Bioengineering a bacterial pathogen to assemble its own particulate vaccine capable of inducing cellular immunity

Many bacterial pathogens naturally form cellular inclusions. Here the immunogenicity of polyhydroxyalkanoate (PHA) inclusions and their use as particulate vaccines delivering a range of host derived antigens was assessed. Our study showed that PHA inclusions of pathogenic Pseudomonas aeruginosa are immunogenic mediating a specific cell-mediated immune response. Protein engineering of the PHA inclusion forming enzyme by translational fusion of epitopes from vaccine candidates outer membrane proteins OprI, OprF, and AlgE mediated self-assembly of PHA inclusions coated by these selected antigens. Mice vaccinated with isolated PHA inclusions produced a Th1 type immune response characterized by antigen-specific production of IFN-γ and IgG2c isotype antibodies. This cell-mediated immune response was found to be associated with the production of functional antibodies reacting with cells of various P. aeruginosa strains as well as facilitating opsonophagocytic killing. This study showed that cellular inclusions of pathogenic bacteria are immunogenic and can be engineered to display selected antigens suitable to serve as particulate subunit vaccines against infectious diseases.

Many bacteria including various human pathogens form polymeric intracellular inclusions such as e.g. polyhydroxyalkanoate (PHA) inclusions which serve as energy and carbon storage material 1,2 . While cell surface structures of pathogens had been the focus of studies towards identifying vaccine candidate antigens, the immunogenicity of intracellular structures had not been studied. However nano-/microsized intracellular structures such as polymer inclusions might serve as particulate vaccines suitable for efficient antigen delivery. Particulate antigen delivery systems are being increasingly considered for vaccine formulations evidenced by recent successful application and commercialization of particle-based vaccines 3,4 . PHA beads had been previously shown to enable delivery of antigens inducing protective immunity in animal models against tuberculosis 5,6 and hepatitis C 7,8 . PHAs are deposited as spherical cytoplasmic inclusions surrounded by proteins 1,9 . Protein engineering of one of these coating proteins, the PHA synthase (PhaC Re ), which catalyzes polyhydroxybutyrate (PHB) formation 10-13 enabled antigen display on PHB beads inducing a specific and protective immune response 5,8,14,15 . Vaccine candidate antigens formulated as particles (< 1 μ m) showed enhanced immunogenicity due to an efficient cellular uptake by professional antigen presenting cells 16 .
Here we selected Pseudomonas aeruginosa as a model human pathogen because it naturally forms PHA inclusions and traditional vaccine development approaches were unsuccessful 17 . Its PHA is composed of medium chain length 3-hydroxy fatty acids (MCL) which polymerization is catalyzed by the MCL-PHA synthase (e.g. PhaC1 Pa ) 1,2 .
P. aeruginosa is one of the leading causes of nosocomial infections and causes serious life-threatening infections due to intrinsic and acquired antibiotic resistances 17 . Immuno-compromised individuals are most at risk, such as those with severe burns and wounds, infected by human immunodeficiency virus (HIV) as well as cystic fibrosis (CF) patients 18 . Vaccines provide a strategy for prevention of the disease caused by P. aeruginosa 19 .
Vaccine candidates include outer membrane proteins (OMPs), flagellin and pilin, toxins as well as killed or live attenuated whole cells 17,20 . The most promising immunogens are the major OMP F (OprF) and outer membrane Scientific RepoRts | 7:41607 | DOI: 10.1038/srep41607 lipoprotein I (OprI) which are highly conserved, serotype independent and well tolerated 21,22 . Vaccination studies in animals have shown long-lived antibody titers and broad protection against all P. aeruginosa serotypes 23 . However high levels of antibodies were associated with more severe lung disease 24 . It has been suggested that a CD4 + Th1 type cell mediated response maybe more protective [24][25][26] , and that OprI vaccination can modulate the immune response from a CD4 + Th2 towards a CD4 + Th1 cell mediated response 27 . OprI vaccination induced protection in mice 28 . OMP AlgE, the alginate pore, may provide an alternative target for vaccine development. AlgE is overproduced in the mucoid alginate overproducing variant found in the lung of CF patients and has been suggested to be immunogenic 29,30 . The crystal structure of AlgE revealed a 18-stranded β -barrel with extended extracellular loops representing possible cell surface exposed antigenic epitopes 31,32 . The use of immunogenic epitopes of OprF fused with OprI have been the main candidates for use in P. aeruginosa vaccine studies 21,22,33 , and have shown synergistic effects 34 .
In this study we investigated the immunogenicity of cellular inclusions formed by the human pathogen, P. aeruginosa. Immunological properties of PHA inclusions encouraged to engineer P. aeruginosa for the production of antigen-displaying PHA inclusions by harnessing its inherent PHA production system. These PHA inclusions were engineered to display selected vaccine antigens of the same host at high density while associated host cell components might serve as additional antigens enhancing the induction of broadly protective immunity and/or having adjuvant properties. This is the first study investigating the immunological properties of cellular polymer inclusions of pathogenic bacteria and to utilize the pathogens own inclusions as carrier of its own antigens to be used as a particulate vaccine.

Results
Bioengineering of P. aeruginosa for self-assembly of antigen-displaying PHA inclusions. To enable the production of antigen-associated PHA MCL inclusions mediated solely by the introduced PHA synthase (PhaC1 Pa = non-engineered wildtype) and its fusion protein derivatives (engineered to incoprarate vaccine candidate antigens), an isogenic PHA MCL deficient strain PAO1 Δ phaC1ZC2 was employed. To promote production of PHA MCL and the vaccine candidate exopolysaccharide (EPS) Psl, essential genes for competing biosynthesis pathways towards the production of alginate and the glucose-rich Pel polysaccharide, respectively, were deleted ( Fig. 1a-c and Supplementary Fig. 1) 35 .
Formation of PHA MCL inclusions mediated by recombinant PhaC1 Pa (natural wildtype inclusions) or PhaC1 Pa -GFP was assessed by fluorescence microscopy, GC/MS, and immunoblot analysis (Fig. 2a-d). In order to assess whether PhaC1 Pa tolerates C terminal translational fusions, GFP was fused to its C terminus. A designed Figure 1. A schematic of the generation of P. aeruginosa knockout mutant PAO1 ΔCΔ8ΔF. In order to promote the production of PHA MCL inclusions and vaccine candidate EPS Psl, site-directed homologous recombination was used to delete major parts of (a) alg8 and (b) pelF genes encoding a glycosyltransferase in the PHA negative mutant PAO1 Δ phaC1ZC2. (c) Resultant triple mutant strain is defective in PHA/alginate/pel polysaccharide was verified by DNA sequencing (see Supplementary Fig. 1).
Scientific RepoRts | 7:41607 | DOI: 10.1038/srep41607 linker 10 was inserted in order to retain functionality of PhaC1 Pa and to display the fusion partner GFP on the surface of PHA MCL beads (Fig. 2a). Colocalization of fluorescent foci for GFP and PHA MCL visualized within PAO1 Δ CΔ 8Δ F cells producing PhaC1 Pa -GFP indicated that the GFP fusion to C terminus of PhaC1 Pa did not abolish PhaC1 Pa activity and implies that GFP fused to the C terminus of this class II PHA synthase was functionally displayed on the surface of the PHA MCL inclusion in vivo (Fig. 2b).
Display of GFP on PHA inclusions anchored via fusion to the C terminus of PhaC1 Pa was observed by fluorescence microscopy expanding the scope of PhaC1 Pa engineering to C terminally fusible antigens. Selected epitopes of the OMPs OprF, OprI (lipoprotein), and AlgE (alginate secretion porin) from P. aeruginosa were used as vaccinate candidates to be immobilized to the surface of PHA inclusions. Selection of antigenic epitopes of OprF and OprI was based on previous studies that demonstrated protective immunity in animal models ( Supplementary Fig. 2a). Antigenic epitopes of AlgE were selected based on its structure using B-cell antigenic epitope prediction method EPCES ( Supplementary Fig. 2b).
Antigenic epitopes of AlgE, OprF, and OprI were combined as a single fusion antigen (OprI/F-AlgE) and translationally fused to either the N or C terminus of PhaC1 Pa and the impact on the production of PHA MCL inclusions as well as the functionality of the fusion partners was analyzed (Fig. 3a-c).
Whole cell lysates and isolated PHA MCL beads contained dominant proteins with an apparent molecular weight of 62.5 kDa (PhaC1 Pa ), 77.75 kDa (Ag-PhaC1 Pa ) and 79.74 kDa (PhaC1 Pa -Ag) (Fig. 4a), and their identity was confirmed (Fig. 4b,c and Supplementary Table 1). Densitometry analysis indicated that PhaC1 Pa , Ag-PhaC1 Pa , and PhaC1 Pa -Ag fusion proteins accounted for 8.8%, 12.3%, and 9.9% of total PHA MCL bead associated protein, respectively (data not shown). Several additional copurifying host cell proteins (HCPs) were detected within the bead material (Fig. 4b,c). The major copurifying eleven proteins (Fig. 4a) were selected (labeled I-XI) for identification and peptides belonging to P. aeruginosa proteins for each protein band ( Fig. 4d and Supplementary Table 2) were ranked based on combined score i.e. -log(P) value. Database hits included the PHA synthase, OMPs (i.e. OprI and OprF), ribosomal proteins, naturally bead associated proteins (i.e. PhaI and PhaF), and heat-shock proteins. Some of these OprI, OprF, and AlgE related protein bands were additionally confirmed by immunoblot analysis (Fig. 4c). Interestingly, tryptic peptides of the PhaC Pa were identified in the majority of protein bands as the best hit (Fig. 4d, bands I-IV and VII-XI) and second best hits (Fig. 4d, bands V-VI). Identified PhaC1 Pa peptides suggested some degradation from the C terminus. Immunoblot analysis using the antibody anti-PhaC1_1 detected co-purifying protein bands I, II, VII, and VIII, while anti-PhaC1_67 detected protein bands I, VII, and VIII indicating some degradation of the fusion protein.
Antibody detected epitopes were aligned with peptides identified by MALDI-TOF MS for each band and showed that anti-PhaC1_1 recognized epitopes exhibited a coverage of 22% for bands I, II and VII and 71% coverage for band VIII, while anti-PhaC1_67 showed full epitope coverage for bands I and VIII and partial coverage of 17% for band VII (Supplementary Table 3). Alignment of the anti-PhaC1_529 recognized epitope with peptides identified by MALDI-TOF MS in the eleven bands showed that the respective epitope was absent.
The recombinant soluble fusion antigen was produced and purified as shown in Supplementary Fig. 3.
Immunological response to vaccination with antigen-displaying PHA MCL beads. C57/BL/6 mice were vaccinated with 20 μ g of PhaC1 Pa attached to beads or 20 μ g OprI/F-AlgE antigen immobilized to beads (Ag-PhaC1 Pa or PhaC1 Pa -Ag) formulated in saline without alum adjuvant. Following vaccination, no obvious adverse effects were observed in any of the animals, with mice gaining weight in all groups. PHA MCL beads stimulated the generation of OprI/F-AlgE antigen specific IgG2c antibodies, indicating a Th1 dominant response (Fig. 5a). The greatest response was obtained in groups vaccinated with 20 μ g of OprI/F-AlgE antigen immobilized to PhaC1 Pa beads or 20 μ g PhaC1 Pa immobilized to beads. No significant difference, but a positive trend was seen between vaccinated groups receiving vaccine formulated with alum compared to their respective groups formulated without alum ( Supplementary Fig. 4a). All beads induced sera antibodies specific for epitopes in bead-associated proteins (HCPs, PhaC Pa and PhaC Pa antigen fusions) while this was not observed in the PBS or soluble antigen His 10 -Ag group (Fig. 5b). Reactivity of sera antibodies to whole cells of different P. aeruginosa strains were tested (Fig. 5c). Results suggest strong reactivity of sera antibodies in the bead vaccinated groups to both nonmucoid and mucoid strains of P. aeruginosa. The PhaC1 Pa vaccinated group showed the strongest overall responses compared to Ag-PhaC1 Pa or PhaC1 Pa -Ag bead vaccinated groups. Minimal responses were seen with PBS and recombinant protein vaccinated groups.
An opsonophagocytic killing assay testing serum antibodies from the vaccinated mice was also conducted (Fig. 5d). Serum antibodies from both PhaC1 Pa and PhaC1 Pa -Ag bead vaccinated groups showed significantly higher killing against strain PAO1 than serum antibodies from the PBS group and only PhaC1Pa vaccinated group showed significantly more killing than soluble antigen from the His 10 -Ag group (p < 0.05).
Cytokine responses of splenocytes to soluble recombinant protein His 10 -Ag ( Fig. 6a,b) showed that Ag-PhaC1 Pa beads induced production of significantly more IFN-γ , IL-10, IL-17a, and IL-6 than found in the PBS group, while the PhaC1 Pa -Ag beads induced significantly more IFN-γ and IL-2 ( Fig. 6a,b). Vaccination with 20 μ g of PhaC1 Pa on beads induced significantly more IFN-γ , IL-4, and IL-6 compared to the PBS group. While the His 10 -Ag group produced significantly more IFN-γ , IL-10, IL-17a, IL-2, and IL-4 compared to the PBS group ( Fig. 6a,b). Minimal cytokine responses were observed when splenocytes were re-stimulated with AlgE or OprI or OprF peptides or a combined pool of all peptides (data not shown). Antigens presented on PHA MCL beads did not induce cytokine responses significantly greater than the His 10 -Ag (Fig. 6a). Only the group vaccinated with 20 μ g of OprI/F-AlgE antigen on Ag-PhaC1 Pa beads produced significantly more IL-6 than the His 10 -Ag group. Notably, a positive trend was observed for cytokines IFN-γ and TNF-α for this Ag-PhaC1 Pa beads group and TNF-α for the 20 μ g PhaC1 Pa beads group when compared with His 10 -Ag group.
Attachment of antigenic proteins to the surface of the beads mediated an enhanced immune response. Significantly more IFN-γ was produced by mice vaccinated with 20 μ g OprI/F-AlgE antigen on Ag-PhaC1 Pa beads compared with the 20 μ g PhaC1 Pa beads group (Fig. 6a,b). Although not significant, a positive trend was observed for IFN-γ with the group receiving 20 μ g OprI/F-AlgE antigen on PhaC1 Pa -Ag beads compared to the Scientific RepoRts | 7:41607 | DOI: 10.1038/srep41607 PhaC1 Pa beads group. Comparatively, the N terminal fusion of OprI/F-AlgE antigen to PhaC1 Pa on Ag-PhaC1 Pa beads induced a greater cytokine response with significantly more IFN-γ , IL-10, IL-6, and TNF-α than its C terminal fusion counterpart, the PhaC1 Pa -Ag beads. No significant dose response difference was observed in mice receiving either 20 μ g or 5 μ g of OprI/F-AlgE antigen immobilized on PhaC1 Pa -Ag beads (Fig. 6a). The addition of adjuvant, alum, generally enhanced the immune response ( Supplementary Fig. 4b).  Supplementary Tables 1 and 2). Detected bands from b immunoblotting were overlaid and matched to specific bands on the Coomassie Blue stained gel (red square). Antibody detected epitopes were aligned with peptides identified by MALDI-TOF MS (see Supplementary Table 3). Major copurified protein bands from PHA MCL beads formed by PHA synthase antigen fusions were identified on SDS-PAGE (see Materials for criteria). (c) Immunoblot analysis of isolated PHA MCL beads using polyclonal antibodies raised against epitopes of OprF or OprI or AlgE. (d) Table  summarizing the Identification of the eleven PHA MCL bead associated HCPs by MALDI-TOF MS (see also Supplementary Table 2 Scientific RepoRts | 7:41607 | DOI: 10.1038/srep41607

Discussion
Many bacteria, including animal and human pathogens, are capable of producing spherical discrete PHA inclusions for carbon and energy storage 37 . Here we utilized the intrinsic PHA MCL synthesis capacity of the disease causing pathogen P. aeruginosa towards production of antigen-displaying PHA MCL beads. The aim was to display selected repeated epitopes of vaccine candidate antigens of P. aeruginosa at high copy number on the surface of the PHA storage granules 5,8,14 . Design and production of PHA beads in the respective pathogen potentially avoids the need for extensive downstream processing in order to remove host cell derived impurities such as HCPs. Impurities originating from the pathogen could be beneficial, by providing additional epitopes, i.e. a large antigen repertoire, and acting as an adjuvant towards enhanced protective immunity to infections caused by P. aeruginosa. P. aeruginosa strain PAO1 was genetically engineered (Fig. 1) by deleting key genes required for the synthesis of PHA, alginate, and pel polysaccharide to enable enhanced recombinant production of its own PHA MCL beads additionally coated with surface epitopes of outer membrane vaccine candidates AlgE, OprF, and OprI as an OprI/F-AlgE fusion antigen 28,30,38 . This was achieved by protein engineering of the P. aeruginosa PhaC1 Pa that catalyzes PHA MCL synthesis mediating PHA MCL bead assembly while remaining covalently attached to the surface of PHA MCL inclusions 39 . Various studies have shown great promise for epitopes of OprF and OprI to be used in vaccine formulations 21,22,34,40,41 .
The successful recognition and uptake of the vaccine PHA beads by professional antigen presenting cells (APCs) may differ due to the mode of display of the OprI/F-AlgE antigen being presented to immune cells. The tolerance of C terminal fusion was assessed by translational fusion with GFP ( Fig. 2a-d), while translational fusion to the N terminus of the PhaC Pa had been previously shown 42 . Hence the OprI/F-AlgE vaccine candidate (a) Antigen-specific IgG1 or IgG2c isotype antibody responses measured by ELISA using a pool of OprI, OprF, and AlgE antigen specific peptides from sera. Results are expressed in reciprocal antibody titers, representing the dilution required to obtain half of the maximal amount of the OD signal (EC 50 ). (b) To identify antigenic proteins on PHA MCL beads, sera obtained was pooled in to their respective groups and used as a primary antibody for detection of epitopes on PHA MCL beads separated by SDS-PAGE. (c) Reactivity of pooled immune sera to different P. aeruginosa strains using a whole-cell ELISA was used. Nonmucoid (grey bars) and mucoid (red bars) strains of P. aeruginosa were tested. (d) Opsonic killing of P. aeruginosa nonmucoid strains by serum from mice immunized with vaccine PHA MCL beads. Bar represents the mean percent killing of three replicates for PAO1 and PA14 or duplicates for PDO300 relative to sera of the PBS vaccinated control group, and error bars represents the s.e.m. Data of graph for IgG are reported as means ± s.e.m (6 mice per group). Statistical significance (p < 0.05) of IgG2c is indicated by 'letter based' representation of pairwise comparisons between groups using Tukey's post-hoc test. IgG1 were not statistically significant. Data of graph for whole-cell ELISA represent means of two replicates of pooled sera ± s.e.m of the replicates. There are insufficient replicates to undertake a statistical analysis. Statistical significance (p < 0.05) for opsonic killing assay is indicated by 'letter based' representation of pairwise comparisons between groups using Tukey's post-hoc test. PA14 were not statistically significant. There are insufficient replicates to undertake a statistical analysis for PDO300. antigen was fused to either the N or C terminus of PhaC1 Pa (Fig. 3a). Translational fusion of the OprI/F-AlgE antigen to the different termini of PhaC1 Pa impacted on PHA MCL bead size suggesting an impact of the fusion partner and fusion site on PHA bead assembly (Fig. 3b). Data also showed fusion to the N terminus or C terminus of PhaC1 Pa did not influence PHA composition i.e. substrate specificity of PhaC1 Pa , but reduced PHA MCL accumulation compared to the wildtype (PhaC1 Pa ) implying an impact of the fusion on in vivo activity (Fig. 3c). This impact had been found for PhaC Re and was dependent on the fusion partner 10,12,13 .
Significantly more fusion protein was produced if the OprI/F-AlgE antigen was fused to the C terminus (PhaC1 Pa -Ag) compared to N terminal fusion (Ag-PhaC1 Pa ) (Fig. 4a). However, the amount of fusion protein did not correlate with the amount of PHA accumulated i.e. PhaC1 Pa -Ag did not mediate greater levels of PHA MCL accumulation (Fig. 3c).
Successful control of disease caused by many extracellular pathogens typically requires an antibody response characterized by the production of IgG1 isotype and production of cytokines IL-4 and IL-5. However, this might not be the case for chronically P. aeruginosa infected CF patients who predominantly show a bias towards a Th2 type immune response when compared to noninfected CF patients or healthy controls 24,43 . Elevated levels of antibodies in CF patients tend to be associated with a poor prognosis. There is increasing evidence that a Th1 type immune response characterized by increased cytokine IFN-γ leads to better pulmonary outcomes and may be the preferred response for the successful control of acute and chronically infected CF patients 24,26,43 .
Here we showed that vaccination of mice with P. aeruginosa derived PHA MCL beads displaying the OprI/F-AlgE antigen fused to the N terminus of PhaC1 Pa (Ag-PhaC1 Pa ) without adjuvants induced a robust T cell immune response with a Th1 pattern. The immune response was characterized by enhanced production of IgG2c isotype titers and antigen-specific cytokine IFN-γ in association with low levels of IL-4 and IgG1 isotype that are both elevated in the Th2 type response. This suggested induction of a Th1 type response through enhanced CD4 + type 1 6 T cell and Toll-like receptor (TLR) activation 44 .
Vaccination with Ag-PhaC1 Pa beads induced significant levels of antigen-specific serum antibodies (Fig. 5a). These antibodies may have resulted from B cell class-switching to IgG2c isotype associated with the activation of Th1 antigen-specific T cells 45 , which conceivably playing a critical role in clearance of acute infection with P. aeruginosa 46 .
Vaccination with plain PhaC1 Pa beads without OprI/F-AlgE antigen also generated an antigen-specific antibody response to epitopes of the OprI/F-AlgE antigen at levels similar to the Ag-PhaC1 Pa bead group (Fig. 5a). This indicated an immune response to the copurified HCPs associated with plain PhaC1 Pa beads. For example, the full-length OMP OprF was identified in band III of the separated copurified proteins (Supplementary Table 2). OprI and OprF are present in high copy numbers in the OM of P. aeruginosa 47 and therefore, more likely to be found as part of the HCP impurities compared to the low copy number AlgE 29 . The detection of antibodies against a wide range of copurifying HCPs supported the concept of the immunogenic delivery of a large antigen repertoire using isolated PHA beads produced by the pathogen (Fig. 5b). Moreover, these serum antibodies in the bead vaccinated groups showed strong reactivity across different P. aeruginosa strains which include nonmucoid and mucoid variants (Fig. 5c). Opsonophagocytic killing activity of serum antibodies as an indication of protective immunity was also tested (Fig. 5d). Sera antibodies from all bead vaccinated goups could mediate around 20-30% killing to nonmucoid and mucoid variants of P. aeruginosa, with lower killing activity seen for soluble His 10 -Ag. The strongest biologically significant killing was mediated by sera antibodies from PhaC1 Pa vaccinated group showing opsonophagocytic killing (≥ 50% killing) against the nonmucoid PAO1 strain, possibly correlating with the strong reactivity of the sera antibodies to antigens of PAO1 (Fig. 5c). Although antigen epitopes were not engineered into the surface of PhaC1 Pa beads, evidence was obtained that outer membrane proteins were associated and hence might have contributed to this immune response (Fig. 4). The relatively low killing shown for all vaccine groups may have been due to high MOI of 100:1 in the assay and greater antibody-mediated enhancement of killing may have been seen at a lower MOI.
Alum added to PhaC1 Pa -Ag beads induced an increase in levels of antigen-specific antibodies ( Supplementary Fig. 4a) and a significant increase in some cytokines ( Supplementary Fig. 4b) suggesting further scope to enhance immunogenicity of PHA MCL beads.
Attachment of OprI/F-AlgE antigen to the PHA MCL beads enhanced the immune response with a bias towards a Th1 response when compared to vaccination with only antigen. Soluble peptides/proteins require addition of a suitable adjuvant and/or delivery system to generate an optimal immune response 48 . Our results showed that vaccination with His 10 -Ag formulated with alum adjuvant induced mainly a humoral Th2 type response ( Supplementary Fig. 4a,b).
Conversely, vaccination with OprI/F-AlgE antigen fused to the C terminus of PhaC1 Pa and displayed on PHA MCL beads induced a response similar to plain PHA MCL beads (PhaC1 Pa ), but a weaker response than observed when vaccinating with Ag-PhaC1 Pa beads (Figs 5a and 6a). This suggests the OprI/F-AlgE antigen fused to the C terminus of PhaC1 Pa may not be fully displayed on the surface of the PHA MCL beads. Due to the inherent orientation of the PHA synthase on the bead surface, the hydrophobic C terminus of PhaC1 Pa was proposed to be attached to the hydrophobic PHA core and hence, required a designed linker to enable surface exposure of the fusion partner 10 . The length of the linker may have not been adequate for the full display of the OprI/F-AlgE antigen on the PHA MCL beads surface compared to the N terminal fusion to the PhaC1 Pa , possibly resulting in reduced OprI/F-AlgE antigen processing by APCs and therefore, leading to a poor antigen-specific immune response 49 .
The reduced immune response seen with PhaC1 Pa -Ag beads compared to Ag-PhaC1 Pa beads could be due to the smaller bead size, resulting in suboptimal antigen uptake compared to the larger Ag-PhaC1 Pa beads. Bead size is a major contributing factor influencing particulate antigen uptake by APCs 16 . The mechanism of antigen uptake can influence the type of immune response, inducing humoral and/or cell-mediated immunity. However, the actual size for the most efficient uptake of particulates by APCs is still controversial as efficiency can be affected by a range of other factors including shape, surface charge, hydrophobicity/hydrophilicity and mode of administration 16 . Therefore, it remains unclear if size was a contributing factor for the reduced response to the PhaC1 Pa -Ag beads. PHA MCL beads produced in this study were all within the generally accepted effective range (< 0.5 μ m) for uptake by professional APCs and induced an antigen-specific immune response 50 .
Vaccination with Ag-PhaC1 Pa beads resulted in significantly increased levels of cytokines IFN-γ , IL-6 and IL-10 with low but significant levels of IL-17a and IL-2 compared to the PBS group suggesting a Th1 and Th17 type immune response (Fig. 6a,b). IL-17a plays a critical role in maintaining control of host defense against extracellular pathogens 51 . Significant level of IL-6 was induced with vaccination using Ag-PhaC1 Pa beads, but this did not correlate with high levels of IL-17a. IL-6 together with cytokines IL-1β or TNF-α during acute inflammation can also result in the recruitment of neutrophils 52 . TNF-α levels were elevated but not significantly in mice vaccinated with Ag-PhaC1 Pa beads. IL-6 and IL-10 may limit damage in the lungs of CF patients caused by hyper inflammation associated with exacerbated recruitment of neutrophils that lead to pulmonary decline.
In conclusion, this study showed that cellular inclusions of bacterial pathogens are immunogenic capable of inducing cell-mediated immune responses. Hence, it is proposed that vaccine research should consider nano-/ microsized cellular inclusions as antigen reservoir and delivery system towards the development of safe and efficient particulate vaccines. We proofed the concept of hijacking the capacity of the pathogen P. aeruginosa to naturally produce PHA MCL inclusions for the design and production of PHA MCL beads displaying selected antigens of the same host as particulate vaccine candidates (Fig. 7). PHA MCL beads with associated HCPs represented as a large antigenic repertoire. PHA MCL beads displaying vaccine candidates AlgE, OprF and OprI without adjuvant induced a dominant Th1 type response required for the control of P. aeruginosa infection 26,43 . Since, a range of pathogens such as e.g. Mycobacterium tuberculosis, Legionella pneumophila, and Bacillus anthracis ( Supplementary Fig. 5 and Supplementary Tables 4 and 5), are able to inherently produce PHA inclusions, the demonstrated concept of producing particulate subunit vaccines within the disease causing pathogen represents a novel approach to subunit vaccine development applicable to a range of infectious diseases.

Methods
Bacterial strains and growth conditions. All bacterial strains and plasmids used are listed in Supplementary Table 6. E. coli strains were grown in Luria broth (LB) medium (Difco, Detroit, MI) at 37 °C unless stated. LB medium was supplemented with 1% NaCl for growth of osmosensitive E. coli strain ClearColi (Lucigen, Middleton, WI). When required, antibiotics were used at the following concentrations: ampicillin, 100 μ g/mL; and gentamycin 10 μ g/mL.
P. aeruginosa strains were grown in LB medium (Difco, Detroit, MI) or mineral salt medium (MSM) 53 at 37 °C and when required, antibiotics were added at the following concentrations: carbenicillin, 300 μ g/mL; and gentamycin, 100 to 300 μ g/mL.
Isolation and manipulation of DNA. General cloning procedures were performed as described previously 54 . Electroporation was used for the transfer of plasmid into P. aeruginosa strains as described elsewhere 55 . All plasmid isolations were performed using High Pure Plasmid Isolation Kit (Roche, BASEL, Switzerland). DNA primers were purchased from Integrated DNA Technologies (Coralville, IA). Taq and platinum pfx polymerases were purchased from Invitrogen (Carlsbad, CA). Synthesized peptides and antibodies were purchased from GenScript (Piscataway, NJ). All newly amplified DNA fragments and final plasmid constructs were confirmed by DNA sequencing.
Construction of alginate-pel-deletion mutant in a PHA negative background. Generation of the isogenic triple mutant (PAO1 Δ CΔ 8Δ F) incapable of PHA/alginate/Pel production is outlined in Fig. 1.
The alginate biosynthesis gene alg8 was disrupted by using the previously described gene-knockout plasmid pEX100T::Δ alg8ΩGm 56 . The plasmid was transferred via electroporation into PHA MCL negative P. aeruginosa strain PAO1 Δ phaC1ZC2 36 and transformants having undergone the first homologous recombination event were selected on LB medium containing 100 μ g/mL of gentamicin. Subsequently, a second homologous recombination event was selected for by plating single cell colonies on LB medium containing 300 μ g/mL of gentamicin and 5% (w/v) sucrose. Insertion of FRT-Gm-FRT cassette was confirmed by PCR with primers Alg8_XUP and Alg8_XDN. Gentamicin cassette was removed by the introduction of Flp recombinase-encoding plasmid pFLP2 57 by electroporation and plated on to LB medium containing carbenicillin (300 μ g/mL). Resistant colonies were then screened on LB medium containing 5% (w/v) sucrose. CFU were subsequently screened for gentamicin (300 μ g/mL) and carbenicillin (300 μ g/mL) sensitivity. PCR with primers Alg8_XUP and Alg8_XDN were used to confirm loss of gentamicin cassette and therefore, P. aeruginosa PAO1 Δ phaC1ZC2 Δ alg8 double mutant was generated.
Disruption of pelF in newly generated strain PAO1 Δ phaC1ZC2 Δ alg8 was achieved similarly as described above with the introduction of previously described gene-knockout plasmid pEX100T::Δ pelFΩGm 58 . PCR with primers PelF_XUP and PelF_XDN was used to confirm the insertion and subsequent removal of the gentamicin cassette. Consequently, P. aeruginosa PAO1 Δ phaC1ZC2 Δ alg8 Δ pelF triple mutant was generated, and form now will be referred to as PAO1 Δ CΔ 8Δ F. PCR products amplified with primers flanking Δ alg8 (Alg8_XUP and Alg8_XDN) and Δ pelF (PelF_XUP and PelF_XDN) were subsequently used to confirm deletion by DNA sequencing (Supplementary Fig. 1).

Analysis of the tolerance of translational fusions to the C terminus of the class II PHA synthase.
A DNA fragment comprising the Shine-Dalgarno (SD) sequence and gene encoding the class II PHA synthase (phaC1 Pa ) was excised from pBHR71 with XbaI and BamHI 59 . The fragment was subsequently ligated into the corresponding sites in pBBR1JO-5. Resultant plasmid pBBR1JO-5_C1 constitutively expresses phaC1 Pa in P. aeruginosa.
Scientific RepoRts | 7:41607 | DOI: 10.1038/srep41607 To assess the ability of the class II PHA synthase to tolerate C terminal fusions, a flexible linker extension fusion with the GFP reporter (Linker-SG-linker-gfp) used previously to assess C terminal fusion to class I PHA synthase from Ralstonia eutropha (PhaC Re ) 10 was adapted for use in this study.
The stop codon of phaC1 Pa was removed by PCR amplification using primers F_phaC1 and R_phaC1_(-)stop_ BamHI with pBHR71 as template. The amplified PCR fragment encoding SD sequence and phaC1 Pa flanked by sites XbaI and BamHI were ligated into vector pGEM-T easy. Resultant plasmid pGEM-T_C1(-) was hydrolyzed with XbaI and BamHI. Excised DNA fragment was ligated into the corresponding sites in vector pBBR1JO-5 giving intermediate plasmid pBBR1JO-5_C1(-). To generate the corresponding insert, primers F_BgIII_LSGLgfp and R_LSGLgfp_BamHI were used to amplify the DNA sequence encoding the Linker-SG-Linker-gfp (LSGLgfp) region of pET-14b PhaC-linker-SG-Linker-gfp. Resultant fragment flanked with newly introduced BgIII and BamHI sites was ligated into vector pGEM-T easy. Following confirmation, LSGLgfp fragment was excised from (1) Plasmid encoding wildtype PHA synthase (phaC1 Pa ) or OprI/F-AlgE fusion antigen fused to the N terminal of PhaC1 Pa or OprI/F-AlgE fusion antigen fused to the C terminal of PhaC1 Pa via linker-SG-linker were transformed in to P. aeruginosa PAO1 Δ CΔ 8Δ F mutant strain. This strain is defective in production of native PHA MCL and of EPS alginate and Pel (see Fig. 1). (2) Plasmid harboring strains are then grown under PHA MCL accumulating conditions to mediate overproduction of the fusion protein and subsequent PHA MCL bead assembly (See Fig. 3a-c). (3) Formation of PHA MCL beads results in the display of fusion antigens covalently linked to the PHA synthase and the incorporation of granule associated and HCPs (See Fig. 4a-d). (4) PHA MCL beads are isolated from the host by mechanical disruption and subsequently purified. (5) C57BL/6 mice were vaccinated with sterilized PHA MCL beads, recombinant His-tagged OprI/F-AlgE, and PBS via subcutaneous route three times at biweekly intervals. (6) Blood and splenocytes were collected from mice euthanized threeweeks after the last vaccination for analysis. Antigen-specific serum antibodies (ELISA) (see Fig. 5a) and cytokines (Cytometric bead array, mouse Th1/Th2/Th17 cytokine kit) (see Fig. 6a,b) were measured. pGEM-T_LSGLgfp with introduced sites and subsequently ligated into the BamHI site in plasmid pBBR1JO-5_ C1(-) downstream and in frame of phaC1 Pa , resulting in plasmid pBBR1JO-5_C1gfp. Ligation resulted in the destruction of the BgIII and BamHI site between phaC1 Pa and linker. Orientation was confirmed by directional PCR.

Construction of plasmids for the production of OprI/F-AlgE antigen-displaying PHA inclusions.
Antigenic epitopes from the outer membrane protein F (OprF 329-342 ), mature outer membrane lipoprotein I (OprI 21-83 ), and outer membrane porin AlgE (AlgE 233-241 -287-303 ) were combined in a single chain fusion antigen (OprI/F-AlgE) and covalently displayed on the surface of the PHA MCL inclusions. Eptitopes of OprF and OprI (Supplementary Fig. 2a) were selected as previously described 22,34,38,41,60,61 . Two epitopes, HLRRPGEEV (L5) and NLTTRIATGKQ (L6), corresponding to amino acids 233-241 and 287-303 of AlgE were identified by B-cell epitope prediction method EPCES 62 (Supplementary Fig. 2b). The OprI/F-AlgE antigen was designed with one copy of AlgE (L5 and L6) and OprI epitopes while including three repeats of the OprF epitope (Fig. 3a). Epitopes in the fusion antigen fragment were arranged as follows: L5-L6-OprF(x3)-OprI for N terminal PhaC1 Pa fusion and OprI-OprF(x3)-L6-L5 for C terminal PhaC1 Pa fusion. All epitope encoding DNA fragments were synthesized with codon usage bias for P. aeruginosa by GenScript (Piscataway, NJ). An arabinose inducible system (pHERD20T) 63 was chosen for the expression of genes required for the production of antigen-displaying PHA MCL inclusions in P. aeruginosa. Modification of pHERD20T vector was required to remove an alternative start site encoded by LacZ. The vector was linearized with NcoI and EcoRI. Resulting cohesive ends of the vector fragment were blunted using T4 DNA polymerase to allow religation, resulting in vector pHERD20T-2.
Generation of the plasmid encoding the N terminal fusion of OprI/F-AlgE antigen to PhaC1 Pa was achieved in two steps. Firstly, the DNA fragment encoding phaC1 Pa was excised from plasmid pBBR1JO5_C1 by hydrolysis with XbaI and HindIII and subsequently ligated into the corresponding sites in vector pHERD20T-2. Secondly, resultant plasmid pHERD20T-2_C1 was linearized by hydrolysis with XbaI and NdeI and OprI/F-AlgE antigen fragment excised from pUC57_Ag(N) was successively ligated upstream of phaC1 Pa with corresponding sites, generating the final plasmid pHERD20T-2_AgC1 which encodes for fusion protein Ag-PhaC1 Pa .
Generation of plasmid encoding C terminal fusion to PhaC1 Pa was achieved in a similar fashion to the above. OprI/F-AlgE antigen fragment from pUC57_Ag(C) was excised with SmaI and EcoRI and linear fragment ligated into corresponding sites of plasmid pBBR1JO-5_C1gfp, replacing GFP reporter. Newly generated plasmid pBBR1JO-5_C1Ag was hydrolyzed with XbaI and HindIII and resultant linear fragment encoding phaC1Ag was ligated into corresponding sites of vector pHERD20T-2, generating final plasmid pHERD20T-2_C1Ag that encodes for fusion protein PhaC1 Pa -Ag.

Production of PHA inclusions and isolation (beads).
To promote PHA inclusion formation, P. aeruginosa strain PAO1 Δ CΔ 8Δ F was grown under nitrogen limitation utilizing sodium gluconate as a carbon source 64 . MSM was modified with the following: 0.05% (w/v) NH 4 Cl; and supplemented with 1% (w/v) sodium gluconate 42 . Antibiotics were added at the following concentrations: carbenicillin, 300 μ g/mL for strains harboring pHERD20T-2 derivatives; and gentamycin, 300 μ g/mL for strains harboring pBBR1MCS-5 derivatives.
A preculture was inoculated from frozen stock and incubated at 37 °C for 10-12 h with agitation. The preculture was then used to inoculate MSM using 5% (v/v) inoculum and grown for further 10-12 h. Main cultures were inoculated with overnight culture giving a starting with OD 600 of 0.5 and were cultivated at 37 °C with agitation. Induction of main cultures with a final concentration of 0.5% (w/v) arabinose was required when OD 600 reached 0.4 for PAO1 Δ CΔ 8Δ F strains harboring pHERD20T-2 derivatives. PAO1 Δ CΔ 8Δ F strains harboring pBBR1MCS-5 derivatives were constitutively expressed in P. aeruginosa and did not require induction. All cultures were cultivated for a further 48 h.
Cells were harvested at 4 °C by centrifugation for 10 min at 9,000 × g. The pellet was washed with 100 mL of 50 mM Tris buffer (pH 8) and then again with 50 mL for a total of two washes. Washed cells were then centrifuged at 9,500 × g for 40 min at 4 °C. The pellet was suspended as a 10% slurry (w/v) in lysis buffer (50 mM Tris buffer [pH 8], 50 mM EDTA, 62.5 μ g/mL lysozyme) and incubated at 37 °C for 35 min with agitation to digest the cell walls. Cells were then sonicated for 30 sec with a power output of 15 W to sheer DNA prior to mechanical lysis by passing the cell suspension through a French press four times at 6,000 Psi. The crude cell lysate was then sonicated for 30 sec with a power output of 15 W, diluted five times in TE buffer (50 mM Tris buffer [pH 8], 50 mM EDTA) and collected by centrifugation at 9,500 × g for 1 h and 4 °C. The pellet containing crude PHA MCL beads and cell debris was washed with 50 mM Tris buffer (pH 8) and pelleted at 9,500 × g for 30 min and 4 °C and then re-suspended in 20 mL of 50 mM Tris buffer (pH 8) and treated with 0.05 mg/mL DNase + 5 mM MgCl 2 for 20 min at 4 °C with mixing. Following DNase treatment, the crude PHA MCL bead suspension was sonicated for 30 sec with a power output of 9 W and subsequently washed two times with 50 mM Tris buffer (pH 8), centrifuging for 30 min at 9,500 × g and 4 °C. The PHA MCL bead material was then suspended as 20% slurry (w/v) in 50 mM Tris buffer (pH 8) with 25% glycerol as a cryoprotectant for storage at − 80 °C. Production and isolation of recombinant protein. E. coli strain ClearColi (Lucigen, Middleton, WI) was transformed with pET16b-HisAg and grown in LB miller medium containing 100 μ g/mL ampicillin. The main culture was inoculated with overnight culture to give a starting OD 600 of 0.1 and cultivated at 37 °C. Induction of main culture was achieved with 1 mM IPTG when cultures had reached OD 600 of 0.3 and further cultivated at a reduced temperature of 30 °C for 15 h with agitation. Cells were harvested at 4 °C by centrifugation for 10 min at 9,000 × g and washed once with 1x PBS (pH 7.4). Sediment The pellet was then suspended as 20% slurry (w/v) in binding buffer (50 mM Tris buffer [pH 7.7], 300 mM NaCl, 10 mM Imidazole). To achieve lysis, cell slurry was sonicated for 10 sec 'on' and 10 sec 'off ' for a total of 10 min 'on' at a power setting of 21 W. Crude cell lysate was centrifuged at 9,500 × g for 5 min and supernatant fraction containing soluble protein was filtered through a 0.45 μ M pore size filter. Zymo (Irvine, CA) His-Spin Protein Miniprep was used for affinity purification of recombinant His-tagged proteins with the following modifications to the manufacturer's instructions: 400 μ L of filtered cell lysate was mixed with 300 μ L of dried His-Affinity Gel per P1 column, and with mixing on a tilting platform left to bind for 5 min. The column was centrifuged at 17,000 × g and sample binding step was repeated several times. Each column was washed twice with 250 μ L of wash buffer (50 mM Tris buffer [p H 7.7], 300 mM NaCl, 50 mM Imidazole) and subsequently eluted with 150 μ L of elution buffer (50 mM Tris buffer [pH 7.7], 300 mM NaCl, 500 mM Imidazole). The eluted protein was dialyzed against 1x PBS (pH 7.4) at 4 °C overnight using dialysis tubing with 10 K MWCO. Insoluble material was removed by centrifugation at 17,000 × g for 10 min. Recombinant protein His 10 -Ag was sterilized by filtration through a 0.22 μ M pore size syringe filter.
Sterilization of PHA beads. PHA MCL beads were thawed and washed two times using 1x PBS (pH 7.4), centrifuging for 1 h at 14,500 × g and 4 °C. PHA MCL beads were suspended to a 20% slurry (w/v) in 1x PBS (pH 7.4). For sterilization, 1 mg/mL carbenicillin was added to PhaC1 Pa PHA beads and 1 mg/mL gentamycin was added to both Ag-PhaC1 Pa and PhaC1 Pa -Ag PHA MCL beads. PHA MCL beads were then distributed into 2 mL screw cap vials and placed in a sonication water bath for 1 h while maintaining a water temperature below 50 °C. Respective PHA MCL bead samples were pooled and washed two times with 1x PBS (pH 7.4), centrifuged at 14,500 × g for 40 min at 4 °C. Beads were suspended as a 10% slurry (w/v) in 1x PBS (pH 7.4) and a representative sample of 200 μ l was plated for each group onto LB agar and incubated at 37 °C to check for CFU.
Nile Red staining and fluorescence microscopy. The presence of PHA MCL inclusions were observed with fluorescent microscopy by staining cells with lipophilic dye Nile Red 65 . MagnaFire imaging software was used to digitally capture images.

TEM.
Transmission electron microscopy (TEM) analysis was used to confirm the accumulation, shape and size of PHA MCL inclusions inside PHA MCL producing recombinant P. aeruginosa and respective PHA MCL bead material. Samples were processed for TEM analysis as previously described 66 . Diameters of PHA MCL inclusions in whole-cells was quantified using ImageJ imaging and analysis software (Wayne Rasband) giving approximately 500 data points for each fusion protein.
Analysis of proteins attached to PHA MCL beads. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used as previously described 67 to assess the protein profiles of PHA MCL beads and recombinant protein. The gels were strained with Coomassie Brilliant Blue G250. The amount of fusion protein was determined by densitometry against known bovine serum albumin (BSA) standards using Gel Doc XR for detection and Image lab software (version 5.2.1) (Bio-Rad, CA) for analysis. Protein bands of interest were excised from the gels and identified by tryptic peptide fingerprint analysis using matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The major co-purified protein bands from PHA MCL beads formed by PHA synthase antigen fusions were identified on SDS-PAGE using the following criteria: Image lab software (BioRad, USA) was used for the identification of dominant bands which had a volume threshold of > 1,000,000 as analyzed by Image lab software (BioRad, USA) and which were not detected using the specific anti-PhaC1_529 antibody. The anti-PhaC1_529 antibody as opposed to anti-PhaC1_1 and anti-PhaC_67 was specifically detecting the proteins PhaC1 Pa (62.5 kDa), Ag-PhaC1 Pa (77.8 kDa) and PhaC1 Pa -Ag (79.7 kDa).
Quantification and analysis of PHA. Typically 10-20 mg of lyophilized cells was subjected to methanolysis as described previously 68 . Methyl esters of the corresponding fatty acid constituents was recovered and analyzed by Gas chromatography-mass spectrometry (GC/MS) for 3-hydroxyalkanoate methyl esters.

Vaccination of mice.
All animal experiments had the approval of the AgResearch Animal Ethics Committee.
Female C57BL/6 mice aged 6 to 8 weeks (obtained from the animal breeding facility of AgResearch, Ruakura, Hamilton, New Zealand) were vaccinated three times subcutaneously with 200 μ l/injection at 2 week intervals (n = 6 per group) with 20 μ g of PHA synthase on PhaC1 Pa PHA MCL beads or 20 μ g of OprI/F-AlgE fusion antigen on Ag-PhaC1 Pa or PhaC1 Pa -Ag PHA MCL beads or 5 μ g OprI/F-AlgE fusion antigen on PhaC1 Pa -Ag PHA MCL beads (low dose). Additional adjuvant formulated groups were included, 20 μ g OprI/F-AlgE fusion antigen on PhaC1 Pa -Ag PHA MCL beads were mixed with 10% (v/v) alum (A8222, Sigma, MO) or 20 μ g soluble recombinant antigen His 10 -Ag protein either alone or mixed with alum to a final concentration of 10% (v/v). PBS-vaccinated control animals were included. Protein concentration was calculated using densitometry and BSA standards.
Immunological assay. Immunological assays were performed as previously described 14 . Briefly, all mice were anesthetized using a mix of ketamine and xylazine hydrochloride three weeks after last vaccinated. Blood was collected by cardiac puncture, allowed to clot and centrifuged before serum was collected. Spleens were removed and single-cell suspensions were prepared by pushing the samples through a sieve and then repeatedly drawn through a 23 g needle. Suspensions were washed with TAC buffer (17 mM Tris-HCl and 140 mM NH 4 Cl), followed by subsequent washes with PBS. Cells were then cultured at 37 °C and 10% CO 2 in Dulbecco's modified Eagle medium supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 μ g/mL streptomycin, 5 × 10 −5 M 2-mercaptoethanol, and 5% (w/v) FCS in 7 well of a flat-bottomed 46-well plate at a concentration of 2 × 10 6 cells/well in a 1 mL volume. Cells were incubated in medium alone or medium containing 5 μ g/mL recombinant protein His 10 -Ag (calculated based on OprI/F-AlgE fusion protein) or 5 μ g/mL synthesized peptide (AlgE 233-241 -Measurement of opsonophagocytic killing activity of serum antibody. Opsonophagocyticic killing assay was performed as previously described 71 with some modifications. In brief, assays were performed in 96-well plates using 25 μ l of the following assay components: mouse serum diluted 1:2.5 (final concentration 1:10), mouse macrophage RAW 264.7 cells at 2 × 10 6 /ml, P. aeruginosa from log-phase culture at 2 × 10 8 /ml, and 4% guinea pig complement as complement (1% final concentration). DMEM medium with 10% heat-inactivated fetal calf serum was used as the diluent. Control reactions, wherein antibody was omitted and substituted with DMEM-10%FCS. Assay was performed at 37 °C with mixing on a plate mixer for 90 mins. Following incubation 25 μ l was removed and diluted in deionized water and the serially in saline as described previously 72 , and plated on LB agar for bacteria counts. Plates were incubated overnight at 37 °C. The percent killing was calculated as follows: [1 -(CFU surviving in immune serum at 90 min/CFU surviving in PBS vaccinated serum at 90 min)] × 100.
Identification of PHA synthases in bacterial human pathogens. eggNOG 4.5 73 , a hierarchical orthology framework with annotations was used for the identification of PHA synthases in bacteria. A sequence search of the database with the amino acid reference sequence of PhaC1 Pa from P. aeruginosa (X66592.1) identified greater then 359 species of bacteria (e-value ranging from 6.56e-185 to 1.6e-07). 33 human pathogens of interest were identified (Supplementary Table 4).
Homology between the PHA synthases from the selected 33 human pathogens was inferred by multiple alignment of the primary amino acid sequence using T-Coffee 74 with BLOSUM ( Supplementary Fig. 5 and Supplementary Table 5).
Statistical analysis. ELISA data were analyzed using SoftMax pro 7 and expressed in titers representing the reciprocal of the serum dilution which gave half the maximal optical density (OD) (EC 50 ).
For IgG anlaysis, data of graph are reported as means ± s.e.m (6 mice per group). Statistical analyses were undertaken on log(e)-transformed IgG1 and IgG2c values. IgG1 was analyzed by Krustal-Wallis nonparametric test, no significant differences found between the groups. IgG2 was analyzed by one-way ANOVA with statistical significance (p < 0.05) indicated by 'letter based' representation of pairwise comparisons between groups using Tukey's post-hoc test.
Analysis of the mean percent kill from triplicate samples of serum were compared by one-way ANOVA with pairwise comparision between groups using Tukey's post-hoc test (p < 0.05).
For analysis of cytokines, results are calculated by subtracting cytokine values of the media-stimulated samples from the cytokine values of the recombinant protein stimulated samples. Data of graphs are reported as means ± s.e.m and each individual mouse are reported as a dot (6 mice per group). Statistical analyses were undertaken on log(e)-transformed IFN-γ , IL-17a, and IL-2 values and square root-transformed IL-4, IL-6, and TNF-α values, while the raw data was analyzed for IL-10. Comparison of multiple groups for statistical significance (p < 0.05) is indicated by 'letter-based' representation of pairwise comparisons between groups, with p-value adjusted by 'Benjamini-Hochberg' method.