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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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Related in: MedlinePlus

Growth curve of S. Typhimurium ATCC 14028 (circles), 14028 ΔyqiC::CAT (triangles), and 14028 ΔyqiC::CAT + pBBR yqiC (squares) at different temperatures. A 1:50 dilution of a saturated culture in LB was incubated at 200 rpm, at the indicated temperature. The OD600 was measured at different time points over 48 hours. The data presented are the results of a representative experiment of three independent repetitions.
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Figure 5: Growth curve of S. Typhimurium ATCC 14028 (circles), 14028 ΔyqiC::CAT (triangles), and 14028 ΔyqiC::CAT + pBBR yqiC (squares) at different temperatures. A 1:50 dilution of a saturated culture in LB was incubated at 200 rpm, at the indicated temperature. The OD600 was measured at different time points over 48 hours. The data presented are the results of a representative experiment of three independent repetitions.

Mentions: The in vivo functions of the members of the COG 2960 are unknown. To investigate the role of YqiC protein in S. Typhimurium, we constructed an S. Typhimurium ATCC 14028 mutant in yqiC through allelic exchange. The resulting strain was named 14028 ΔyqiC::CAT. The gene yqiC is encoded divergently to the ribB gene and convergent to the glgS gene in the S. Typhimurium chromosome. Thus, it appears that yqiC is transcribed as a monocistronic element, and polar effects upon allelic exchange are not expected. The successful elimination of the yqiC gene was corroborated by PCR analysis and a western blot assay of cell lysates of 14028 ΔyqiC::CAT and its complemented derivative (bearing plasmid pBBR-yqiC, which encodes intact yqiC gene), using a polyclonal antibody raised against YqiC (data not shown). As a first approach to assess the effect of the mutation in the physiology of Salmonella, we tested the effect of temperature in the replication of yqiC mutant strain in LB. No difference in the growth pattern of the yqiC mutant strain compared with the WT was detected at 28°C (average generation time 44.9 +/- 1.4). However, an increased generation time at 37°C was observed for 14028 ΔyqiC::CAT, where the average generation time was 22.5 +/- 0.7 minutes for S. Typhimurium 14028 and 48 minutes for 14028 ΔyqiC::CAT (Figure 5). This difference in growth was enhanced when the strains were incubated at 42°C, where the average generation time was 30.2 +/- 0.68 minutes for the WT strain and 78.9 +/- 0.7 minutes for the ΔyqiC::CAT mutant strain. At both temperatures, trans complementation with the plasmid encoding yqiC restored the wild-type growth curve pattern to 14028 ΔyqiC::CAT. These results indicate that the mutation of yqiC affects the ability of S. Typhimurium to replicate at physiological and high temperatures. No growth curve pattern alteration was observed for the 14028 ΔyqiC::CAT strain when incubated in M9 minimal media or acid LB (pH = 4.0) at 28°C (data not shown), which indicates that the yqiC mutant is neither auxotrophic nor acid sensitive.


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)

Growth curve of S. Typhimurium ATCC 14028 (circles), 14028 ΔyqiC::CAT (triangles), and 14028 ΔyqiC::CAT + pBBR yqiC (squares) at different temperatures. A 1:50 dilution of a saturated culture in LB was incubated at 200 rpm, at the indicated temperature. The OD600 was measured at different time points over 48 hours. 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 5: Growth curve of S. Typhimurium ATCC 14028 (circles), 14028 ΔyqiC::CAT (triangles), and 14028 ΔyqiC::CAT + pBBR yqiC (squares) at different temperatures. A 1:50 dilution of a saturated culture in LB was incubated at 200 rpm, at the indicated temperature. The OD600 was measured at different time points over 48 hours. The data presented are the results of a representative experiment of three independent repetitions.
Mentions: The in vivo functions of the members of the COG 2960 are unknown. To investigate the role of YqiC protein in S. Typhimurium, we constructed an S. Typhimurium ATCC 14028 mutant in yqiC through allelic exchange. The resulting strain was named 14028 ΔyqiC::CAT. The gene yqiC is encoded divergently to the ribB gene and convergent to the glgS gene in the S. Typhimurium chromosome. Thus, it appears that yqiC is transcribed as a monocistronic element, and polar effects upon allelic exchange are not expected. The successful elimination of the yqiC gene was corroborated by PCR analysis and a western blot assay of cell lysates of 14028 ΔyqiC::CAT and its complemented derivative (bearing plasmid pBBR-yqiC, which encodes intact yqiC gene), using a polyclonal antibody raised against YqiC (data not shown). As a first approach to assess the effect of the mutation in the physiology of Salmonella, we tested the effect of temperature in the replication of yqiC mutant strain in LB. No difference in the growth pattern of the yqiC mutant strain compared with the WT was detected at 28°C (average generation time 44.9 +/- 1.4). However, an increased generation time at 37°C was observed for 14028 ΔyqiC::CAT, where the average generation time was 22.5 +/- 0.7 minutes for S. Typhimurium 14028 and 48 minutes for 14028 ΔyqiC::CAT (Figure 5). This difference in growth was enhanced when the strains were incubated at 42°C, where the average generation time was 30.2 +/- 0.68 minutes for the WT strain and 78.9 +/- 0.7 minutes for the ΔyqiC::CAT mutant strain. At both temperatures, trans complementation with the plasmid encoding yqiC restored the wild-type growth curve pattern to 14028 ΔyqiC::CAT. These results indicate that the mutation of yqiC affects the ability of S. Typhimurium to replicate at physiological and high temperatures. No growth curve pattern alteration was observed for the 14028 ΔyqiC::CAT strain when incubated in M9 minimal media or acid LB (pH = 4.0) at 28°C (data not shown), which indicates that the yqiC mutant is neither auxotrophic nor acid sensitive.

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