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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

Cell permeability assay of S. Typhi STH2370 WT, and S. Typhimurium 14028s WT and derivatives, through H-T29 human cell line monolayers. The arrow indicates the time at which gentamicin was added. The experiments were performed in three full biological replicates, each time in technical triplicate. The values are expressed as the mean ± standard deviation of three independent experiments. *p < 0.005 (Student’s-test).
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Fig2: Cell permeability assay of S. Typhi STH2370 WT, and S. Typhimurium 14028s WT and derivatives, through H-T29 human cell line monolayers. The arrow indicates the time at which gentamicin was added. The experiments were performed in three full biological replicates, each time in technical triplicate. The values are expressed as the mean ± standard deviation of three independent experiments. *p < 0.005 (Student’s-test).

Mentions: S. Typhi exhibits a high cytotoxicity towards eukaryotic cells compared to S. Typhimurium, clearly affecting the permeability of the infected cells. This phenotype can be explained because of the presence of cytolytic factors in the serovar Typhi and/or because of the presence of proteins that diminishes the cell cytotoxicity in the serovar Typhimurium [13–15]. Since the stg operon is only present in the serovar Typhi and absent from the serovar Typhimurium, we assessed whether the presence of the stg operon can also affect the cell permeability of a monolayer of epithelial cells. For that, polarized HT-29 monolayers were cultured in Transwells and incubated for 1 week to allow cell polarization with an apical zone (upper chamber) and a basolateral zone (lower chamber). Cell polarization was confirmed by a gradual increase of transepithelial resistance (TER) (data not shown). The polarized monolayers were subsequently infected with S. Typhimurium 14028s WT, S. Typhimurium 14028s WT/pSstg, and S. Typhimurium 14028s WT/pSU19. As control, we used S. Typhi STH2370 WT. The infected monolayers were used to perform a modified transepithelial migration assay that included addition of gentamicin (after 1 h of infection) into the upper chamber as previously described [14, 15]. As shown in Figure 2, the recovered CFU/mL represented bacteria that migrated to the lower chamber and survived the presence of the gentamicin leaking through the cell monolayer. If bacteria disrupt the integrity of the monolayer, gentamicin will leak through from the upper chamber to the lower chamber, killing bacteria in the lower chamber and decreasing the recovered CFU/mL. On the other hand, if the monolayer is not disrupted, the recovered CFU/mL should remain essentially constant over the same time course since gentamicin cannot permeate through cellular membranes [16]. As observed in Figure 2, the recovered CFU/mL corresponding to S. Typhimurium 14028s presented a slight decline over the time course of the assay (black squares), showing that the monolayer integrity is not largely affected by bacteria in this case, accordingly to previous published studies [14, 15]. In contrast, the CFU/mL of S. Typhi STH2370 recovered from the lower chamber abruptly decreased after the gentamicin addition until they became undetectable, showing that the gentamicin leaked into the lower chamber due to a monolayer disruption (black diamonds). Most importantly, when S. Typhimurium 14028s was complemented with the S. Typhi stg operon (pSstg, white squares) we observed that the corresponding recovered CFU/mL clearly decreased, marking a sharp difference with the otherwise isogenic S. Typhimurium 14028s WT strain and resembling the S. Typhi phenotype (compare the black squares and white squares) (Figure 2). All these results were corroborated by measuring the transepithelial electrical resistance (TER) of the infected HT-29 monolayer as previously described [14] (data not shown).Figure 2


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)

Cell permeability assay of S. Typhi STH2370 WT, and S. Typhimurium 14028s WT and derivatives, through H-T29 human cell line monolayers. The arrow indicates the time at which gentamicin was added. The experiments were performed in three full biological replicates, each time in technical triplicate. The values are expressed as the mean ± standard deviation of three independent experiments. *p < 0.005 (Student’s-test).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Cell permeability assay of S. Typhi STH2370 WT, and S. Typhimurium 14028s WT and derivatives, through H-T29 human cell line monolayers. The arrow indicates the time at which gentamicin was added. The experiments were performed in three full biological replicates, each time in technical triplicate. The values are expressed as the mean ± standard deviation of three independent experiments. *p < 0.005 (Student’s-test).
Mentions: S. Typhi exhibits a high cytotoxicity towards eukaryotic cells compared to S. Typhimurium, clearly affecting the permeability of the infected cells. This phenotype can be explained because of the presence of cytolytic factors in the serovar Typhi and/or because of the presence of proteins that diminishes the cell cytotoxicity in the serovar Typhimurium [13–15]. Since the stg operon is only present in the serovar Typhi and absent from the serovar Typhimurium, we assessed whether the presence of the stg operon can also affect the cell permeability of a monolayer of epithelial cells. For that, polarized HT-29 monolayers were cultured in Transwells and incubated for 1 week to allow cell polarization with an apical zone (upper chamber) and a basolateral zone (lower chamber). Cell polarization was confirmed by a gradual increase of transepithelial resistance (TER) (data not shown). The polarized monolayers were subsequently infected with S. Typhimurium 14028s WT, S. Typhimurium 14028s WT/pSstg, and S. Typhimurium 14028s WT/pSU19. As control, we used S. Typhi STH2370 WT. The infected monolayers were used to perform a modified transepithelial migration assay that included addition of gentamicin (after 1 h of infection) into the upper chamber as previously described [14, 15]. As shown in Figure 2, the recovered CFU/mL represented bacteria that migrated to the lower chamber and survived the presence of the gentamicin leaking through the cell monolayer. If bacteria disrupt the integrity of the monolayer, gentamicin will leak through from the upper chamber to the lower chamber, killing bacteria in the lower chamber and decreasing the recovered CFU/mL. On the other hand, if the monolayer is not disrupted, the recovered CFU/mL should remain essentially constant over the same time course since gentamicin cannot permeate through cellular membranes [16]. As observed in Figure 2, the recovered CFU/mL corresponding to S. Typhimurium 14028s presented a slight decline over the time course of the assay (black squares), showing that the monolayer integrity is not largely affected by bacteria in this case, accordingly to previous published studies [14, 15]. In contrast, the CFU/mL of S. Typhi STH2370 recovered from the lower chamber abruptly decreased after the gentamicin addition until they became undetectable, showing that the gentamicin leaked into the lower chamber due to a monolayer disruption (black diamonds). Most importantly, when S. Typhimurium 14028s was complemented with the S. Typhi stg operon (pSstg, white squares) we observed that the corresponding recovered CFU/mL clearly decreased, marking a sharp difference with the otherwise isogenic S. Typhimurium 14028s WT strain and resembling the S. Typhi phenotype (compare the black squares and white squares) (Figure 2). All these results were corroborated by measuring the transepithelial electrical resistance (TER) of the infected HT-29 monolayer as previously described [14] (data not shown).Figure 2

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