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stg fimbrial operon from S. Typhi STH2370 contributes to association and cell disruption of epithelial and macrophage-like cells.

Berrocal L, Fuentes JA, Trombert AN, Jofré MR, Villagra NA, Valenzuela LM, Mora GC - Biol. Res. (2015)

Bottom Line: We compared S.We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells.The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Microbiología, Facultad de Ciencias Biológicas, Universidad Andres Bello, República 217, Santiago, Chile. lberrocal@uft.cl.

ABSTRACT

Background: Salmonella enterica serovar Typhi (S. Typhi) stg operon, encoding a chaperone/usher fimbria (CU), contributes to an increased adherence to human epithelial cells. However, one report suggests that the presence of the Stg fimbria impairs the monocyte--bacteria association, as deduced by the lower level of invasion to macrophage-like cells observed when the stg fimbrial cluster was overexpressed. Nevertheless, since other CU fimbrial structures increase the entry of S. Typhi into macrophages, and considering that transcriptomic analyses revealed that stg operon is indeed expressed in macrophages, we reassessed the role of the stg operon in the interaction between S. Typhi strain STH2370 and human cells, including macrophage-like cells and mononuclear cells directly taken from human peripheral blood.

Results: We compared S. Typhi STH2370 WT, a Chilean clinical strain, and the S. Typhi STH2370 Δstg mutant with respect to association and invasion using epithelial and macrophage-like cells. We observed that deletion of stg operon reduced the association and invasion of S. Typhi, in both cellular types. The presence of the cloned stg operon restored the WT phenotype in all the cases. Moreover, we compared Salmonella enterica sv. Typhimurium 14028s (S. Typhimurium, a serovar lacking stg operon) and S. Typhimurium heterologously expressing S. Typhi stg. We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells.

Conclusions: S. Typhi stg operon encodes a functional adhesin that participates in the interaction bacteria-eukaryotic cells, including epithelial cells and macrophages-like cells. The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption.

No MeSH data available.


Related in: MedlinePlus

Association and invasion of HEp-2 epithelial cells. The strains used include S. Typhi STH2370 WT (black), S. Typhi STH2370 ΔstgABCD::FRT (Δstg) (white), S. Typhi STH2370 ΔstgABCD::FRT/pSstg (Δstg/pSstg) (dark grey), and S. Typhi STH2370 ΔstgABCD::FRT/pSU19 (Δstg/pSU19) (light grey) (a); and S. Typhimurium 14028s WT (black), S. Typhimurium 14028s WT/pSstg (white), and S. Typhimurium 14028s/pSU19 (dark grey) (b). The figure shows values expressed as the mean ± standard deviation of three full biological replicates, each time in technical triplicate. *p < 0.05 (Student’s-test) compared with the WT in the corresponding group.
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Fig1: Association and invasion of HEp-2 epithelial cells. The strains used include S. Typhi STH2370 WT (black), S. Typhi STH2370 ΔstgABCD::FRT (Δstg) (white), S. Typhi STH2370 ΔstgABCD::FRT/pSstg (Δstg/pSstg) (dark grey), and S. Typhi STH2370 ΔstgABCD::FRT/pSU19 (Δstg/pSU19) (light grey) (a); and S. Typhimurium 14028s WT (black), S. Typhimurium 14028s WT/pSstg (white), and S. Typhimurium 14028s/pSU19 (dark grey) (b). The figure shows values expressed as the mean ± standard deviation of three full biological replicates, each time in technical triplicate. *p < 0.05 (Student’s-test) compared with the WT in the corresponding group.

Mentions: Considering that one of the first steps in the S. enterica infection involves the interaction with human epithelial cells, the contribution of the stg operon to cell adherence was assessed using HEp-2 cells. For that, the strains to be tested were cultured in LB to OD600 = 0.2 in microaerophilia without shaking prior to determining the number of bacteria associated to eukaryotic cells and the number of bacteria that invaded as described in “Methods”. Associated bacteria can be defined as adherent bacteria plus bacteria that invaded during the early stage of the interaction between bacteria and eukaryotic cells. As observed in Figure 1a, S. Typhi ΔstgABCD (i.e. Δstg) exhibited a significantly lower level of association to HEp-2 compared to the otherwise isogenic S. Typhi STH2370 WT, a highly virulent Chilean strain [12]. The complementation with the S. Typhi stg whole operon cloned into the pSU19 plasmid (pSstg) restored the WT phenotype, whereas the empty vector pSU19 exerted no effect (Figure 1a; Additional file 1: Table S1). On the other hand, S. Typhimurium naturally lacks the stg operon [7]. Thus, to test the contribution of the stg operon in a heterologous system, we transformed pSstg into S. Typhimurium 14028s WT prior to testing the bacterial association to the HEp-2 cells. As shown in Figure 1b (and Additional file 1: Table S1), S. Typhimurium 14028s WT/pSstg exhibited an increased association compared with the respective S. Typhimurium 14028s WT.Figure 1


stg fimbrial operon from S. Typhi STH2370 contributes to association and cell disruption of epithelial and macrophage-like cells.

Berrocal L, Fuentes JA, Trombert AN, Jofré MR, Villagra NA, Valenzuela LM, Mora GC - Biol. Res. (2015)

Association and invasion of HEp-2 epithelial cells. The strains used include S. Typhi STH2370 WT (black), S. Typhi STH2370 ΔstgABCD::FRT (Δstg) (white), S. Typhi STH2370 ΔstgABCD::FRT/pSstg (Δstg/pSstg) (dark grey), and S. Typhi STH2370 ΔstgABCD::FRT/pSU19 (Δstg/pSU19) (light grey) (a); and S. Typhimurium 14028s WT (black), S. Typhimurium 14028s WT/pSstg (white), and S. Typhimurium 14028s/pSU19 (dark grey) (b). The figure shows values expressed as the mean ± standard deviation of three full biological replicates, each time in technical triplicate. *p < 0.05 (Student’s-test) compared with the WT in the corresponding group.
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Related In: Results  -  Collection

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Fig1: Association and invasion of HEp-2 epithelial cells. The strains used include S. Typhi STH2370 WT (black), S. Typhi STH2370 ΔstgABCD::FRT (Δstg) (white), S. Typhi STH2370 ΔstgABCD::FRT/pSstg (Δstg/pSstg) (dark grey), and S. Typhi STH2370 ΔstgABCD::FRT/pSU19 (Δstg/pSU19) (light grey) (a); and S. Typhimurium 14028s WT (black), S. Typhimurium 14028s WT/pSstg (white), and S. Typhimurium 14028s/pSU19 (dark grey) (b). The figure shows values expressed as the mean ± standard deviation of three full biological replicates, each time in technical triplicate. *p < 0.05 (Student’s-test) compared with the WT in the corresponding group.
Mentions: Considering that one of the first steps in the S. enterica infection involves the interaction with human epithelial cells, the contribution of the stg operon to cell adherence was assessed using HEp-2 cells. For that, the strains to be tested were cultured in LB to OD600 = 0.2 in microaerophilia without shaking prior to determining the number of bacteria associated to eukaryotic cells and the number of bacteria that invaded as described in “Methods”. Associated bacteria can be defined as adherent bacteria plus bacteria that invaded during the early stage of the interaction between bacteria and eukaryotic cells. As observed in Figure 1a, S. Typhi ΔstgABCD (i.e. Δstg) exhibited a significantly lower level of association to HEp-2 compared to the otherwise isogenic S. Typhi STH2370 WT, a highly virulent Chilean strain [12]. The complementation with the S. Typhi stg whole operon cloned into the pSU19 plasmid (pSstg) restored the WT phenotype, whereas the empty vector pSU19 exerted no effect (Figure 1a; Additional file 1: Table S1). On the other hand, S. Typhimurium naturally lacks the stg operon [7]. Thus, to test the contribution of the stg operon in a heterologous system, we transformed pSstg into S. Typhimurium 14028s WT prior to testing the bacterial association to the HEp-2 cells. As shown in Figure 1b (and Additional file 1: Table S1), S. Typhimurium 14028s WT/pSstg exhibited an increased association compared with the respective S. Typhimurium 14028s WT.Figure 1

Bottom Line: We compared S.We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells.The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Microbiología, Facultad de Ciencias Biológicas, Universidad Andres Bello, República 217, Santiago, Chile. lberrocal@uft.cl.

ABSTRACT

Background: Salmonella enterica serovar Typhi (S. Typhi) stg operon, encoding a chaperone/usher fimbria (CU), contributes to an increased adherence to human epithelial cells. However, one report suggests that the presence of the Stg fimbria impairs the monocyte--bacteria association, as deduced by the lower level of invasion to macrophage-like cells observed when the stg fimbrial cluster was overexpressed. Nevertheless, since other CU fimbrial structures increase the entry of S. Typhi into macrophages, and considering that transcriptomic analyses revealed that stg operon is indeed expressed in macrophages, we reassessed the role of the stg operon in the interaction between S. Typhi strain STH2370 and human cells, including macrophage-like cells and mononuclear cells directly taken from human peripheral blood.

Results: We compared S. Typhi STH2370 WT, a Chilean clinical strain, and the S. Typhi STH2370 Δstg mutant with respect to association and invasion using epithelial and macrophage-like cells. We observed that deletion of stg operon reduced the association and invasion of S. Typhi, in both cellular types. The presence of the cloned stg operon restored the WT phenotype in all the cases. Moreover, we compared Salmonella enterica sv. Typhimurium 14028s (S. Typhimurium, a serovar lacking stg operon) and S. Typhimurium heterologously expressing S. Typhi stg. We found that the latter presents an increased cell disruption of polarized epithelial cells and an increased association in both epithelial and macrophage-like cells.

Conclusions: S. Typhi stg operon encodes a functional adhesin that participates in the interaction bacteria-eukaryotic cells, including epithelial cells and macrophages-like cells. The phenotypes associated to stg operon include increased association and consequent invasion in bacteria-eukaryotic cells, and cell disruption.

No MeSH data available.


Related in: MedlinePlus