Anna M. Stenkova

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease

Graves’ disease results from thyroid-stimulating Abs (TSAbs) activating the thyrotropin receptor (TSHR). How TSAbs arise from early precursor B cells has not been established. Genetic and environmental factors may contribute to pathogenesis, including the bacterium Yersinia enterocolitica. We developed two pathogenic monoclonal TSAbs from a single experimental mouse undergoing Graves’ disease, which shared the same H and L chain germline gene rearrangements and then diversified by numerous somatic hypermutations. To address the Ag specificity of the shared germline precursor of the monoclonal TSAbs, we prepared rFab germline, which showed negligible binding to TSHR, indicating importance of somatic hypermutation in acquiring TSAb activity. Using rFab chimeras, we demonstrate the dominant role of the H chain V region in TSHR recognition. The role of microbial Ags was tested with Y. enterocolitica proteins. The monoclonal TSAbs recognize 37-kDa envelope proteins, also recognized by rFab germline. MALDI-TOF identified the proteins as outer membrane porin (Omp) A and OmpC. Using recombinant OmpA, OmpC, and related OmpF, we demonstrate cross-reactivity of monoclonal TSAbs with the heterogeneous porins. Importantly, rFab germline binds recombinant OmpA, OmpC, and OmpF confirming reactivity with Y. enterocolitica. A human monoclonal TSAb, M22 with similar properties to murine TSAbs, also binds recombinant porins, showing cross-reactivity of a spontaneously arising pathogenic Ab with Y. enterocolitica. The data provide a mechanistic framework for molecular mimicry in Graves’ disease, where early precursor B cells are expanded by Y. enterocolitica porins to undergo somatic hypermutation to acquire a cross-reactive pathogenic response to TSHR.

Trends of the Major Porin Gene (ompF) Evolution: Insight from the Genus Yersinia

OmpF is one of the major general porins of Enterobacteriaceae that belongs to the first line of bacterial defense and interactions with the biotic as well as abiotic environments. Porins are surface exposed and their structures strongly reflect the history of multiple interactions with the environmental challenges. Unfortunately, little is known on diversity of porin genes of Enterobacteriaceae and the genus Yersinia especially. We analyzed the sequences of the ompF gene from 73 Yersinia strains covering 14 known species. The phylogenetic analysis placed most of the Yersinia strains in the same line assigned by 16S rDNA-gyrB tree. Very high congruence in the tree topologies was observed for Y. enterocolitica, Y. kristensenii, Y. ruckeri, indicating that intragenic recombination in these species had no effect on the ompF gene. A significant level of intra- and interspecies recombination was found for Y. aleksiciae, Y. intermedia and Y. mollaretii. Our analysis shows that the ompF gene of Yersinia has evolved with nonrandom mutational rate under purifying selection. However, several surface loops in the OmpF porin contain positively selected sites, which very likely reflect adaptive diversification Yersinia to their ecological niches. To our knowledge, this is a first investigation of diversity of the porin gene covering the whole genus of the family Enterobacteriaceae. This study demonstrates that recombination and positive selection both contribute to evolution of ompF, but the relative contribution of these evolutionary forces are different among Yersinia species.

Adaptive responses of outer membrane porin balance of Yersinia ruckeri under different incubation temperature, osmolarity, and oxygen availability.

The capability of Yersinia ruckeri to survive in the aquatic systems reflects its adaptation (most importantly through the alteration of membrane permeability) to the unfavorable environments. The nonspecific porins are a key factor contributing to the permeability. Here we studied the influence of the stimuli, such as temperature, osmolarity, and oxygen availability on regulation of Y. ruckeri porins. Using qRT‐PCR and SDS‐PAGE methods we found that major porins are tightly controlled by temperature. Hyperosmosis did not repress OmpF production. The limitation of oxygen availability led to decreased expression of both major porins and increased transcription of the minor porin OmpY. Regulation of the porin balance in Y. ruckeri, in spite of some similarities, diverges from that system in Escherichia coli. The changes in porin regulation can be adapted in Y. ruckeri in a species‐specific manner determined by its aquatic habitats.

Diversity and adaptive evolution of a major porin gene (ompF) in Yersinia pseudotuberculosis

We analyzed the molecular diversity and evolution of the allelic variants in Yersinia pseudotuberculosis, Yersinia similis, and Yersinia pestis ompF gene. The average evolutionary divergence of ompF sequences was 0.041 ± 0.004. We revealed 30 unique ompF nucleotide alleles, which clustered into seven groups on the tree. We found that the Y. pseudotuberculosis ompF gene has evolved with nonrandom mutational rate under purifying selection in overall. Some surface loops (L1, L6, and L8) of the OmpF porin contain sites evolving under positive selection that indicates diversification (possibly by adaptive evolution) of the ompF gene of Y. pseudotuberculosis before emergence of Y. pestis. The species most likely have undergone different selective and evolutionary pressures, and thus evolved into diverse Y. pseudotuberculosis OmpF groups.

Engineering of Chimeric Protein Based on E Protein Domain III of Tick- Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis

BACKGROUND Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. OBJECTIVES The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. METHODS The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. RESULTS The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. CONCLUSION The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.

Regulation of Yersinia pseudotuberculosis Major Porin Expression in Response to Antibiotic Stress

The expression of the OmpF porin gene in Yersinia pseudotuberculosis in response to antibiotics of two different classes (kanamycin and nalidixic acid) was analyzed using a quantitative PCR and a fluorescence reporter system. Both antibiotics downregulated the expression of the ompF gene. The nalidixic acid significantly reduced the ompF expression, while kanamycin, for which porins are considered to be an alternative transport route, only slightly reduced the ompF level.

The Molecular Phylogeny of the gyrB Gene: A Molecular Marker for Systematic Characterization of the Genus Yersinia

Phylogenetic analysis of 198 Yersinia strains belonging to 16 species was conducted using 430 bp of the gyrB gene to determine inter-species relatedness and to aid in rapid and accurate species identification. Based on the phylogenetic data, a genetic distance threshold value of 0.025 ± 0.001 was proposed for future prediction of strains of different species or genomospecies in the genus Yersinia. We have demonstrated that partial gyrB gene sequencing can be used to accurately and insightfully construct the phylogeny of the genus Yersinia.

Permanent culture and parasitic impact of the microalga Coccomyxa parasitica, isolated from horse mussel Modiolus kurilensis

Animals with deformed shells and microalgal invasion have been identified in the natural population of the horse mussel species Modiolus kurilensis of Peter the Great Bay in the Sea of Japan. The haemolymph is initially infested with algae, followed by the rectum, siphons, mantles and gonads located in the posterior body areas. Mantles, which are primarily exposed to light, are major depots for algae. The microscopic analysis of algal cells has revealed the absence of flagella and pyrenoids, the presence of single chloroplast, and reproduction by autosporulation, with dispores prevailing over tetraspores. These results, together with the nearly complete sequence analysis of small subunit (SSU) 18S rDNA (1728bp), have confirmed that these cells are Coccomyxa parasitica. A newly developed method of isolating microalgae from mollusk tissues has facilitated the continuous pure - probably axenic - culture of C. parasitica, thereby providing a description of the time course of each life stage. Histological analyses have revealed significant haemocyte infiltration into the mantles, gonads, kidneys and digestive gland tissues infested with microalgae and the gill tissues, in which the intruder was not identified. Algal encapsulation with major focal areas of fibrosis and amorphic necrosis has been revealed in these infested organs. The spaces between the gonad follicles and digestive gland tubules were significantly widened as these areas were filled with a mass of algae and phagocytic haemocytes, showing acini with a thickened basement membrane. The mantles and kidneys of Modiolus displayed significant morphological deviations of different cells in epithelial, connective and muscle tissues, resulting in the dysfunction of the infested organs. Therefore, C. parasitica, which reproduces in the culture, regardless of the host, is a facultative parasite, causing major pathological alterations, such as anomalous histomorphological patterns and infested organ dysfunctions.

Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC)

The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system.

Carrageenanolytic enzymes from marine bacteria associated with the red alga Tichocarpus crinitus

One hundred six bacterial strains (106 strains) were isolated from the Pacific red alga Tichocarpus crinitus, collected from the Troitsa Bay of Gulf of Peter the Great (Sea of Japan, Russia), and studied on their ability to degrade different samples of carrageenan as substrates. Some of the studied strains exhibited hydrolytic activity selectively to a total polysaccharide (kappa and lambda) from Chondrus armatus, other strains, to κ/β-carrageenan from T. crinitus, which did not possess the same effect to a total polysaccharide from T. crinitus. The strains with the highest enzyme activity (28 strains) were identified using 16S rRNA gene sequence techniques and classified to the phyla Bacteroidetes and Proteobacteria. Most (75%) of the studied carrageenase producers belong to phylum Bacteroidetes (21 strains). These strains were related with 7 phylotypes of the genera Aquimarina, Cellulophaga, Maribacter, and Zobellia (family Flavobacteriaceae of the class Flavobacteriia). Cellulophaga strains were the dominant group (35.7% of total) and closely related to species C. baltica and C. lytica with 99.8 and 99.9% sequence similarity, respectively. The remaining strains (7 strains) were represented by members of the genera Altererythrobacter (the family Erythrobacteraceae), Phaeobacter, Sulfitobacter, Tateyamaria (the family Rhodobacteraceae), and Sphingomonas (the family Sphingobacteriaceae) of the class Alphaproteobacteria and Psychrobacter (the family Moraxellaceae) and Vibrio (the family Vibrionaceae) of the class Gammaproteobacteria.