Identification of smooth and rough forms in cultures of Brucella melitensis strains by flow cytometry.

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.

Highly effective generic adjuvant systems for orphan or poverty-related vaccines.

Safe and effective adjuvants are needed for many vaccines with limited commercial appeal, such as vaccines to infrequent (orphan) diseases or to neglected and poverty-related diseases. Here we found that three nonproprietary liposome formulations containing monophosphoryl lipid A each induced 3-fold to 5-fold increased titers of binding and neutralizing antibodies to anthrax protective antigen compared to aluminum hydroxide-adsorbed antigen in monkeys. All vaccinated monkeys were protected against lethal challenge with aerosolized Ames strain spores.

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice.

BACKGROUND: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. CONCLUSIONS/SIGNIFICANCE: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.

Interactions between Brucella melitensis and human phagocytes: bacterial surface O-Polysaccharide inhibits phagocytosis, bacterial killing, and subsequent host cell apoptosis.

Brucellae are gram-negative intracellular pathogens that survive and multiply within host phagocytic cells. Smooth organisms present O-polysaccharides (OPS) on their surface. 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. Unlike B. abortus, both smooth and rough strains of B. melitensis are resistant to complement-mediated killing. To determine the role of surface OPS in the interactions of B. melitensis with monocytes/macrophages (M/M), 16M and WRR51 were transformed with the plasmid pBBR1MCS-6y encoding green fluorescent protein, and the transformants were used to infect human mononuclear phagocytes with and without fresh human serum as a source of complement. Human monocytes were cultured in the presence of macrophage colony-stimulating factor to allow their differentiation into macrophages during the course of infection. Intracellular bacteria were easily visualized using fluorescence microscopy. Infection in M/M, identified by surface staining and fate of infected phagocytes, was quantitated by flow cytometry. Rough bacteria were internalized, with no requirement for opsonization by serum, at a higher rate than smooth organisms. Smooth B. melitensis survived and multiplied for at least 6 days inside M/M, but rough organisms were eliminated by death of the infected cells. In human monocytes cultured for 1 day without serum in order to trigger the apoptotic pathway, infection by rough brucellae accelerated phagocyte death; smooth brucellae inhibited apoptosis. This study suggests that the presence of surface OPS on live B. melitensis benefits the bacterium by preventing the death of macrophages, Brucella's preferred target for intracellular replication.