Genome-wide characterization of Salmonella Typhimurium genes required for the fitness under iron restriction

Abstract

Background: Iron is a crucial element for bacterial survival and virulence. During Salmonella infection, the host utilizes a variety of mechanisms to starve the pathogen from iron. However, Salmonella activates distinctive defense mechanisms to acquire iron and survive in iron-restricted host environments. Yet, the comprehensive set of the conditionally essential genes that underpin Salmonella survival under iron-restricted niches has not been fully explored.

Results: Here, we employed transposon sequencing (Tn-seq) method for high-resolution elucidation of the genes in Salmonella Typhimurium (S. Typhimurium) 14028S strain required for the growth under the in vitro conditions with four different levels of iron restriction achieved by iron chelator 2,2'-dipyridyl (Dip): mild (100 and 150 μM), moderate (250 μM) and severe iron restriction (400 μM). We found that the fitness of the mutants reduced significantly for 28 genes, suggesting the importance of these genes for the growth under iron restriction. These genes include sufABCDSE, iron transport fepD, siderophore tonB, sigma factor E ropE, phosphate transport pstAB, and zinc exporter zntA. The siderophore gene tonB was required in mild and moderate iron-restricted conditions, but it became dispensable in severe iron-restricted conditions. Remarkably, rpoE was required in moderate and severe iron restrictions, leading to complete attenuation of the mutant under these conditions. We also identified 30 genes for which the deletion of the genes resulted in increased fitness under iron-restricted conditions.

Conclusions: The findings broaden our knowledge of how S. Typhimurium survives in iron-deficient environments, which could be utilized for the development of new therapeutic strategies targeting the pathways vital for iron metabolism, trafficking, and scavenging.

Keywords: Conditionally essential genes; Salmonella Typhimurium; Tn-seq; iron-restriction.

Fig. 1

Effect of varying concentrations of 2,2`-Dipyridyl on the growth of S. Typhimurium 14028S. S. Typhimurium 14028S wild-type strain was grown in LB broth supplemented with different levels of Dip (0, 100, 125, 250, or 400 μM). The cultures were incubated in a 96-well plate and OD₆₀₀ was measured with Tecan Infinite 200 microplate reader for 24 hr. at 37 °C. The collected data were used to calculate the growth rate (A) and to obtain maximum OD₆₀₀ (B). Data represent at least three replicates

Fig. 2

sufS is required for the optimal growth of S. Typhimurium under iron-restricted conditions. S. Typhimurium 14028S wild-type and ΔsufS mutant were grown in LB broth, supplemented with 0 (control), 100 or 150 μM 2,2`-Dipyridyl (Dip). OD₆₀₀ were recorded every 15 minutes during incubation at 37 °C for 24 hr. in a 96-well plate to show the growth responses (A) and to calculate the doubling times (B). Data represent at least three replicates. Statistical significance of doubling times was determined by unpaired two-tailed t test, ***p < 0.001

Fig. 3

degS and rpoE are required for the growth S. Typhimurium growth under iron-restricted conditions. Spot dilution assay was performed with S. Typhimurium 14,028 s wild-type, ΔdegS and ΔrpoE mutants. The serial dilutions (10⁰–10^− 7 dilutions) of the cultures of wild-type, ∆ degS and ∆ rpoE, were spotted on the surface of LB agar plates containing 0 (control), 100, 200, 300, and 400 μM 2,2`-Dipyridyl (Dip). The plates were incubated at 37 °C and results were recorded after 24 hr.

Fig. 4

zntA is required for the growth of S. Typhimurium under iron-restricted conditions. S. Typhimurium 14028S wild-type and ΔzntA mutant were grown in LB broth supplemented with 0 (control), 100 or 150 μM 2,2`-Dipyridyl (Dip). OD₆₀₀ was recorded every 15 minutes during incubation at 37 °C for 24 h in a 96-well plate. Statistical significance was determined by unpaired two-tailed t test, *P < 0.05