and topoisomerase IV (Gram-positive bacteria) enzymes, by interacting with double stranded DNA, covalently binding to cleavaged DNA [45
] and stopping strand rejoining during DNA replication [45
]. Additional quinolone bactericidal irreversible effects are induced by the generation of harmful hydroxyl radicals or ROS [46
]. The above-mentioned topoisomerases are essential in bacteria but absent in higher eukaryotes, making them an attractive possibility for the present purposes. The best studied gyrase is that from E. coli
, which has A and B subunits. The A subunit cleaves and covalently binds DNA strands, while the B subunit rejoins the strands. Inhibition of further strand cleavage/rejoining by stabilisation of the covalent gyrase-DNA complex (gyrase poisoning) shows concentration-dependent bacteriostatic or bactericidal effects [45
]. For instance, the Ciprofloxacin (CPFX) quinolone exhibits a bacteriostatic reversible activity at minimal concentrations and an irreversible bactericidal activity at higher concentrations [47
]. First-generation quinolones derived from nalidixic/oxolinic acids are rarely used today because of their toxicity to eukaryotic cells. Second (i.e., Ciprofloxacin), third (i.e., Levofloxacin) and fourth (i.e., Gemifloxacin) generation quinolones are clinically used. After numerous experiments, Ciprofloxacin was selected for this work because of its high activity at low concentrations, its covalent linking to cleavaged DNA strands, the induction of irreversible DNA-damage (bactericidal) and its low cost. Because this is an area of intensive research, new quinolones may appear in the future to cause irreversible DNA-damage of recombinant bacteria with even lower concentrations which will reduce possible concerns about the use of antibiotics to inactivate bacteria. Among the reasons mentioned above, CPFX was preferentially chosen because it cleaves the double-stranded DNA by covalently linking itself to the DNA strands [45
] in a related mechanism to that of formaldehyde/paraformaldehyde crosslinking. The low concentration required and the final washing steps that remove any excess of CPFX, reduces the possibility of free CPFX being released to the environment, very much like it does with crosslinked fish vaccines. In addition, CPFX induces a ROS-dependent bacterial killing effect without affecting antigenicity, in contrast to crosslinking [21
]. To enhance safety, it may be possible to add formaldehyde/paraformaldehyde at low concentrations to some the spinycterins or to the CPFX-inactivated spinycterins, provided a crosslinking-resistant anchor-motif is used (for instance, in the case of paraformaldehyde with Nmistic+frgC91–220
spinycterins). Therefore, in the case the use of CPFX may be rejected because of being an antibiotic, other linking/crosslinking compounds could be further explored as alternatives for spinycterin inactivation. In each particular combination of anchor-motif and immunogen care should be taken as to preserve surface display and antigenicity. In this work, we have focused on describing a minimal proof-of-concept prototype of a fish vaccine alternative platform which should be further studied by other inactivation procedures, alternative mass delivery techniques and including host innate and adaptive immune responses. In its present state-of-the-art, the described spinycterin adjuvant-less vehicles require further work to be practical.
Even though some fish vaccines based in eukaryotic expression plasmids (i.e., DNA vaccines) have been recently approved, their use is still highly controversial in Europe [49
]. Therefore, using immune-relevant viral protein antigens rather than DNA may still be an alternative. Furthermore, protein antigens coded in prokaryotic rather than eukaryotic plasmid vectors (like those employed for DNA vaccines), offer safer environmental possibilities. Both of the above commented properties and the maintenance of bacterial morphology in the spinycterins described here for substituting oil-adjuvants- [50
] allow easier mass delivery and lower production costs. Because of the lack of cysteins in the displayed antigen, the spinycterins described here may be also looked as a method to reduce the generation of low-immunogenicity inclusion bodies during manufacturing very often found when expressing whole heterologous proteins in recombinant bacteria. Although recent results suggested that isolated nanopellets derived from bacterial inclusion bodies may also be immunogenic [54
], their practical use would need additional purification steps, losing their bacterial morphology and their adjuvant properties. Most probably, the use of inclusion bodies as fish vaccines will require too high concentrations. Recently, however, intact recombinant bacteria carrying their inherent adjuvanticity and coding for the whole C VNNV protein have been reported to induce partial protection against VNNV together with very low levels of antibodies when orally delivered to sea bass [20
]. Furthermore, injection of extracts corresponding to 1010
cfu of such recombinant bacteria per fish fully protected against VNNV challenge [20
]. In this context, there are some practical advantages of the DNA-damaged recA- E. coli
alternative described here when coding for surface-displayed downsized viral antigens (spinycterins) in comparison with the recombinant wild-type E. coli
coding for the whole C VNNV protein [20
]. For instance, as shown in this work, the injection of only 108
cfu per fish of morphologically intact YBEL + frgC91–220
spinycterins fully protected against VNNV challenge in the absence of any adjuvants. Furthermore, spinycterins may be better accepted in aquaculture because they are safer due to their DNA-repair deficient E. coli
vehicle. Other advantages may be due to the antigen downsizing concept providing a higher immunorelevant epitope density for a given mass of bacteria, and the future possibility to use spinycterins expressing mixes of different pathogen antigens in a single delivery. Additionally, because of its isoleucine deficiency, the BLR(DE3) E. coli
strain opens up the possibility of future developing of antibiotic-free selection methods to eliminate any antibiotic resistance sequences from the vaccine vehicles and thus further increase their environmental safety.
AccNum, Gene Bank accession numbers. FrgC91–220,
amino acid residues 91–220 from the C coat protein of viral nervous necrosis virus VNNV (sequence accession number AY284959) [22
]. The anchor-motif + GSGS + frgC91–220
+ GSGS + polyH DNA sequences were designed, synthesized, cloned into pRSET, used to transform E. coli
and autoinduced in SB medium. KDa, expected molecular weight of the recombinant proteins. Mistic and N-mistic, 110 and 33 N-terminal amino acids anchor-motif from the Mistic gene from Bacillus subtilis.
NTD, N-terminal domain of 21 amino acids anchor-motif of the exosporal BclA protein from Bacillus anthracis
. P9, 90 amino acid anchor-motif from the coat-protein of bacteriophage ϕ6. YAIN, 91 amino acid anchor-motif from the hydrophilic regulatory protein of the frmR operon of E. coli
YBEL, 160 amino acid anchor-motif from the hydrophilic HTH-type transcriptional regulator DUF1451 family protein from E. coli
(A) Hydropathicity plot of the coat protein C from the D. labrax
encephalitis virus isolate DL-040899-IL (AY284969) obtained using Clone Manager vs 9. Shell (S, blue rectangle) and protrusion (P, red rectangle) domains were located at amino acid residues 52–213 and 221–338, respectively, according to X-ray data [31
]. Blue top rectangle, frgC91–220
(amino acid residues 91–220). Blue horizontal lines inside the plot, neutralizing B-cell epitopes localized by pepscan mapping targeted by sera from sea bass surviving VNNV infection and by anti-VNNV neutralizing monoclonal antibodies [23
] or by alanine-scanning mutagenesis [22
]. Red circles, cysteine positions which were mutated to serines in the recombinant frgC91–220
. Green squares, Ca++
binding sites for subunit-subunit interactions in the betanodavirus shell structure [31
]. Grey hatched rectangle, highest protein sequence variability among betanodavirus isolates corresponding to the 223-331 amino acid positions [5
]. (B) Scheme of the tridimensional structure of the C protein of the AY284969 isolate. The automatically predicted modelled structure of the C protein of the AY284969 isolate (using the Swiss model server), selected the 4WIZ.3.A sequence as the best template. The template was derived from a Grouper Nervous Necrosis Virus isolate [31
] with a 99.11% of amino acid sequence identity.
The nucleotide sequences corresponding to the frgC91–220
of VNNV were fused downstream to each of the 6 bacterial membrane anchor-motif sequences described in Table 2
, bracketed by an arbitrarily chosen flexible linker (coding for amino acids GliSerGlicSer, GSGS). All the corresponding synthetically fused DNA sequences (GeneArt, Regensburg, Germany) were cloned into the multiple cloning site of the pRSET prokaryotic expression plasmid using the NdeI and HindIII restriction sites and adding 6 Histidine tails (polyH) at their C-terminal ends. Red, FrgC91–220
. Blue triangles, Anchor-motifs. Yellow, schematic bacterial membrane. Blue lines, C-terminal polyH tail.
(A) BL21 (DE3) E. coli
coding for anchor-motif+frgC91–220
+ polyH recombinant proteins were grown in autoinduction SB medium overnight. Bacteria were pelleted and their extracts analysed by Coomassie-blue staining. One representative experiment is represented. (B). Densitometry of the anchor-motif+frgC91–220
recombinant bands stained by Coomassie by Image J 1.41o software (http://rsb.info.nih.gov/ij
). Means and standard deviations are shown (n
= 3). (C) Western blotting of gels transferred to nitrocellulose membranes, stained with peroxide-labeled anti-polyH monoclonal antibody and visualized with DAB as described [21
]. One of 3 experiments was represented. Numbers to the right of the gels, KDa positions of molecular weight markers. The anchor-motifs of the recombinant E. coli
in A and C corresponded to lanes: 1, empty plasmid. 2, frgC91–220
. 3, Mistic + frgC91–220
. 4, Nmistic + frgC91–220
. 5, NTD + frgC91–220
. 6, P9 + frgC91–220
, 7, YAIN + frgC91–220
. 8, YBEL + frgC91–220
To select for a suitable drug to inactivate E. coli BL21(DE3) without altering their immunogenicity, several antibiotics and/or base analogs were tested for YBEL + frgC91–220 spinycterin replication. The recombinant bacteria (3 × 1010 cfu/mL) were exposed overnight to several concentrations of the selected compounds in 150 µL of TB with continuous agitation. After 2 washes with PBS, 10 µL of the suspensions were inoculated into 100 µL of fresh TB medium. Bacterial growth was estimated by absorbance at 540 nm after overnight incubation with agitation at 37 °C. Upper-half open red circles, Oxolinic acid. Left-half open red circles, Levofloxacin. Solid red circles, Ciprofloxacin. Open squares, 5-Bromo deoxiuridine. Solid blue squares, 5-Fluoracin. Open triangles, 6-Thioguanine. Open stars, Rifampicin. Solid starts, Mitomicin C.
coding for anchor-motif + GSGS+frgC91–220
+ GSGS + polyH recombinant proteins was obtained in either BL21 (DE3) or in the repair-deficient recA-
BLR(DE3) E. coli
strains and grown in SB medium overnight. The E. coli
BL21 (DE3) were grown at 37 °C and induced with IPTG at 24 °C for 2 h. The E. coli
BLR(DE3) were grown and autoinduced for additional 4 days at 37 °C. The resulting suspensions were incubated at 24 °C for 2 h with 125 µg/mL of Ciprofloxacin (CPFX) for irreversible inactivation to generate spinycterins. Extracts were analysed as described in Figure 3
. One of 2 experiments was represented. Numbers to the right of the gels, KDa positions of Coomassie blue stained molecular weight markers. Lanes: 1, frgC91–220
spinycterins. 2, empty pRSET plasmid spinycterins. 3, Nmistic + frgC91–220
spinycterins. 4, YBEL + frgC91–220
(A) To assay by polyacrylamide gel electrophoresis (PAGE), the amounts of the corresponding stained bands were compared between trypsin-digested and control undigested BLR(DE3) spinycterins. The optical densities were first normalized by the formula, optical density of the recombinant bands/total optical density of each spinycterin extract. The normalized optical densities were then calculated relative to the optical density obtained in the frgC91–220 spinycterin bands by the formula, 100 × (% of anchor + frgC91–220/% of frgC91–220). Means and standard deviations (n = 3) were represented. (B) To assay by ELISA, 96-well plates were coated with trypsin-digested or control undigested BLR(DE3) spinycterins. The amount of exposed polyH tails was estimated by binding to peroxidase-conjugated anti-polyH monoclonal antibodies. Percentage of polyH-binding was calculated by the formula, 100 × (Absorbance after trypsin digestion/Absorbance of undigested samples). The percentage of absorbance was then calculated relative to the frgC91–220 spinycterins by the formula, 100 × (absorbance of anchor + frgC91–220/absorbance of frgC91–220. Means and standard deviations (n = 3) are presented. Red horizontal dashed lines, mean optical density (A) and absorbance (B) of frgC91–220 spinycterins *, significatively different from frgC91–220 spinycterins as determined by the Student t-test (p < 0.05).
Two independent aquaria per group, each containing 15–20 fingerling sea bass (D. labrax) of ~10 g of body weight were injected with 108 cfu of frgC91–220, Nmistic + frgC91–220 or YBEL + frgC91–220 spinycterins. Non-infected, injected with empty pRSET plasmid and non-immunized controls were also included. Viral challenge was performed by intramuscular injection of VNNV (2 × 104 TCID50/mL). Mortality in the non-immunized VNNV-challenged group controls was 25.7%. Relative percentages of survival were calculated by the formula 100–(percentage of mortality in the spinycterin-immunized VNNV-challenged fish/percentage of mortality in the non-immunized VNNV-challenged fish). *, significantly different from the frgC91–220 survival by the Log-Rank (Mantel-Cox) test at the p < 0.05 level. Open circles, fish injected with frgC91–220 spinycterins. Closed triangles, fish injected with Nmistic + frgC91–220 spinycterins. Closed circles, fish injected with YBEL + frgC91–220 spinycterins.
To preliminary experiment with some drugs to inactivate E. coli BL21(DE3) some quinolones (Ciprofloxacin, Levofloxacin, Oxolinic acid) and rifampicin were tested for colony formation of IPTG-induced YBEL + frgC91–220 spinycterins on 1.5% agar plates in TB medium containing ampicillin (A) and for preservation of the level of recombinant protein by polyacrylamide gel electrophoresis stained with Coomassie blue (B). The recombinant bacteria (3 × 1010 cfu/mL) were exposed during 2 h to 20 or 200 µg/mL of the selected quinolones and 10-fold lower concentrations of rifampicin. The plates were divided in two halves, plated with 5 (left) or 50 (right) µL of antibiotic-treated bacterial suspensions, incubated overnight at 37 °C and photographed. Red left arrow, YBEL + frgC91–220 recombinant protein.
Suspensions of 0.5 × 109 cfu/mL of BLR(DE3) spinycterins in phosphate buffered saline (PBS) were agitated with a final concentration of 4% of paraformaldehyde for 20 h at 4 °C. Parallel suspensions of spinycterins were treated with PBS. The spinycterins suspensions were then quenched for 2 h with saturated glycine in PBS. Several concentrations of the resulting spinycterins were used to coat polyLys(D) 96-well plate solid-phases. (A), photomicrography of solid-phase bound spinycterins at 100 × 106 cfu/mL (each bacteria length corresponded to ~2 µm). (B) The amount of exposed polyH tails was estimated by binding to peroxidase-conjugated anti-polyH monoclonal antibodies. The absorbance at 492 nM reflects the colour conversion of the OPD substrate. The absorbance at 620 nm was used to correct for individual well differences. Representative results of one of two experiments have been represented. Solid circles, YBEL + frgC91–220 spinycterins. Open circles, YBEL + frgC91–220 spinycterins treated with paraformaldehyde. Solid triangles, Nmistic + frgC91–220. Open triangles, Nmistic+frgC91–220 spinycterins treated with paraformaldehyde. Anti-polyH binding of spinycterins transformed with the empty pRSET plasmid showed Absorbances of ~0.5.