Attenuation of Yersinia pestis fyuA Mutants Caused by Iron Uptake Inhibition and Decreased Survivability in Macrophages

Abstract

Yersinia pestis is the etiological agent of plague, a deadly infectious disease that has caused millions of deaths throughout history. Obtaining iron from the host is very important for bacterial pathogenicity. Y. pestis possesses many iron uptake systems. Yersiniabactin (Ybt) plays a major role in iron uptake in vivo and in vitro, and in virulence toward mice as well. FyuA, a β-barrel TonB-dependent outer membrane protein, serves as the receptor for Ybt. In this study, we examined the role of the fyuA gene in Y. pestis virulence using different challenging ways and explored the underlying mechanisms. The BALB/c mouse infection assay showed that the virulence of the mutant strains (ΔfyuA and ΔfyuA_GCAdel) was lower when compared with that of the wild-type (WT) strain 201. Furthermore, the attenuation of virulence of the mutant strains via subcutaneous and intraperitoneal challenges was far greater than that via intravenous injection. Iron supplementation restored lethality during subcutaneous challenge with the two mutants. Thus, we speculated that the attenuated virulence of the mutant strains toward the mice may be caused by dysfunctional iron uptake. Moreover, ΔfyuA and ΔfyuA_GCAdel strains exhibited lower survival rates in murine RAW264.7 macrophages, which might be another reason for the attenuation. We further explored the transcriptomic differences between the WT and mutant strains at different temperatures and found that the expressions of genes related to Ybt synthesis and its regulation were significantly downregulated in the mutant strains. This finding indicates that fyuA might exert a regulatory effect on Ybt. Additionally, the expressions of the components of the type III secretion system were unexpectedly upregulated in the mutants, which is inconsistent with the conventional view that the upregulation of the virulence genes enhances the virulence of the pathogens.

Keywords: Ybt system; Yersinia pestis; fyuA; iron uptake system; pathogenicity; virulence.

Figure 1

Growth curves of Y. pestis. Growth curve of WT, ΔfyuA, and ΔfyuA-Comp at 37°C in LB (A), at 26°C in defined TMH with 0.1 mM FeSO₄ (C), or at 26°C in deferrated TMH with 10 μM FeCl₃ (E). The bacterial growth was monitored by measuring the absorbance at OD_{620 nm} at 2 h intervals. The areas under the growth curve were applied for the statistical analysis of different conditions (B, D, F).

Figure 2

Real-time cell analysis assay curves of HeLa cells infected with WT and the two mutants. (A) Normalized CI value curves. The bacteria were added to the cell culture at 10h. (B) Normalized CI values of 14h (4h post infection). (C) Normalized CI values of 18h (8h post infection). (D) Normalized CI values of 21h (11h post infection). **, p < 0.01. ***, p < 0.001.

Figure 3

Y. pestis ΔfyuA and ΔfyuA _GCAdel mutants showed virulence attenuation in mice, and iron accelerated the mouse lethality during subcutaneous infection with ΔfyuA and ΔfyuA _GCAdel mutants. Each mouse of each group (n = 10) was challenged with 10⁴ CFU of bacteria. (A) Intraperitoneal challenges with WT, ΔfyuA, or ΔfyuA _GCAdel. (B) Intravenous challenges with WT, ΔfyuA, or ΔfyuA _GCAdel. (C) Subcutaneous challenges with WT, ΔfyuA, ΔfyuA _GCAdel, and the complemented strains of the two mutants. (D) The WT, ΔfyuA, and ΔfyuA _GCAdel groups were subcutaneously challenged with WT, ΔfyuA, or ΔfyuA _GCAdel respectively. The FeCl₂ groups were challenged with the mutants and were supplemented with 50 μg of FeCl₂ intraperitoneally or subcutaneously plus intraperitoneally. ***, p < 0.001.

Figure 4

Comparison of the viabilities of WT and the mutants in blood (A), serum (B) and RAW264.7 cells (C, D). The survival rates of ΔfyuA and ΔfyuA _GCAdel mutant strains were lower than that of strain 201 in RAW264.7 cells but showed no significant differences in blood and serum. The experiments were performed three times independently. **, p < 0.01. NS, not significant.

Figure 5

Comparative transcriptomics of strain 201 and ΔfyuA _GCAdel cultured at 26°C or 37°C. (A) Venn diagrams showing overlapping of the gene expression profiles between different comparison groups. (B) Clustering analysis of differential gene expression between WT and mutant strains. Different colors show different gene expression levels, with high expression being colored red and low expression being colored green. (C) Correlations among the expression levels of 17 genes measured with RNA-seq and qRT-PCR were analyzed using linear regression.