Limits...
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 monocytes U937 (a) or mononuclear cells directly extracted from human blood (b). The strains used include S. Typhi STH2370 WT (black), S. Typhi STH2370 ΔstgABCD::FRT (Δstg) (white), S. Typhi STH2370 ΔstgABCD::FRT/pSstg (Δstg/pSstg) (fark 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.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4494162&req=5

Fig3: Association and invasion of monocytes U937 (a) or mononuclear cells directly extracted from human blood (b). The strains used include S. Typhi STH2370 WT (black), S. Typhi STH2370 ΔstgABCD::FRT (Δstg) (white), S. Typhi STH2370 ΔstgABCD::FRT/pSstg (Δstg/pSstg) (fark 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: Macrophage-like cells play an important role in the dissemination of Salmonella enterica in the systemic phase of the disease [9]. In a previous report, it has been stated that deletion of stg increased uptake of serovar Typhi by human macrophages, and overexpression of stg operon in S. Typhi and S. Typhimurium strains reduced phagocytosis by human macrophages [10]. In that study, S. Typhi strain ISP1820 and derivatives were grown to stationary phase under aerobic conditions, prior to performing the infection [10]. Nevertheless, other studies reported that the stg operon increases its expression during an infection of macrophages [17, 18], suggesting that this operon may contribute to S. Typhi invasion in these cells. Considering that other CU fimbriae have an active role in bacterial invasion of host phagocytic cells [11, 19], we reassessed the role of the stg operon in the macrophage-like cell interaction. The first approach was to determine the role of stg in the bacterial association to human macrophage-like cells (monocytes). For that, the human monocyte cell line U937 was infected with S. Typhi STH2370 WT, S. Typhi STH2370 Δstg, S. Typhi STH2370 Δstg/pSstg, or S. Typhi STH2370 Δstg/pSU19, previously cultured in LB to OD600 = 0.2 under microaerophilic conditions, to perform the adherence and invasion assays. As shown in Figure 3a and in Additional file 1: Table S2, S. Typhi Δstg exhibited a significant impaired association to U937 cells compared to the WT strain. Trans-complementing with the pSstg plasmid reverted the mutant phenotype (Figure 3a). Considering that a lower association could lead to a decreased bacterial invasion, we determined the bacterial uptake by U937 monocytes using the gentamicin protection assay. As observed in Figure 3a, the S. Typhi Δstg also presented a decreased invasion compared with the WT. Again, when invasion efficiency was calculated (see data in Additional file 1: Table S2), no significant differences were observed, suggesting that the contribution of stg to the invasion depends on the association (plausibly on the adherence) in this case.Figure 3


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 monocytes U937 (a) or mononuclear cells directly extracted from human blood (b). The strains used include S. Typhi STH2370 WT (black), S. Typhi STH2370 ΔstgABCD::FRT (Δstg) (white), S. Typhi STH2370 ΔstgABCD::FRT/pSstg (Δstg/pSstg) (fark 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.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494162&req=5

Fig3: Association and invasion of monocytes U937 (a) or mononuclear cells directly extracted from human blood (b). The strains used include S. Typhi STH2370 WT (black), S. Typhi STH2370 ΔstgABCD::FRT (Δstg) (white), S. Typhi STH2370 ΔstgABCD::FRT/pSstg (Δstg/pSstg) (fark 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: Macrophage-like cells play an important role in the dissemination of Salmonella enterica in the systemic phase of the disease [9]. In a previous report, it has been stated that deletion of stg increased uptake of serovar Typhi by human macrophages, and overexpression of stg operon in S. Typhi and S. Typhimurium strains reduced phagocytosis by human macrophages [10]. In that study, S. Typhi strain ISP1820 and derivatives were grown to stationary phase under aerobic conditions, prior to performing the infection [10]. Nevertheless, other studies reported that the stg operon increases its expression during an infection of macrophages [17, 18], suggesting that this operon may contribute to S. Typhi invasion in these cells. Considering that other CU fimbriae have an active role in bacterial invasion of host phagocytic cells [11, 19], we reassessed the role of the stg operon in the macrophage-like cell interaction. The first approach was to determine the role of stg in the bacterial association to human macrophage-like cells (monocytes). For that, the human monocyte cell line U937 was infected with S. Typhi STH2370 WT, S. Typhi STH2370 Δstg, S. Typhi STH2370 Δstg/pSstg, or S. Typhi STH2370 Δstg/pSU19, previously cultured in LB to OD600 = 0.2 under microaerophilic conditions, to perform the adherence and invasion assays. As shown in Figure 3a and in Additional file 1: Table S2, S. Typhi Δstg exhibited a significant impaired association to U937 cells compared to the WT strain. Trans-complementing with the pSstg plasmid reverted the mutant phenotype (Figure 3a). Considering that a lower association could lead to a decreased bacterial invasion, we determined the bacterial uptake by U937 monocytes using the gentamicin protection assay. As observed in Figure 3a, the S. Typhi Δstg also presented a decreased invasion compared with the WT. Again, when invasion efficiency was calculated (see data in Additional file 1: Table S2), no significant differences were observed, suggesting that the contribution of stg to the invasion depends on the association (plausibly on the adherence) in this case.Figure 3

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