Adaptation of the binding domain of Lactobacillus acidophilus S-layer protein as a molecular tag for affinity chromatography development.

Introduction: The S-layer proteins are a class of self-assembling proteins that form bi-dimensional lattices named S-Layer on the cell surface of bacteria and archaea. The protein SlpA, which is the major constituent of the Lactobacillus acidophilus S-layer, contains in its C-terminus region (SlpA284 - 444), a protein domain (named here as SLAPTAG) responsible for the association of SlpA to the bacterial surface. SLAPTAG was adapted for the development of a novel affinity chromatography method: the SLAPTAG-based affinity chromatography (SAC). Methods: Proteins with different molecular weights or biochemical functions were fused in-frame to the SLAPTAG and efficiently purified by a Bacillus subtilis-derived affinity matrix (named Bio-Matrix or BM). Different binding and elution conditions were evaluated to establish an optimized protocol. Results: The binding equilibrium between SLAPTAG and BM was reached after a few minutes of incubation at 4°C, with an apparent dissociation constant (KD) of 4.3µM. A reporter protein (H6-GFP-SLAPTAG) was used to compare SAC protein purification efficiency against commercial immobilized metal affinity chromatography. No differences in protein purification performance were observed between the two methods. The stability and reusability of the BM were evaluated, and it was found that the matrix remained stable for more than a year. BM could be reused up to five times without a significant loss in performance. Additionally, the recovery of bound SLAP-tagged proteins was explored using proteolysis with a SLAP-tagged version of the HRV-3c protease (SLAPASE). This released the untagged GFP while the cut SLAPTAG and the SLAPASE were retained in the BM. As an alternative, iron nanoparticles were linked to the BM, resulting in BMmag. The BMmag was successfully adapted for a magnetic SAC, a technique with potential applications in high-throughput protein production and purification. Discussion: The SAC protocol can be adapted as a universal tool for the purification of recombinant proteins. Furthermore, the SAC protocol utilizes simple and low-cost reagents, making it suitable for in-house protein purification systems in laboratories worldwide. This enables the production of pure recombinant proteins for research, diagnosis, and the food industry.

Biochemical and functional characterization of Brucella abortus cyclophilins: So similar, yet so different.

Brucella spp. are the etiological agent of animal and human brucellosis. We have reported previously that cyclophilins of Brucella (CypA and CypB) are upregulated within the intraphagosomal replicative niche and required for stress adaptation and host intracellular survival and virulence. Here, we characterize B. abortus cyclophilins, CypA, and CypB from a biochemical standpoint by studying their PPIase activity, chaperone activity, and oligomer formation. Even though CypA and CypB are very similar in sequence and share identical chaperone and PPIase activities, we were able to identify outstanding differential features between them. A series of differential peptide loops were predicted when comparing CypA and CypB, differences that might explain why specific antibodies (anti-CypA or anti-CypB) were able to discriminate between both cyclophilins without cross-reactivity. In addition, we identified the presence of critical amino acids in CypB, such as the Trp134 which is responsible for the cyclosporin A inhibition, and the Cys128 that leads to CypB homodimer formation by establishing a disulfide bond. Here, we demonstrated that CypB dimer formation was fully required for stress adaptation, survival within HeLa cells, and mouse infection in B. abortus. The presence of Trp134 and the Cys128 in CypB, which are not present in CypA, suggested that two different kinds of cyclophilins have evolved in Brucella, one with eukaryotic features (CypB), another (CypA) with similar features to Gram-negative cyclophilins.

Development of a Salmonella-based oral vaccine to control intestinal colonization of Shiga-toxin-producing Escherichia coli (STEC) in animals.

Shiga-toxin-producing Escherichia coli (STEC) is an important food-borne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. Since no vaccines are available and antibiotic treatment is not recommended because promotes the appearance of HUS symptoms, the control of STEC intestinal colonization in cows, which is an important environmental reservoir, is crucial to control this zoonosis. Here, we evaluated the adaptation of an attenuated strain of Salmonella enterica serovar Typhimurium (ΔaroA mutant) as a vaccine platform for preventing STEC intestinal colonization that was studied in a mouse model. A chimeric antigen formed by the combination of the STEC peptides EspA36-192, Intimin653-935, Tir 258-361, and H7 flagellin352-374 (EITH7) was constructed and fused to the ß-lactamase signal sequence (bla SS) that drives the secretion of the chimeric antigen to the bacterial periplasmic space. Oral administration of ΔaroA-ST(EITH7) in a regime of three doses of immunization elicited both mucosal and humoral immune responses that protect mice against a STEC oral experimental infection. Remarkably, serum antibodies not only were able to bind the chimeric antigen EITH7 but also to block actin pedestal formation triggered by the type three secretion system (T3SS) in Enteropathogenic Escherichia coli (EPEC). Furthermore, a single-dose protocol was evaluated, and mice were orally immunized with ΔaroA-ST(EITH7). Interestingly, although with this protocol of immunization only fecal α-EITH7 IgA antibodies were induced and no α-EITH7 in sera were detected, mice were able to efficiently control an oral experimental infection with 1010 STEC (strain Escherichia coli O157:H7), suggesting that mucosal immune response was necessary and sufficient to control STEC intestinal colonization.

Development of an Antigen Delivery Platform Using Lactobacillus acidophilus Decorated With Heterologous Proteins: A Sheep in Wolf's Clothing Story.

S-layers are bacterial structures present on the surface of several Gram-positive and Gram-negative bacteria that play a role in bacterial protection. In Lactobacillus acidophilus (L. acidophilus ATCC 4356), the S-layer is mainly composed of the protein SlpA. A tandem of two copies of the protein domain SLP-A (pfam: 03217) was identified at the C-terminal of SlpA, being this double SLP-A protein domain (in short dSLP-A) necessary and sufficient for the association of the protein to the L. acidophilus cell wall. A variety of proteins fused to the dSLP-A domain were able to spontaneously associate with high affinity to the cell wall of L. acidophilus and Bacillus subtilis var. natto, in a process that we termed decoration. Binding of dSLP-A-containing-proteins to L. acidophilus was stable at conditions that mimic the gastrointestinal transit in terms of pH, proteases, and bile salts. To evaluate if protein decoration of L. acidophilus can be adapted to generate an oral vaccine platform, a chimeric antigen derived from the bacterial pathogen Shiga-toxin-producing Escherichia coli (STEC) was constructed by fusing the sequences encoding the polypeptides EspA36-192, Intimin653-953, Tir240-378, and H7 flagellin352-374 (EITH7) to the dSLP-A domain (EITH7-dSLP-A). Recombinantly expressed EITH7-dSLP-A protein was affinity purified and combined with L. acidophilus cultures to allow the association of the chimeric antigen to the bacterial surface. EITH7-decorated L. acidophilus was orally administered to BALB/c mice and the induction of anti-EITH7 specific antibodies in sera and feces determined by ELISA. Mice presenting significantly higher anti-EITH7 antibodies titers were able to control more efficiently an experimental STEC infection than mice that received the non-decorated L. acidophilus carrier, indicating that antigen-decorated L. acidophilus can be adapted as a mucosal immunization delivery platform to elicit a protective immune response for vaccine purposes.

Omp19 Enables Brucella abortus to Evade the Antimicrobial Activity From Host's Proteolytic Defense System.

Pathogenic microorganisms confront several proteolytic events in the molecular interplay with their host, highlighting that proteolysis and its regulation play an important role during infection. Microbial inhibitors, along with their target endogenous/exogenous enzymes, may directly affect the host's defense mechanisms and promote infection. Omp19 is a Brucella spp. conserved lipoprotein anchored by the lipid portion in the Brucella outer membrane. Previous work demonstrated that purified unlipidated Omp19 (U-Omp19) has protease inhibitor activity against gastrointestinal and lysosomal proteases. In this work, we found that a Brucella omp19 deletion mutant is highly attenuated in mice when infecting by the oral route. This attenuation can be explained by bacterial increased susceptibility to host proteases met by the bacteria during establishment of infection. Omp19 deletion mutant has a cell division defect when exposed to pancreatic proteases that is linked to cell-cycle arrest in G1-phase, Omp25 degradation on the cell envelope and CtrA accumulation. Moreover, Omp19 deletion mutant is more susceptible to killing by macrophage derived microsomes than wt strain. Preincubation with gastrointestinal proteases led to an increased susceptibility of Omp19 deletion mutant to macrophage intracellular killing. Thus, in this work, we describe for the first time a physiological function of B. abortus Omp19. This activity enables Brucella to better thrive in the harsh gastrointestinal tract, where protection from proteolytic degradation can be a matter of life or death, and afterwards invade the host and bypass intracellular proteases to establish the chronic infection.

Combinatorial control of adhesion of Brucella abortus 2308 to host cells by transcriptional rewiring of the trimeric autotransporter btaE gene.

Regulatory network plasticity is a key attribute underlying changes in bacterial gene expression and a source of phenotypic diversity to interact with the surrounding environment. Here, we sought to study the transcriptional circuit of HutC, a regulator of both metabolic and virulence genes of the facultative intracellular pathogen Brucella. Using in silico and biochemical approaches, we identified a novel functional HutC-binding site upstream of btaE, a trimeric-autotransporter adhesin involved in the attachment of Brucella to host extracellular matrix components. Moreover, we identified two additional regulators, one of which, MdrA, acts in concert with HutC to exert a combinatorial control of both btaE promoter activity and attachment of Brucella to HeLa cells. Analysis of btaE promoter sequences of different species indicated that this HutC-binding site was generated de novo by a single point mutation in a virulent Brucella strain, indicative of a transcriptional rewiring event. In addition to major domain organization differences existing between BtaE proteins within the genus Brucella, our analyses revealed that sequences upstream of btaE display high variability probably associated to intrinsic promoter structural features, which may serve as a substrate for reciprocal selection during co-evolution between this pathogen and its mammalian host.

Development of a dual vaccine for prevention of Brucella abortus infection and Escherichia coli O157:H7 intestinal colonization.

Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH7) from STEC, was constructed and expressed in B. abortus Δpgm vaccine strain (BabΔpgm). Mice orally immunized with BabΔpgm(EITH7) elicited an immune response with the induction of anti-EITH7 antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t=4 days) than mice immunized with BabΔpgm carrier strain (t=12 days). As expected, mice immunized with BabΔpgm(EITH7) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles-the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC.

Brucella cyclic ß-1,2-glucan plays a critical role in the induction of splenomegaly in mice.

Brucella, the etiological agent of animal and human brucellosis, is a bacterium with the capacity to modulate the inflammatory response. Cyclic ß-1,2-glucan (CßG) is a virulence factor key for the pathogenesis of Brucella as it is involved in the intracellular life cycle of the bacteria. Using comparative studies with different CßG mutants of Brucella, cgs (CßG synthase), cgt (CßG transporter) and cgm (CßG modifier), we have identified different roles for this polysaccharide in Brucella. While anionic CßG is required for bacterial growth in low osmolarity conditions, the sole requirement for a successful Brucella interaction with mammalian host is its transport to periplasmic space. Our results uncover a new role for CßG in promoting splenomegaly in mice. We showed that CßG-dependent spleen inflammation is the consequence of massive cell recruitment (monocytes, dendritics cells and neutrophils) due to the induction of pro-inflammatory cytokines such as IL-12 and TNF-α and also that the reduced splenomegaly response observed with the cgs mutant is not the consequence of changes in expression levels of the characterized Brucella PAMPs LPS, flagellin or OMP16/19. Complementation of cgs mutant with purified CßG increased significantly spleen inflammation response suggesting a direct role for this polysaccharide.

FixL-like sensor FlbS of Brucella abortus binds haem and is necessary for survival within eukaryotic cells.

Replication of Brucella inside eukaryotic cells is essential for pathogenesis, and successful infection requires rapid adaptation to the intracellular milieu. Close relatives of Brucella use the two-component system FixLJ to survive inside the host. We aimed to identify a homologous sensor in Brucella abortus. A predicted protein with transmembrane and conserved histidine kinase domains was identified as the Fix-like Brucella sensor, FlbS. Although it lacks the PAS domain, recombinant FlbS binds haem in vitro. An internal in-frame deletion in flbS severely decreased B. abortus survival inside professional and non-professional phagocytes. This phenotype was reverted by genetic complementation. These results indicate the critical role of this haemoprotein in the intracellular lifestyle of Brucella.

The Aggregation of Brucella abortus Occurs Under Microaerobic Conditions and Promotes Desiccation Tolerance and Biofilm Formation.

Brucella abortus causes brucellosis mainly in cattle. The infection is transmitted to humans by ingestion of animal products or direct contact with infected material. While the intracellular lifestyle of Brucella is well characterized, its extracellular survival is poorly understood. In nature, bacterial persistence is associated with biofilms, where aggregated cells are protected from adversity. The inability of Brucella abortus to aggregate under aerobiosis and that fact that the replicative niche of Brucella is characterized by microaerobic conditions prompted us to investigate the capacity of this pathogen to aggregate and grow in biofilms under microaerobiotic conditions. The results show that B. abortus aggregates and produces biofilms. The aggregates tolerate desiccation better than planktonic cells do, adhere and displace even in the absence of the lipopolysaccharide-O antigen, flagella, the transcriptional regulator VjbR, or the enzymes that synthesize, transport, and modify cyclic ß (1,2) glucan.

A Brucella virulence factor targets macrophages to trigger B-cell proliferation.

Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA-treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified nonmuscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homologue of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen.

Intracellularly induced cyclophilins play an important role in stress adaptation and virulence of Brucella abortus.

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells.

The Brucella abortus cyclic beta-1,2-glucan virulence factor is substituted with O-ester-linked succinyl residues.

Brucella periplasmic cyclic beta-1,2-glucan plays an important role during bacterium-host interaction. Nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and DEAE-Sephadex chromatography were used to characterize Brucella abortus cyclic glucan. In the present study, we report that a fraction of B. abortus cyclic beta-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic beta-1,2-glucan. The oligosaccharide backbone is substituted at C-6 positions with an average of two succinyl residues per glucan molecule. This O-ester-linked succinyl residue is the only substituent of Brucella cyclic glucan. A B. abortus open reading frame (BAB1_1718) homologous to Rhodobacter sphaeroides glucan succinyltransferase (OpgC) was identified as the gene encoding the enzyme responsible for cyclic glucan modification. This gene was named cgm for cyclic glucan modifier and is highly conserved in Brucella melitensis and Brucella suis. Nucleotide sequencing revealed that B. abortus cgm consists of a 1,182-bp open reading frame coding for a predicted membrane protein of 393 amino acid residues (42.7 kDa) 39% identical to Rhodobacter sphaeroides succinyltransferase. cgm null mutants in B. abortus strains 2308 and S19 produced neutral glucans without succinyl residues, confirming the identity of this protein as the cyclic-glucan succinyltransferase enzyme. In this study, we demonstrate that succinyl substituents of cyclic beta-1,2-glucan of B. abortus are necessary for hypo-osmotic adaptation. On the other hand, intracellular multiplication and mouse spleen colonization are not affected in cgm mutants, indicating that cyclic-beta-1,2-glucan succinylation is not required for virulence and suggesting that no low-osmotic stress conditions must be overcome during infection.

Identification of active site residues of the inverting glycosyltransferase Cgs required for the synthesis of cyclic beta-1,2-glucan, a Brucella abortus virulence factor.

Brucella abortus cyclic glucan synthase (Cgs) is a 320-kDa (2868-amino acid) polytopic integral inner membrane protein responsible for the synthesis of the virulence factor cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. Cgs functions as an inverting processive beta-1,2-autoglucosyltransferase and has the three enzymatic activities required for the synthesis of the cyclic glucan: initiation, elongation, and cyclization. To gain further insight into the protein domains that are essential for the enzymatic activity, we have compared the Cgs sequence with other glycosyltransferases (GTs). This procedure allowed us to identify in the Cgs region (475-818) the widely spaced D, DxD, E/D, (Q/R)xxRW motif that is highly conserved in the active site of numerous GTs. By site-directed mutagenesis and in vitro and in vivo activity assays, we have demonstrated that most of the amino acid residues of this motif are essential for Cgs activity. These sequence and site-directed mutagenesis analyses also indicate that Cgs should be considered a bi-functional modular GT, with an N-terminal GT domain belonging to a new GT family related to GT-2 (GT-84) followed by a GH-94 glycoside hydrolase C-terminal domain. Furthermore, over-expression of inactive mutants results in wild-type (WT) production of cyclic glucan when bacteria co-express the mutant and the WT form, indicating that Cgs may function in the membrane as a monomeric enzyme. Together, these results are compatible with a single addition model by which Cgs acts in the membrane as a monomer and uses the identified motif to form a single center for substrate binding and glycosyl-transfer reaction.

Membrane topology analysis of cyclic glucan synthase, a virulence determinant of Brucella abortus.

Brucella abortus cyclic glucan synthase (Cgs) is a 316-kDa (2,831-amino-acid) integral inner membrane protein that is responsible for the synthesis of cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. B. abortus Cgs uses UDP-glucose as a sugar donor and has the three enzymatic activities necessary for synthesis of the cyclic polysaccharide (i.e., initiation, elongation, and cyclization). Cyclic glucan is required in B. abortus for effective host interaction and complete expression of virulence. To gain further insight into the structure and mechanism of action of B. abortus Cgs, we studied the membrane topology of the protein using a combination of in silico predictions, a genetic approach involving the construction of fusions between the cgs gene and the genes encoding alkaline phosphatase (phoA) and beta-galactosidase (lacZ), and site-directed chemical labeling of lysine residues. We found that B. abortus Cgs is a polytopic membrane protein with the amino and carboxyl termini located in the cytoplasm and with six transmembrane segments, transmembrane segments I (residues 419 to 441), II (residues 452 to 474), III (residues 819 to 841), IV (residues 847 to 869), V (residues 939 to 961), and VI (residues 968 to 990). The six transmembrane segments determine four large cytoplasmic domains and three very small periplasmic regions.

Molecular cloning and characterization of cgt, the Brucella abortus cyclic beta-1,2-glucan transporter gene, and its role in virulence.

The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic beta-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic beta-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic beta-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic beta-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor.