Jan Machold

Characterization of New Members of the Group 3 Outer Membrane Protein Family of Brucella spp.

ABSTRACT Impairment of the omp25 gene in Brucella spp. leads to attenuated strains and confers protection to the host. Omp25 and Omp31, whose functions remain unknown, were the first characterized members of group 3 outer membrane proteins (Omps) (25 to 34 kDa). Recently, genomic and proteomic approaches identified five new putative members of this family, some of which are produced in B. melitensis or B. abortus. In the present study, using protein microsequencing, we identified new members of group 3 Omps proteins produced in B. suis. Since several monoclonal antibodies (MAbs) against Omp25 cross-reacted with other members of group 3 Omps, we also performed Western immunoblotting to compare wild-type B. suis with mutants systematically having B. suis omp25-related genes knocked out. We demonstrate the production of three paralogs of Omp31 and/or Omp25 in B. suis, and the existence of a common site of signal peptide cleavage (AXAAD), which is very similar to that present in the five homologous Omps of Bartonella quintana. The seven group 3 Omps were classified in four-subgroups on the basis of percentage amino acid sequence identities: Omp25 alone, the Omp25b-Omp25c-Omp25d cluster, the Omp31/31b subgroup, and the less related Omp22 protein (also called Omp3b). Together with previous data, our results demonstrate that all new members of group 3 Omps are produced in B. suis or in other Brucella species and we propose a nomenclature that integrates all of these proteins to facilitate the understanding of future Brucella interspecies study results.

Release of periplasmic proteins of Brucella suis upon acidic shock involves the outer membrane protein Omp25

ABSTRACT The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B. suis released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens. The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In B. suis, this protein contained 9 repeats, while 12 were present in the B. melitensis orthologue. B. suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product. However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.

The Brucella suis homologue of the Agrobacterium tumefaciens chromosomal virulence operon chvE is essential for sugar utilization but not for survival in macrophages.

Brucella strains possess an operon encoding type IV secretion machinery very similar to that coded by the Agrobacterium tumefaciens virB operon. Here we describe cloning of the Brucella suis homologue of the chvE-gguA-gguB operon of A. tumefaciens and characterize the sugar binding protein ChvE (78% identity), which in A. tumefaciens is involved in virulence gene expression. B. suis chvE is upstream of the putative sugar transporter-encoding genes gguA and gguB, also present in A. tumefaciens, but not adjacent to that of a LysR-type transcription regulator. Although results of Southern hybridization experiments suggested that the gene is present in all Brucella strains, the ChvE protein was detected only in B. suis and Brucella canis with A. tumefaciens ChvE-specific antisera, suggesting that chvE genes are differently expressed in different Brucella species. Analysis of cell growth of B. suis and of its chvE or gguA mutants in different media revealed that ChvE exhibited a sugar specificity similar to that of its A. tumefaciens homologue and that both ChvE and GguA were necessary for utilization of these sugars. Murine or human macrophage infections with B. suis chvE and gguA mutants resulted in multiplication similar to that of the wild-type strain, suggesting that virB expression was unaffected. These data indicate that the ChvE and GguA homologous proteins of B. suis are essential for the utilization of certain sugars but are not necessary for survival and replication inside macrophages.

Matrix-Assisted Laser Desorption Ionization (MALDI) and Post-Source Decay (PSD) Product Ion Mass Analysis Localize a Photolabel Crosslinked to the Delta-Subunit of nAChR Protein by Neurotoxin II

In an attempt to probe the molecular topology of the ion gate in the nicotinic AChR protein, the high affinity (Kd > 10−11 molar) ligand neurotoxin II (NT II), a 61 residue peptide from the venom of Naja naja oxiana, was photocrosslinked to the nAChR of Torpedo californica. The photoactivatable group was a (125I)-p-azidosalicylamidoethyl-1,3-dithiopropyl (ASED) moiety located at the K25 residue of NT II. Specific labeling occurred at the delta-subunit which, after SDS-PAGE, was subjected to tryptic digestion and reverse phase high performance liquid chromatography (rpHPLC) separation. Although the radioactivity profile of the collected HPLC fractions exhibited two sharp maxima suggesting rather specific crosslinking site(s), attempts to localize these sites by Edman degradation failed. Thus, matrix-assisted laser desorption ionization (MALDI) mass spectrometry was employed in the hope of circumventing the difficulties encountered with the conventional protocols. This technique, in conjunction with the option to perform mass spectrometric peptide sequencing by so-called post-source decay (PSD) product ion analysis, (even with subpicomoles of analyte loads in heterogeneous samples) allowed us not only to determine the labeled tryptic peptide but also to localize the site of the crosslink down to the A268 position of the delta-subunit. It is felt that these results demonstrate the capability of the MALDI technique to overcome some of the constraints inherent in the analytical tool of photolabeling, especially in cases where large molecular probes (such as NT II) are employed.

Synthesis of nitrodiazirinyl derivatives of neurotoxin II fromNaja naja oxiana and their interaction with theTorpedo californica nicotinic acetylcholine receptor

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) fromNaja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of {2-nitro-4 [3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy}acetic acid followed by Chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [125I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane bound nicotinic acetylcholine receptor (AChR) fromTorpedo californica. The pattern of labeling the receptorsα, β, γ, orδ subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series ofp-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.