Protein Crystallization of Two Recombinant Lpt Proteins.

The prerequisite for 3D structure determination of macromolecules via X-ray crystallography is well-ordered, diffracting crystals. Here, we report the recombinant production, biophysical/biochemical protein sample characterization, and vapor diffusion sitting drop crystallization protocols for two lipopolysaccharide transport proteins: LptH from Pseudomonas aeruginosa (Pa-LptH) and an inactive LptC mutant (G153R) from Escherichia coli (EcLptC24-191G153R).

Solution Structure of the BPSL1445 Protein of Burkholderia pseudomallei Reveals the SYLF Domain Three-Dimensional Fold.

The SYLF domain is an evolutionary conserved protein domain with phosphatidylinositol binding ability, whose three-dimensional structure is unknown. Here, we present the solution structure and the dynamics characterization of the SYLF domain of the bacterial BPSL1445 protein. BPSL1445 is a seroreactive antigen and a diagnostic marker of Burkholderia pseudomallei, the etiological agent of melioidosis, a severe infectious disease in the tropics. The BPSL1445 SYLF domain (BPSL1445-SYLF) consists of a ß-barrel core, with two flexible loops protruding out of the barrel and three helices packing on its surface. Our structure allows for a more precise definition of the boundaries of the SYLF domain compared to the previously reported one and suggests common ancestry with bacterial EipA domains. We also demonstrate by phosphatidyl-inositol phosphate arrays and nuclear magnetic resonance titrations that BPSL1445-SYLF weakly interacts with phosphoinositides, thus supporting lipid binding abilities of this domain also in prokaryotes.

Epidemic Preparedness-Leishmania tarentolae as an Easy-to-Handle Tool to Produce Antigens for Viral Diagnosis: Application to COVID-19.

To detect and prevent emerging epidemics, discovery platforms are urgently needed, for the rapid development of diagnostic assays. Molecular diagnostic tests for COVID-19 were developed shortly after the isolation of SARS-CoV-2. However, serological tests based on antiviral antibody detection, revealing previous exposure to the virus, required longer testing phases, due to the need to obtain correctly folded and glycosylated antigens. The delay between the identification of a new virus and the development of reliable serodiagnostic tools limits our readiness to tackle future epidemics. We suggest that the protozoan Leishmania tarentolae can be used as an easy-to-handle microfactory for the rapid production of viral antigens to face emerging epidemics. We engineered L. tarentolae to express the SARS-CoV-2 receptor-binding domain (RBD) and we recorded the ability of the purified RBD antigen to detect SARS-CoV-2 infection in human sera, with a sensitivity and reproducibility comparable to that of a reference antigen produced in human cells. This is the first application of an antigen produced in L. tarentolae for the serodiagnosis of a Coronaviridae infection. On the basis of our results, we propose L. tarentolae as an effective system for viral antigen production, even in countries that lack high-technology cell factories.

Producing natural vanilla extract from green vanilla beans using a ß-glucosidase from Alicyclobacillus acidiphilus.

Current methods for the production of natural vanilla extract are long and tedious, and the efficiency of the vanillin extraction is usually conditioned by different factors during the traditional curing process (temperatures and weather conditions). As an important fraction of vanillin is present in the form of glucovanillin in green beans, endogenous ß-glucosidases contribute to its hydrolysis; however, these enzymes lose efficiency during the curing process. The use of extremophilic organisms as a source of an appropriate exogenous enzyme can offer a valid alternative when producing natural vanillin. Here, a ß-glucosidase from the thermo-acidophilic organism Alicyclobacillus acidiphilus (AacGH1) was cloned, expressed in E. coli BL21, and fully characterized in respect to both function and crystal structure. Notably, AacGH1 was stable at a temperature up to 50 °C and exhibited good tolerance to glucose, fructose and organic solvents, in particular it maintained full activity in the presence of up to 20 % (v/v) ethanol. The enzyme was then successfully applied to an ethanol-water (20 % (v/v)) extract of green vanilla beans and the complete hydrolysis of glucovanillin (1.7 mM) to vanillin, and other flavour compounds commonly found in vanilla, was achieved using 0.5 mg/mL of enzyme in just 15 min at 30 °C.

Detecting the nature and solving the crystal structure of a contaminant protein from an opportunistic pathogen.

The unintentional crystallization of contaminant proteins in the place of target recombinant proteins is sporadically reported, despite the availability of stringent expression/purification protocols and of software for the detection of contaminants. Typically, the contaminant protein originates from the expression organism (for example Escherichia coli), but in rare circumstances contaminants from different sources have been reported. Here, a case of contamination from a Serratia bacterial strain that occurred while attempting to crystallize an unrelated protein from Burkholderia pseudomallei (overexpressed in E. coli) is presented. The contamination led to the unintended crystallization and structure analysis of a cyanase hydratase from a bacterial strain of the Serratia genus, an opportunistic enterobacterium that grows under conditions similar to those of E. coli and that is found in a variety of habitats, including the laboratory environment. In this context, the procedures that were adopted to identify the contaminant based on crystallographic data only are presented and the crystal structure of Serrata spp. cyanase hydratase is briefly discussed.

An (R)-Selective Transaminase From Thermomyces stellatus: Stabilizing the Tetrameric Form.

The identification and 3D structural characterization of a homolog of the (R)-selective transaminase (RTA) from Aspergillus terreus (AtRTA), from the thermotolerant fungus Thermomyces stellatus (TsRTA) is here reported. The thermostability of TsRTA (40% retained activity after 7 days at 40°C) was initially attributed to its tetrameric form in solution, however subsequent studies of AtRTA revealed it also exists predominantly as a tetramer yet, at 40°C, it is inactivated within 48 h. The engineering of a cysteine residue to promote disulfide bond formation across the dimer-dimer interface stabilized both enzymes, with TsRTA_G205C retaining almost full activity after incubation at 50°C for 7 days. Thus, the role of this mutation was elucidated and the importance of stabilizing the tetramer for overall stability of RTAs is highlighted. TsRTA accepts the common amine donors (R)-methylbenzylamine, isopropylamine, and d-alanine as well as aromatic and aliphatic ketones and aldehydes.

Widely applicable background depletion step enables transaminase evolution through solid-phase screening.

Directed evolution of transaminases is a widespread technique in the development of highly sought-after biocatalysts for industrial applications. This process, however, is challenged by the limited availability of effective high-throughput protocols to evaluate mutant libraries. Here we report a rapid, reliable, and widely applicable background depletion method for solid-phase screening of transaminase variants, which was successfully applied to a transaminase from Halomonas elongata (HEWT), evolved through rounds of random mutagenesis towards a series of diverse prochiral ketones. This approach enabled the identification of transaminase variants in viable cells with significantly improved activity towards para-substituted acetophenones (up to 60-fold), as well as tetrahydrothiophen-3-one and related substrates. Rationalisation of the mutants was assisted by determination of the high-resolution wild-type HEWT crystal structure presented herein.

Strategic single point mutation yields a solvent- and salt-stable transaminase from Virgibacillus sp. in soluble form.

A new transaminase (VbTA) was identified from the genome of the halotolerant marine bacterium Virgibacillus 21D. Following heterologous expression in Escherichia coli, it was located entirely in the insoluble fraction. After a single mutation, identified via sequence homology analyses, the VbTA T16F mutant was successfully expressed in soluble form and characterised. VbTA T16F showed high stability towards polar organic solvents and salt exposure, accepting mainly hydrophobic aromatic amine and carbonyl substrates. The 2.0 Å resolution crystal structure of VbTA T16F is here reported, and together with computational calculations, revealed that this mutation is crucial for correct dimerisation and thus correct folding, leading to soluble protein expression.

BPSL1626: Reverse and Structural Vaccinology Reveal a Novel Candidate for Vaccine Design against Burkholderia pseudomallei.

Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel of 104 candidate antigens from the Gram-negative bacterial pathogen Burkholderia pseudomallei, which is responsible for the disease melioidosis. B. pseudomallei can cause fatal sepsis in endemic populations in the tropical regions of the world and treatment with antibiotics is mostly ineffective. With the aim of identifying potential vaccine candidates, we report the experimental validation of predicted antigen and type I fimbrial subunit, BPSL1626, which we show is able to recognize and bind human antibodies from the sera of Burkholderia infected patients and to stimulate T-lymphocytes in vitro. The prerequisite for a melioidosis vaccine, in fact, is that both antibody- and cell-mediated immune responses must be triggered. In order to reveal potential antigenic regions of the protein that may aid immunogen re-design, we also report the crystal structure of BPSL1626 at 1.9 Å resolution on which structure-based epitope predictions were based. Overall, our data suggest that BPSL1626 and three epitope regions here-identified can represent viable candidates as potential antigenic molecules.

A stereospecific carboxyl esterase from Bacillus coagulans hosting nonlipase activity within a lipase-like fold.

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of ß-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a "lid" domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under accession numbers 5O7G (apo-BCE) and 5OLU (glycerol-bound BCE).

Designing Probes for Immunodiagnostics: Structural Insights into an Epitope Targeting Burkholderia Infections.

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 on the basis of 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 the early detection of infectious diseases.

Boosting of post-exposure human T-cell and B-cell recall responses in vivo by Burkholderia pseudomallei-related proteins.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with high incidence and mortality in South East Asia and northern Australia. To date there is no protective vaccine and antibiotic treatment is prolonged and not always effective. Most people living in endemic areas have been exposed to the bacteria and have developed some immunity, which may have helped to prevent disease. Here, we used a humanized mouse model (hu-PBL-SCID), reconstituted with human peripheral blood mononuclear cells from seropositive donors, to illustrate the potential of three known antigens (FliC, OmpA and N-PilO2) for boosting both T-cell and B-cell immune responses. All three antigens boosted the production of specific antibodies in vivo, and increased the number of antibody and interferon-γ-secreting cells, and induced antibody affinity maturation. Moreover, antigen-specific antibodies isolated from either seropositive individuals or boosted mice, were found to enhance phagocytosis and oxidative burst activities from human polymorphonuclear cells. Our study demonstrates that FliC, OmpA and N-PilO2 can stimulate human memory T and B cells and highlight the potential of the hu-PBL-SCID system for screening and evaluation of novel protein antigens for inclusion in future vaccine trials against melioidosis.

Immunisation with proteins expressed during chronic murine melioidosis provides enhanced protection against disease.

There is an urgent need for an effective vaccine against human disease caused by Burkholderia pseudomallei, and although a wide range of candidates have been tested in mice none provide high level protection. We considered this might reflect the inability of these vaccine candidates to protect against chronic disease. Using Q-RT PCR we have identified 6 genes which are expressed in bacteria colonising spleens and lungs of chronically infected mice. Three of the genes (BPSL1897, BPSL3369 and BPSL2287) have been expressed in Escherichia coli and the encoded proteins purified. We have also included BPSL2765, a protein known to induce immune responses associated with a reduced incidence of chronic/recurrent disease in humans. Immunisation of mice with a combination of these antigens resulted in the induction of antibody responses against all of the proteins. Compared with mice immunised with capsular polysaccharide or LolC protein, mice immunised with the combination of chronic stage antigens showed enhanced protection against experimental disease in mice.

Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.

The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using ß-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.

Sequence- and Structure-Based Immunoreactive Epitope Discovery for Burkholderia pseudomallei Flagellin.

Burkholderia pseudomallei is a Gram-negative bacterium responsible for melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management. One of the main subunit vaccine candidates is flagellin of Burkholderia pseudomallei (FliCBp). Here, we present the high resolution crystal structure of FliCBp and report the synthesis and characterization of three peptides predicted to be both B and T cell FliCBp epitopes, by both structure-based in silico methods, and sequence-based epitope prediction tools. All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells. Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils. The epitopes derived from this study may represent potential melioidosis vaccine components.

Backbone and side-chain (1)H, (15)N, (13)C assignment and secondary structure of BPSL1445 from Burkholderia pseudomallei.

BPSL1445 is a lipoprotein produced by the Gram-negative bacterium Burkholderia pseudomallei (B. pseudomallei), the etiological agent of melioidosis. Immunodetection assays against sera patients using protein microarray suggest BPSL1445 involvement in melioidosis. Herein we report backbone, side chain NMR assignment and secondary structure for the recombinant protein.

Crystal structure of LptH, the periplasmic component of the lipopolysaccharide transport machinery from Pseudomonas aeruginosa.

UNLABELLED: Lipopolysaccharide (LPS) is the main glycolipid present in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria, where it modulates OM permeability, therefore preventing many toxic compounds from entering the cell. LPS biogenesis is an essential process in Gram-negative bacteria and thus is an ideal target pathway for the development of novel specific antimicrobials. The lipopolysaccharide transport (Lpt) system is responsible for transporting LPS from the periplasmic surface of the inner membrane, where it is assembled, to the cell surface where it is then inserted in the OM. The Lpt system has been widely studied in Escherichia coli, where it consists of seven essential proteins located in the inner membrane (LptBCFG), in the periplasm (LptA) and in the OM (LptDE). In the present study, we focus our attention on the Pseudomonas aeruginosa PAO1 Lpt system. We identified an LptA orthologue, named LptH, and solved its crystal structure at a resolution of 2.75 Å. Using interspecies complementation and site-directed mutagenesis of a conserved glycine residue, we demonstrate that P. aeruginosa LptH is the genetic and functional homologue of E. coli LptA, with whom it shares the ß-jellyroll fold identified also in other members of the canonical E. coli Lpt model system. Furthermore, we modeled the N-terminal ß-jellyroll domain of P. aeruginosa LptD, based on the crystal structure of its homologue from Shigella flexneri, aiming to provide more general insight into the mechanism of LPS binding and transport in P. aeruginosa. Both LptH and LptD may represent new targets for the discovery of next generation antibacterial drugs, targeting specific opportunistic pathogens such as P. aeruginosa. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank under accession number PDB 4uu4.

From crystal structure to in silico epitope discovery in the Burkholderia pseudomallei flagellar hook-associated protein FlgK.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a potentially fatal infection that is endemic in Southeast Asia and Northern Australia that is poorly controlled by antibiotics. Research efforts to identify antigenic components for a melioidosis vaccine have led to the identification of several proteins, including subunits forming the flagella that mediate bacterial motility, host colonization, and virulence. This study focuses on the B. pseudomallei flagellar hook-associated protein (FlgK(Bp)), and provides the first insights into the 3D structure of FlgK proteins as targets for structure-based antigen engineering. The FlgK(Bp) crystal structure (presented here at 1.8-Å resolution) reveals a multidomain fold, comprising two small ß-domains protruding from a large elongated α-helical bundle core. The evident structural similarity to flagellin, the flagellar filament subunit protein, suggests that, depending on the bacterial species, flagellar hook-associated proteins are likely to show a conserved, elongated α-helical bundle scaffold coupled to a variable number of smaller domains. Furthermore, we present immune serum recognition data confirming, in agreement with previous findings, that recovered melioidosis patients produce elevated levels of antibodies against FlgK(Bp), in comparison with seronegative and seropositive healthy subjects. Moreover, we show that FlgK(Bp) has cytotoxic effects on cultured murine macrophages, suggesting an important role in bacterial pathogenesis. Finally, computational epitope prediction methods applied to the FlgK(Bp) crystal structure, coupled with in vitro mapping, allowed us to predict three antigenic regions that locate to discrete protein domains. Taken together, our results point to FlgK(Bp) as a candidate for the design and production of epitope-containing subunits/domains as potential vaccine components.

Redefining the PF06864 Pfam family based on Burkholderia pseudomallei PilO2(Bp) S-SAD crystal structure.

Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2(Bp), a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2(Bp) belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2(Bp) (N-PilO2(Bp)), here reported, is the first structural representative of the PF06864 family. N-PilO2(Bp) presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery.

Exploiting the Burkholderia pseudomallei acute phase antigen BPSL2765 for structure-based epitope discovery/design in structural vaccinology.

We solved the crystal structure of Burkholderia pseudomallei acute phase antigen BPSL2765 in the context of a structural vaccinology study, in the area of melioidosis vaccine development. Based on the structure, we applied a recently developed method for epitope design that combines computational epitope predictions with in vitro mapping experiments and successfully identified a consensus sequence within the antigen that, when engineered as a synthetic peptide, was selectively immunorecognized to the same extent as the recombinant protein in sera from melioidosis-affected subjects. Antibodies raised against the consensus peptide were successfully tested in opsonization bacterial killing experiments and antibody-dependent agglutination tests of B. pseudomallei. Our strategy represents a step in the development of immunodiagnostics, in the production of specific antibodies and in the optimization of antigens for vaccine development, starting from structural and physicochemical principles.