Iron-Sulfur Cluster Repair Contributes to Yersinia pseudotuberculosis Survival within Deep Tissues

Abstract

To successfully colonize host tissues, bacteria must respond to and detoxify many different host-derived antimicrobial compounds, such as nitric oxide (NO). NO has direct antimicrobial activity through attack on iron-sulfur (Fe-S) cluster-containing proteins. NO detoxification plays an important role in promoting bacterial survival, but it remains unclear if repair of Fe-S clusters is also important for bacterial survival within host tissues. Here we show that the Fe-S cluster repair protein YtfE contributes to the survival of Yersinia pseudotuberculosis within the spleen following nitrosative stress. Y. pseudotuberculosis forms clustered centers of replicating bacteria within deep tissues, where peripheral bacteria express the NO-detoxifying gene hmp. ytfE expression also occurred specifically within peripheral cells at the edges of microcolonies. In the absence of ytfE, the area of microcolonies was significantly smaller than that of the wild type (WT), consistent with ytfE contributing to the survival of peripheral cells. The loss of ytfE did not alter the ability of cells to detoxify NO, which occurred within peripheral cells in both WT and ΔytfE microcolonies. In the absence of NO-detoxifying activity by hmp, NO diffused across ΔytfE microcolonies, and there was a significant decrease in the area of microcolonies lacking ytfE, indicating that ytfE also contributes to bacterial survival in the absence of NO detoxification. These results indicate a role for Fe-S cluster repair in the survival of Y. pseudotuberculosis within the spleen and suggest that extracellular bacteria may rely on this pathway for survival within host tissues.

Subpopulations of Stressed Yersinia pseudotuberculosis Preferentially Survive Doxycycline Treatment within Host Tissues

“…The genetic tools available for Y. pseudotuberculosis allow us to easily construct fluorescent reporters to detect changes in the host environment at the single-bacterium level ( 22 – 24 ). Here, we introduce a tetracycline-responsive fluorescent reporter based on the tet operon that enables detection of the diffusion of tetracycline derivatives, including doxycycline (Dox), within host tissues.…”

“…Bacteria at the periphery of the microcolony respond to NO by expressing the NO-detoxifying gene, hmp (22). hmp expression at the periphery prevents NO diffusion into the center of microcolonies, establishing an example of cooperative behavior in which the peripheral bacteria protect the interior bacteria from the antimicrobial action of NO (22,23). It remains unclear if the protective expression of hmp by peripheral cells comes at a fitness cost, and if this stress response is sufficient to alter the antibiotic susceptibility of this subpopulation.…”

Structural basis of RICs iron donation for iron-sulfur cluster biogenesis

“…Lanes 1-4: control samples from extracts expressing only one protein. Lanes 5-8: co-purification of IscS with IscU (5); co-purification of YtfE and IscU with IscS (6); co-purification of IscS with YtfE (7); and co-purification of IscU with YtfE (8); c . In BiCF of YtfE and ISC proteins, cells were co-transformed with vectors expressing YtfE and IscS or IscU – GFP fusions.…”

Yersinia pseudotuberculosis: Cultivation, Storage, and Methods for Introducing DNA

“…Typically, we use this protocol to delete a target gene through recombination with the suicide vector by generating a PCR product that fuses the upstream and downstream regions of DNA together and inserting this into the pSR47s MCS ( Fig. 1) (Davis, Krupp, Clark, & Isberg, 2019;Davis, Mohammadi, & Isberg, 2015). We also use this protocol to introduce fluorescent protein genes into specific sites in the Yptb chromosome by fusing the target upstream and downstream regions around the fluorescent protein gene and inserting this into the pSR47s MCS.…”