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YqiC of Salmonella enterica serovar Typhimurium is a membrane fusogenic protein required for mice colonization.

Carrica MC, Craig PO, García-Angulo VA, Aguirre A, García-Véscovi E, Goldbaum FA, Cravero SL - BMC Microbiol. (2011)

Bottom Line: We found that YqiC shares biophysical and biochemical properties with Brucella abortus BMFP, the only previously characterized member of this group, such as a high alpha helix content, a coiled-coil domain involved in trimerization and a membrane fusogenic activity in vitro.In addition, we demonstrated that YqiC localizes at cytoplasmic and membrane subcellular fractions, that a S.This work firstly demonstrates the importance of a COG2960 member for pathogen-host interaction, and suggests a common function conserved among members of this group.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biotecnología, CICVyA-INTA Castelar, Los Reseros y Las Cabañas s/n, Buenos Aires, Argentina. mcarrica@leloir.org.ar

ABSTRACT

Background: Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen which can colonize a variety of hosts, including human, causing syndromes that vary from gastroenteritis and diarrhea to systemic disease.

Results: In this work we present structural information as well as insights into the in vivo function of YqiC, a 99-residue protein of S. Typhimurium, which belongs to the cluster of the orthologous group 2960 (COG2960). We found that YqiC shares biophysical and biochemical properties with Brucella abortus BMFP, the only previously characterized member of this group, such as a high alpha helix content, a coiled-coil domain involved in trimerization and a membrane fusogenic activity in vitro. In addition, we demonstrated that YqiC localizes at cytoplasmic and membrane subcellular fractions, that a S. Typhimurium yqiC deficient strain had a severe attenuation in virulence in the murine model when inoculated both orally and intraperitoneally, and was impaired to replicate at physiological and high temperatures in vitro, although it was still able to invade and replicate inside epithelial and macrophages cell lines.

Conclusion: This work firstly demonstrates the importance of a COG2960 member for pathogen-host interaction, and suggests a common function conserved among members of this group.

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In vitro liposome aggregation and fusion induced by YqiC. (A) Time course of DPPC/DPPA SUV aggregation monitored by light scattering and (B) time course of aqueous content mixing was measured after addition of YqiC protein. Equimolar amounts of terbium (Tb)- and dipicolinic acid (DPA)-loaded SUV were premixed in 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1 mM EDTA. The fluorescence of the Tb(DPA)3 complex formed after the mixing of aqueous contents by protein addition was measured at 545 nm over incubation time. The measurements were taken in 50 mM Tris-HCl buffer (pH 8.0) (open circles) or 50 mM sodium acetate buffer (pH 4.0) (close circles) at 25°C. The liposomes were composed of DPPC and DPPA in a molar ratio of 75:25. The lipid:protein molar ratio was 100: 1. The data presented are the results of a representative experiment of three independent repetitions.
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Figure 3: In vitro liposome aggregation and fusion induced by YqiC. (A) Time course of DPPC/DPPA SUV aggregation monitored by light scattering and (B) time course of aqueous content mixing was measured after addition of YqiC protein. Equimolar amounts of terbium (Tb)- and dipicolinic acid (DPA)-loaded SUV were premixed in 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1 mM EDTA. The fluorescence of the Tb(DPA)3 complex formed after the mixing of aqueous contents by protein addition was measured at 545 nm over incubation time. The measurements were taken in 50 mM Tris-HCl buffer (pH 8.0) (open circles) or 50 mM sodium acetate buffer (pH 4.0) (close circles) at 25°C. The liposomes were composed of DPPC and DPPA in a molar ratio of 75:25. The lipid:protein molar ratio was 100: 1. The data presented are the results of a representative experiment of three independent repetitions.

Mentions: As YqiC shares structural properties with BMFP, we investigated if this protein also conserves the membrane fusion activity reported for BMFP [9]. With this aim, we measured the increase in the size and aqueous content mixing of phospholipids vesicles produced after YqiC addition. Changes in the size and aggregation state of vesicles were evaluated by turbidity measurements at 400 nm whereas the aqueous content mixing was evaluated by measuring the fluorescence of the Tb-DPA complex produced upon fusion of vesicles containing TbCl3 or DPA encapsulated in their aqueous interior phase, and the percentage of mixing was calculated as described in materials and methods. Experiments were carried out on small unilamellar vesicles composed of a mixture of DPPC and DPPA in a 75:25 molar ratio, both at acid or neutral pH. YqiC produced both a significant increase in the turbidity (Figure 3A) and aqueous content mixing (Figure 3B) in the vesicle solutions, mainly at acid pH, after addition of YqiC. These results indicate that YqiC has a pH-dependent in vitro fusogenic activity.


YqiC of Salmonella enterica serovar Typhimurium is a membrane fusogenic protein required for mice colonization.

Carrica MC, Craig PO, García-Angulo VA, Aguirre A, García-Véscovi E, Goldbaum FA, Cravero SL - BMC Microbiol. (2011)

In vitro liposome aggregation and fusion induced by YqiC. (A) Time course of DPPC/DPPA SUV aggregation monitored by light scattering and (B) time course of aqueous content mixing was measured after addition of YqiC protein. Equimolar amounts of terbium (Tb)- and dipicolinic acid (DPA)-loaded SUV were premixed in 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1 mM EDTA. The fluorescence of the Tb(DPA)3 complex formed after the mixing of aqueous contents by protein addition was measured at 545 nm over incubation time. The measurements were taken in 50 mM Tris-HCl buffer (pH 8.0) (open circles) or 50 mM sodium acetate buffer (pH 4.0) (close circles) at 25°C. The liposomes were composed of DPPC and DPPA in a molar ratio of 75:25. The lipid:protein molar ratio was 100: 1. The data presented are the results of a representative experiment of three independent repetitions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3107778&req=5

Figure 3: In vitro liposome aggregation and fusion induced by YqiC. (A) Time course of DPPC/DPPA SUV aggregation monitored by light scattering and (B) time course of aqueous content mixing was measured after addition of YqiC protein. Equimolar amounts of terbium (Tb)- and dipicolinic acid (DPA)-loaded SUV were premixed in 10 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 1 mM EDTA. The fluorescence of the Tb(DPA)3 complex formed after the mixing of aqueous contents by protein addition was measured at 545 nm over incubation time. The measurements were taken in 50 mM Tris-HCl buffer (pH 8.0) (open circles) or 50 mM sodium acetate buffer (pH 4.0) (close circles) at 25°C. The liposomes were composed of DPPC and DPPA in a molar ratio of 75:25. The lipid:protein molar ratio was 100: 1. The data presented are the results of a representative experiment of three independent repetitions.
Mentions: As YqiC shares structural properties with BMFP, we investigated if this protein also conserves the membrane fusion activity reported for BMFP [9]. With this aim, we measured the increase in the size and aqueous content mixing of phospholipids vesicles produced after YqiC addition. Changes in the size and aggregation state of vesicles were evaluated by turbidity measurements at 400 nm whereas the aqueous content mixing was evaluated by measuring the fluorescence of the Tb-DPA complex produced upon fusion of vesicles containing TbCl3 or DPA encapsulated in their aqueous interior phase, and the percentage of mixing was calculated as described in materials and methods. Experiments were carried out on small unilamellar vesicles composed of a mixture of DPPC and DPPA in a 75:25 molar ratio, both at acid or neutral pH. YqiC produced both a significant increase in the turbidity (Figure 3A) and aqueous content mixing (Figure 3B) in the vesicle solutions, mainly at acid pH, after addition of YqiC. These results indicate that YqiC has a pH-dependent in vitro fusogenic activity.

Bottom Line: We found that YqiC shares biophysical and biochemical properties with Brucella abortus BMFP, the only previously characterized member of this group, such as a high alpha helix content, a coiled-coil domain involved in trimerization and a membrane fusogenic activity in vitro.In addition, we demonstrated that YqiC localizes at cytoplasmic and membrane subcellular fractions, that a S.This work firstly demonstrates the importance of a COG2960 member for pathogen-host interaction, and suggests a common function conserved among members of this group.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Biotecnología, CICVyA-INTA Castelar, Los Reseros y Las Cabañas s/n, Buenos Aires, Argentina. mcarrica@leloir.org.ar

ABSTRACT

Background: Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen which can colonize a variety of hosts, including human, causing syndromes that vary from gastroenteritis and diarrhea to systemic disease.

Results: In this work we present structural information as well as insights into the in vivo function of YqiC, a 99-residue protein of S. Typhimurium, which belongs to the cluster of the orthologous group 2960 (COG2960). We found that YqiC shares biophysical and biochemical properties with Brucella abortus BMFP, the only previously characterized member of this group, such as a high alpha helix content, a coiled-coil domain involved in trimerization and a membrane fusogenic activity in vitro. In addition, we demonstrated that YqiC localizes at cytoplasmic and membrane subcellular fractions, that a S. Typhimurium yqiC deficient strain had a severe attenuation in virulence in the murine model when inoculated both orally and intraperitoneally, and was impaired to replicate at physiological and high temperatures in vitro, although it was still able to invade and replicate inside epithelial and macrophages cell lines.

Conclusion: This work firstly demonstrates the importance of a COG2960 member for pathogen-host interaction, and suggests a common function conserved among members of this group.

Show MeSH
Related in: MedlinePlus