Biomedical subjects
Publications and source records attributed to Mikeljon P Nikolich.
Here, we describe the genomes of six Enterobacter bacteriophages (phages) of the genus Karamvirus. The range of genome length, GC content, and number of predicted protein-coding sequences were, respectively, 171,900-175,675 bp, 39.54-39.82%, and 298-316. Genomic analysis indicates that these phages have a lytic lifestyle and are suitable for therapeutic use.
Brucella melitensis strains may occur as either smooth or rough variants depending on the expression of O-polysaccharides (OPS) as a component of the bacterial outer membrane lipopolysaccharide (LPS). The wboA gene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain in Brucella. Deletion of wboA in smooth virulent B. melitensis 16M results in a rough mutant designated WRR51. We developed a flow cytometric method to determine the proportion of B. melitensis cells displaying surface O-polysaccharide (OPS) in liquid culture. OPS was detected using polyclonal antibodies from rabbits immunized with smooth (S) or rough (R) Brucella LPS. First, we evaluated the binding of these antibodies to 16M (S), WRR51 (R) and complemented WRR51 expressing the wboA gene (S) as well as to their corresponding GFP-expressing derivative strains 16M/GFP, WRR51/GFP and WRR51/GFP+wboA. The rough mutants did not react with anti-S-LPS nor did the smooth strains react with anti-R-LPS. Second, using different ratios of 16M/GFP and WRR51/GFP, we were able to detect the presence of 1% rough bacteria spiked into a sample of smooth organisms. Third, we evaluated the purity of cultures of B. melitensis strains grown in a fermenter. These flow cytometric methods may be useful for quality control of process development for large-scale vaccine production.
The recent description of the complete genomes of the two most pathogenic species of Brucella opens the way for genome-based analysis of the antigenicity of their proteins. In the present report, we describe a bench-level high-efficiency cloning and expression system (HECES) that allow expression of large numbers of Brucella proteins based on genomic sequence information. Purified proteins are produced with high efficiency in a microarray format conducive to analysis of their sero-reactivity against serum from immunized animals. This method is applicable at either small or large scale of protein processing. While it does not require robotics, the format is amenable to robotic implementation for all aspects of the process and subsequent analysis of protein characteristics. This method will allow selection of new reagents for diagnosis of brucellosis and development of vaccine against Brucella, an important zoonotic disease and biothreat agent.
Groups of mice were immunized either subcutaneously or intranasally with purified Brucella melitensis lipopolysaccharide (LPS) or with LPS as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (LPS-GBOMP). Control mice were inoculated with sterile saline. Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally with virulent B. melitensis strain 16M 4 weeks after the second dose of vaccine. Sera, spleens, lungs, and livers of mice were harvested 8 weeks after challenge. The bacterial loads in the organs were determined by culture on brucella agar plates. Protective efficacy was determined by comparing the clearance of bacteria from organs of immunized mice with the clearance of bacteria from organs of control mice. At 8 weeks postchallenge there was significant protection from disseminated infection of spleens and livers of mice intranasally immunized with either vaccine compared to infection of control mice (P < 0.01). There was no significant difference in clearance of bacteria from the lungs of immunized mice and control mice. However, mice immunized subcutaneously with either LPS or LPS-GBOMP vaccine showed significant protection against infection of the spleen (P < 0.001), liver (P < 0.001), and lungs (P < 0.05). These results show that intranasal immunization of mice with either vaccine provided significant protection against disseminated infection of the spleen and liver but subcutaneous immunization of mice with the vaccines conferred significant protection against infection of the spleen, liver, and lungs.
Yersinia pestis is the causative agent of plague, which diverged from Yersinia pseudotuberculosis within the past 20,000 years. Although these two species share a high degree of homology at the DNA level (>90%), they differ radically in their pathogenicity and transmission. In this review, we briefly outline the known virulence factors that differentiate these two species and emphasize genetic studies that have been conducted comparing Y. pestis and Y. pseudotuberculosis. These comparisons have led to a better understanding of the genetic contributions to the differences in the virulence and pathogenicity between these two organisms and have generated information that can be applied in future diagnostic and vaccine development. Comparison of the genetic differences between Y. pestis and Y. pseudotuberculosis has also lent insight into the emergence of acute pathogens from organisms causing milder diseases.
Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into Escherichia coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum.