The stg operon contributes to the association, invasion, and to an increased permeability of HEp-2 human epithelial cells in S. enterica
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.
Considering that an increased association between bacteria and epithelial cells conceivably may affect the subsequent bacterial internalization, we assessed whether stg was implicated in the invasion of epithelial cells. The invasion is a critical step in the normal infection cycle of S. enterica. Since the invasion is promoted by effectors injected into host cells through a type-III secretory system, an increased association bacteria—epithelial cell might affect this process. To test this hypothesis, we performed a gentamicin protection assay comparing the invasion rates of S. Typhi STH2370 WT and S. Typhi Δstg. As shown in Figure 1a, S. Typhi Δstg presented an impaired invasion compared with the WT strain, consistent with the results obtained for the association bacteria—epithelial cells. Again, the pSstg plasmid, and not the vector alone, restored the WT phenotype (Figure 1a, Additional file 1: Table S1). Nevertheless, when invasion efficiency was calculated by determining the ratio of invaded/associated bacteria using the raw data (Additional file 1: Table S1), no significant differences were observed. This result suggests that the decreased invasion is a consequence of a decreased association, probably mediated by an effect in adherence, and not to an effect in invasion specifically. On the other hand, the pSstg plasmid apparently produced no effects in the invasion of the S. Typhimurium heterologous strain (Figure 1b), showing that the stg operon is not specifically contributing to the invasion of S. Typhimurium 14028s under the tested conditions.
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).
All these results together show that the stg operon contributes to increase the interaction bacteria—epithelial cell and the invasion in S. Typhi. Moreover, when heterologously expressed, stg contributes to increase the adherence, and cell disruption in S. Typhimurium.
The stg operon contributes to increase the association of bacteria and human macrophage-like cells in S. enterica
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.
To assess the contribution of the stg operon in macrophage-like—bacteria interaction in an independent way, we performed association and invasion assays using S. Typhimurium heterologously expressing S. Typhi stg from the pSstg plasmid. In this case, we tested the S. Typhimurium/pSstg with a mononuclear cell fraction obtained directly from human peripheral blood isolated with HISTOPAQUE®-1077. As observed in Figure 3b and in Additional file 1: Table S3, the presence of the stg operon in S. Typhimurium significantly contributes to the bacterial uptake compared with the control. In all cases, the empty vector exerted no effects (Figure 3). The invasion efficiency (Additional file 1: Table S3) presented no significant differences among the strains, suggesting that also in this case the contribution of stg to invasion is dependent on the bacteria—eukaryotic cells association.
All these results together show that the stg operon contributes to the macrophage-like—bacteria interaction, contributing to the association and the invasion in a line of human monocytes (i.e. U937) and in mononuclear cells directly extracted from human blood.