Designing Probes for Immunodiagnostics: Structural Insights into an

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Designing probes for immunodiagnostics: structural insights into an epitope targeting Burkholderia infections Riccardo Capelli, Elena Matterazzo, Marco Amabili, Claudio Peri, Alessandro Gori, Paola Gagni, Marcella Chiari, Ganjana Lertmemongkolchai, Marina Cretich, Martino Bolognesi, Giorgio Colombo, and LOUISE JANE GOURLAY ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00080 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 16, 2017

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ACS Infectious Diseases

Designing probes for immunodiagnostics: structural insights into an epitope targeting Burkholderia infections

AUTHOR LIST: Riccardo Capellia,bψ, Elena Matterazzocψ, Marco Amabilia,c, Claudio Peria, Alessandro Goria, Paola Gagnia, Marcella Chiaria, Ganjana Lertmemongkolchaid, Marina Creticha, Martino Bolognesic,e, Giorgio Colomboa* and Louise J. Gourlayc*.

AUTHOR INFORMATION: a

Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle

Ricerche, Via Mario Bianco 9, 20131 Milano, Italy b

Center for Complexity and Biosystems and Dipartimento di Fisica, Università degli

Studi di Milano and INFN, Via Celoria 16, 20133 Milano, Italy c

Department of Biosciences, Università degli Studi di Milano, Via Celoria 26,

Milano, 20133, Italy d

Center for Research and Development of Medical Diagnostic Laboratories (CMDL),

Faculty of Associated Medical Sciences, Khon Kaen University, 40002, Khon Kaen, Thailand e

Pediatric Clinical Research Center “Romeo ed Enrica Invernizzi”, Cryo Electron-

Microscopy Laboratory, Università degli Studi di Milano, Milano, 20133, Italy

ψ

These authors contributed equally to this work

*Corresponding authors: [email protected], [email protected]

1 ACS Paragon Plus Environment


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Structure-based epitope prediction drives the design of diagnostic peptidic probes to reveal specific antibodies elicited in response to infections. We previously identified a highly immunoreactive epitope from the peptidoglycan-associated lipoprotein (Pal) antigen from Burkholderia pseudomallei, which could also diagnose Burkholderia cepacia infections. Here, considering the high phylogenetic conservation within Burkholderia species, we ask whether cross-reactivity can be reciprocally displayed by the synthetic epitope from B. cenocepacia. We perform comparative analyses of the conformational preferences and diagnostic performances of the corresponding epitopes from the two Burkholderia species when presented in the context of the full-length proteins or as isolated peptides. The effects of conformation on the diagnostic potential and cross-reactivity of Pal peptide epitopes are rationalized based on the 1.8 Å crystal structure of B. cenocepacia Pal, and through computational analyses. Our results are discussed in the context of designing new diagnostic molecules for early detection of infectious diseases.

KEYWORDS: immunodiagnostics, antigen, Burkholderia spp., epitope, microarray, crystal structure.


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Bacteria from the Burkholderia genus are opportunistic Gram-negative pathogens that cause life threatening lung infections.1 Antimicrobial therapies are severely limited by the lack of efficient diagnostic methods able to detect the infectious agent in a timely fashion. The high phylogenetic correlation among members of the Burkholderia species have led to the investigation of conserved protein antigens, but also to the design and exploitation of epitope peptides as potential probes for the detection of Burkholderia infections.2-3 There are numerous advantages of using epitope peptides rather than recombinant antigens, such as the reduced cost practical simplicity of peptide synthesis, and the ability to easily introduce chemical modifications that can block the peptide in the desired conformation that may improve its recognition profile.

4-5

Furthermore, the use of

cross-reactive epitopes renders the possibility of utilizing a single test in significantly diverse diagnostic or therapeutic contexts. In particular, efforts have focused on the analysis of epitope conservation between B. pseudomallei (Bp) and B. cenocepacia (Bc). The former is the etiologic agent of melioidosis, a severe endemic disease in South-East Asia, and an emerging threat in Australia, in the Indian subcontinent and in South America. Melioidosis can cause septicemia and organ failure, with a high mortality rate; treatment with antibiotics is largely ineffective, due to multi-drug resistance.6 B. cenocepacia is an opportunistic pathogen that colonizes the respiratory apparatus of Cystic Fibrosis (CF) patients, causing lung infections that often have fatal consequences.1, 7 Genomic similarities between the two bacteria raise the possibility of designing cross-reactive epitopes for simultaneous diagnosis of Burkholderia spp. Such designed molecules, once shown to be immunoreactive in serological tests, may be further developed into



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components of protective vaccines, thus opening new and long sought perspectives for the therapeutic treatment of Burkholderia infections. In this context, detailed structural information on the antigens and epitopes of the pathogen offers prime opportunities to engineer biomolecules with specific immunological and recognition properties. In previous studies, starting from the Xray structure of the peptidoglycan-associated lipoprotein from B. pseudomallei (PalBp), we predicted and designed an epitope peptide (BpEp3, comprising PalBp residues 72 to 91) using in silico methods.8 BpEp3 showed improved immunological properties with respect to the initial recombinant antigen, as well as cross-reactivity and significant diagnostic performances for Burkholderia cenocepacia infections in CF patients, demonstrating that rational epitope engineering can be an effective strategy to deliver better immuno-reagents.2,

9

The conformational flexibility of

BpEp3 was found to be a key property affecting its diagnostic potential; a cyclic, more rigid form of the peptide performed better.4 In the present study, we targeted the Pal antigen from B. cenocepacia (PalBc) as the basis for comparative and structure-based epitope discovery, design and immunodiagnostic studies. PalBc and PalBp are highly conserved proteins (sequence identity of 84%) with only 27/170 differing amino acids (Figure S1). Four such residue differences fall in the region corresponding to Ep3 in PalBp, with the most significant substitution from a structural point-of-view being an Ala to Pro replacement (residue 81). The presence of Pro81 raises the possibility that the region corresponding to BpEp3 in Bc may influence the dynamic states of the epitope when synthesized and used as an isolated peptide, as previously carried out on BpEp3.9 Such residue substitution could ultimately provide different diagnostic properties for BpEp3 and BcEp3, despite two otherwise very similar epitope sequences, thus


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providing new insights into the design requirements for efficient immunoreactive probes. To investigate the above issues, we solved the crystal structure of PalBc at 1.8 Å resolution for subsequent in silico epitope predictions and epitope design (Figure 1). Epitope predictions carried out on the crystal structure identified the region of PalBc corresponding to BpEp3 as a potential candidate for epitope design. We synthesized BcEp3 as a free peptide and carried out comparative functional and structural analyses with its Bp counterpart. Specifically, we probed sera from individuals affected by Bp and Bc infections on a microarray platform, comparing immunodiagnostic performances of the BcEp3 and BpEp3 epitopes both in the context of their corresponding recombinant full antigens (PalBc and PalBp) and as isolated synthetic peptides. Our analyses suggest that sequence and structure-based conservation of the full-length antigen alone may not be enough to correlate with immunodiagnostic properties. Attention must be paid to the conformational dynamics of the epitope sequence and to the main ensembles that this may determine.

RESULTS AND DISCUSSION PalBc crystal structure. PalBc (residues 19-170) was expressed as a N-terminal Histagged fusion protein and crystallized as described in the Methods section. A single crystal was used to collect data at a resolution of 1.8Å and the structure of PalBc was solved by molecular replacement (see Methods). Three identical PalBc chains (A-C; RMSD values 0.24-0.42 Å over the traced Cα chains) were present in the crystal asymmetric unit; such trimeric arrangement is not proposed to be biologically relevant since there are no significantly extended interaction surfaces between chains (Figure



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1A). Electron density was visible for residues 52 to 170 (for chains A and B) and for residues 52 to 169 (chain C), but was absent for the first 62 (29 pertaining to the vector) N-terminal residues, indicating that this region is flexible and lost to the solvent (Figure S2). The overall 3D structure conforms to the canonical Pal α-β sandwich fold, organized in a helix-strand-helix topology (Figure 1B). Structural comparisons were made between the A chains of PalBc and PalBp using the Superpose module available under the CCP4 suite.10 As expected, the two structures are highly similar (RMSD 0.43Å; sequence identity of 84 %) (Figure 1B). Among the 27 residues that differ between the primary structures of PalBc and PalBp, four (positions 76, 78, 81 and 83) are located in the region corresponding to the highly antigenic BpEp3; at residue 81 an Ala to Pro replacement occurs in the region encompassing an α-helix in BpEp3 (Figure S1). Despite the conformational restraints posed by Pro residues that typically cause distortions in polypeptide chains, such as kinks in α-helices, BpEp3 and BcEp3 maintain the same backbone conformation in the full-length proteins (Figure 1B). There is one oxalate ion bound to each PalBc chain. Hydrogen bonds are formed between main- and side-chain atoms contributed from residues D71, D105, R107, N113, and R120. As reported in the literature, an acetate ion was found bound to each PalBp chain, interacting with equivalent residues present in PalBc. The cavity that houses these anions is the proposed peptidoglycan-binding pocket.9,11

Structure-based computational epitope mapping of PalBc epitopes and immunoreactivity of recombinant PalBc. We previously identified the B-cell epitope BpEp3 using the Matrix of Local Coupling Energies (MLCE) computational epitope


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prediction method applied to the crystal structure of PalBp.9, 12 In peptide form, BpEp3 elicited bactericidal antibodies (Abs), triggered Ab-dependent agglutination and was preferentially recognized by recovery melioidosis IgGs, in comparison with healthy controls and seropositive individuals, thus implying potential diagnostic and therapeutic applications.9 T-cell epitopes conserved between Bc and Bp have been observed for the flagellin antigen.3 Furthermore, B-cell epitope conservation was also demonstrated in a separate microarray study that showed that a panel of Bp epitope peptides could specifically detect Bc infections in CF patients.2 These results suggested the possibility of rationally designing cross-reactive probes for the diagnosis of Burkholderia spp. infections in general. In this context, to investigate the potential effects of sequence and structural properties on immunoreactivity, an analogous strategy using the MLCE epitope prediction approach was applied to the PalBc crystal structure (see Methods).

9, 12

Following in silico analysis, PalBc displayed two main epitope regions largely overlapping with the previously determined PalBp putative epitopes; one is equivalent to BpEp3, and here labeled BcEp3, while the second region maps a conformational epitope (Figure 2), strongly suggesting that PalBc and PalBp share immunoreactive regions.

Probing human antibody response to BpEp3 and BcEp3 in Burkholderia affected individuals. As previously mentioned, BpEp3 isolated peptide was found to be crossreactive and immunodiagnostic for Bc infections in CF patients.2 Here we investigate the immunoreactivity of the Bc counterpart against Bc patient sera, and assess its cross-reactivity against melioidosis patient sera samples. To this aim we compared the



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serodiagnostic capability of the two epitopes to identify individuals affected by Bc and Bp infections, both in the context of Pal full-antigens and as synthetized free peptides. A protein microarray displaying recombinant PalBc and PalBp antigens was probed with 12 serum samples from Bc-positive CF patients, diagnosed by microbiological culture and MALDI-TOF spectrometry, and with 20 melioidosis patient serum samples (10 healthy seropositive sera, and 10 recovered melioidosis cases, as judged by indirect hemagglutinin assay (IHA) Ab titers (Khon Kaen University and Srinakarin Hospital, Thailand).2 As a control group, seronegative serum samples from healthy donors were used (12 samples for the Bc patients, and 10 sera samples for melioidosis patients). The antigen-specific IgG content in each serum was evaluated by fluorescence detection using an anti-human IgG labeled with the Cy3 dye. The ability of each antigen to distinguish controls and patients was evaluated performing the unpaired t-test (significant if p values

Designing Probes for Immunodiagnostics: Structural Insights into an

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