Identification and characteristics of a novel aminoglycoside phosphotransferase, APH(3')-IId, from an MDR clinical isolate of Brucella intermedia.

OBJECTIVES: To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3')-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. METHODS: Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. RESULTS: APH(3')-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3')-IIb. Expression of aph(3')-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3')-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3')-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3')-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from ∼105 to 107 M-1 s-1. Genetic environment and comparative genomic analyses suggested that aph(3')-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. CONCLUSIONS: APH(3')-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3')-IId represents the fourth characterized example of an APH(3')-II enzyme.

Restoration of antitumor immunity through anti-MICA antibodies elicited with a chimeric protein.

BACKGROUND: Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. METHODS: We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. RESULTS: Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. CONCLUSIONS: Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors.

Lethality of Brucella microti in a murine model of infection depends on the wbkE gene involved in O-polysaccharide synthesis.

Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

RNA-seq reveals the critical role of Lon protease in stress response and Brucella virulence.

The Brucella spp encounter stressful environment inside their host cells. The Lon protein is an important protease related to cellular protein degradation and resistance to stress in Brucella. However, the molecular mechanism between Lon protein and stress response was still unknown. In this study, it was found that the lon mutant exhibited obvious growth defect in TSB medium, compared with its parent strain. In addition, our results indicated that Lon protein was involved in resistance to various stress conditions and all the ß-lactam antibiotics tested. Although deletion of this protease did not affect Brucella virulence in macrophage, the mutant strain was significantly attenuated in mice infection model at 1 week post infection, and the expression level of several cytokine genes was significantly changed in vivo. To gain insight into the genetic basis for the distinctive phenotypic properties exhibited by the lon mutant strain, RNA-seq was performed, and the result showed that various genes involved in stress response, quorum sensing and transcriptional regulation were significantly altered in Δlon strain. Overall, these studies have preliminary uncovered the molecular mechanism between Lon protease, stress response and bacterial virulence.

Brucella spp. Lumazine Synthase Induces a TLR4-Mediated Protective Response against B16 Melanoma in Mice.

Brucella Lumazine Synthase (BLS) is a highly immunogenic decameric protein which can accept the fusion of foreign proteins at its ten N-termini. These chimeras are very efficient to elicit systemic and oral immunity without adjuvants. BLS signaling via Toll-Like Receptor 4 (TLR4) regulates innate and adaptive immune responses, inducing dendritic cell maturation and CD8(+) T-cell cytotoxicity. In this work we study the effect induced by BLS in TLR4-expressing B16 melanoma. In order to evaluate the effectiveness of BLS as a preventive vaccine, C57BL/6J mice were immunized with BLS or BLS-OVA, and 35 days later were subcutaneously inoculated with B16-OVA melanoma. BLS or BLS-OVA induced a significant inhibition of tumor growth, and 50% of mice immunized with the highest dose of BLS did not develop visible tumors. This effect was not observed in TLR4-deficient mice. For treatment experiments, mice were injected with BLS or BLS-OVA 2 days after the inoculation of B16 cells. Both treatments induced significant and equal tumor growth delay and increased survival. Moreover, BLS and BLS-OVA stimulation were also effective in TLR4-deficient mice. In order to study whether BLS has a direct effect on tumor cells, B16 cells were preincubated with BLS, and after 48h, cells were inoculated. Tumors induced by BLS-stimulated cells had inhibited growth and survival was increased. In the BLS group, 40% of mice did not develop tumors. This effect was abolished by the addition of TLR4/MD2 blocking antibody to cells before BLS stimulation. Our work demonstrates that BLS immunization induces a preventive antitumor response that depends on mice TLR4. We also show that BLS generates a therapeutic effect in mice inoculated with B16 cells. Our results show that BLS acts directly in cultured tumor cells via TLR4, highly suggesting that BLS elicits its therapeutic effects acting on the TLR4 from B16 melanoma cells.

Identification of a single-nucleotide insertion in the promoter region affecting the sodC promoter activity in Brucella neotomae.

Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli ß-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella.

Brucella spp. lumazine synthase as a bovine rotavirus antigen delivery system.

Brucella spp. lumazine synthase (BLS) is a highly immunogenic decameric protein. It has been previously evaluated as a carrier to increase the immunogenicity of peptides fused to its N-termini. VP8 is a sialic acid binding domain of rotavirus external capsid protein VP4, which is involved in virus adhesion to host cells. In this work, the C486 bovine rotavirus (BRV) VP8 core protein (VP8d) was fused to the structure of BLS with the aim to produce an enhancement of the immune response against BRV VP8 and to evaluate the possible use of this antigen for vaccine development. The feasibility of using BLS as an antigen delivery system of polypeptides larger in size than those previously tested was also evaluated. Groups of female mice were immunized with BLS-VP8d fusion protein, VP8d or an equimolar mixture of purified VP8d and BLS (BLS+VP8d). Dams immunized with BLS-VP8 induced 97.5-100% protection against homologous challenge with C486 BRV; while pups born to dams immunized either with VP8d or BLS+VP8d presented a significant lower level of protection. The neutralizing antibody pattern was also significantly different among these experimental groups, and in concordance with challenge experiment. Overall, these results demonstrate that the BLS-VP8d chimeric protein is properly folded and stable, and that the BLS scaffold is a potent antigen delivery system that enhances the antibody response against BRV and elicits complete homotypic passive protection in a suckling mouse model.

Brucella spp. lumazine synthase: a novel adjuvant and antigen delivery system to effectively induce oral immunity.

Brucella lumazine synthase (BLS) has been previously used with success as a delivery system for systemic immunization against murine cysticercosis. We herein determined the usefulness of BLS as a new antigen-delivery system and mucosal-adjuvant using KETc1, one of the peptides of the anti-cysticercosis vaccine. A protection of up to 98% was induced when KETc1 was used as a chimera fused to BLS. Used as adjuvant of KETc1, BLS also induced a high level of protection (79%), which did not significantly differ from that induced by the cholera toxin (74%). KETc1 and BLS administered separately also reduced the parasite load. KETc1 administered orally as a chimera, and to a lesser extent with BLS as adjuvant, elicited IgG and IgA specific antibodies, which were detectable both in fecal extracts and in sera, and increased B and CD4 activated cells. BLS-KETc1 also increased the levels of transcription of TNF-alpha, IL-2 and IFNgamma in Peyer's patches, and in spleen, only increased TNF-alpha was observed. Overall, these results showed that BLS can be used as both an antigen-carrier and as an adjuvant in the design of new oral subunit vaccines.

Crystallographic studies on decameric Brucella spp. Lumazine synthase: a novel quaternary arrangement evolved for a new function?

The enzyme lumazine synthase (LS) catalyzes the penultimate step of riboflavin biosynthesis in plants, fungi and bacteria. The quaternary structure of the polypeptide differs between species, existing as pentamers or as icosahedrally arranged dodecamers of pentamers with 60 subunits. The pathogen Brucella spp. expresses two proteins that exhibit LS activity, RibH1 and RibH2. The latter enzyme belongs to a novel third category of quaternary arrangement for LS, that of a decameric structure assembled as a head-to-head oriented dimer of pentamers. In contrast, the RibH1 enzyme is assembled as a pentamer, as noted for several other LS enzymes. RibH1 appears to be the functional LS in Brucella spp., whereas RibH2, an enzyme of lower catalytic activity, is a virulence factor presumably acting in response to oxidative stress. The latter observation prompted us to further investigate the structural and catalytic properties of RibH2 in order to clarify the biological function of this enzyme. Here, we present a detailed analysis of two new crystallographic forms of RibH2 that explain the low catalytic activity of this enzyme in comparison with RibH1 and other LSs. Additionally, we analyze the effect of pH on the structure of this enzyme, and the binding of riboflavin and 6,7-dimethyl-8-ribityllumazine to its active site.

Brucella spp. lumazine synthase: a novel antigen delivery system.

Lumazine synthase from Brucella spp. (BLS) was evaluated as a protein carrier to improve antigen delivery of KETc1, one of the peptides of the anti-cysticercosis vaccine. KETc1 becomes antigenic, preserved its immunogenicity and its protective capacity when expressed as a recombinant chimeric protein using Brucella spp. lumazine synthase. KETc1 and BLS-KETc1 were not MHC H-2(d), H-2(k) nor H-2(b) haplotype-restricted albeit KETc1 is preferentially presented in the H-2(b) haplotype. These findings support that BLS is a potent new delivery system for the improvement of subunit vaccines.

Functional analysis of the ClpATPase ClpA of Brucella suis, and persistence of a knockout mutant in BALB/c mice.

The protein ClpA belongs to a diverse group of polypeptides named ClpATPases, which are highly conserved, and which include several molecular chaperones. In this study the gene encoding the 91 kDa protein b-ClpA of the facultative intracellular pathogen Brucella suis, which showed 70% identity to ClpA of Rhodobacter blasticus, was identified and sequenced. Following heterologous expression in Escherichia coli strains SG1126 (DeltaclpA) and SG1127 (Deltalon DeltaclpA), b-ClpA replaced the function of E. coli ClpA, participating in the degradation of abnormal proteins. A b-clpA null mutant of B. suis was constructed, and growth experiments at 37 and 42 degrees C showed reduced growth rates for the null mutant, especially at the elevated temperature. The mutant complemented by b-clpA and overexpressing the gene was even more impaired at 37 and 42 degrees C. In intracellular infection of human THP-1 or murine J774 macrophage-like cells, the clpA null mutant and, to a lesser extent, the strain of B. suis overexpressing b-clpA behaved similarly to the wild-type strain. In a murine model of infection, however, the absence of ClpA significantly increased persistence of B. suis. These results showed that in B. suis the highly conserved protein ClpA by itself was dispensable for intramacrophagic growth, but was involved in temperature-dependent growth regulation, and in bacterial clearance from infected BALB/c mice.