Proteomic analysis of iron acquisition, metabolic and regulatory responses of Yersinia pestis to iron starvation

Abstract

Background: The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26 degrees C and 37 degrees C).

Results: Differential protein display was performed for three Y. pestis subcellular fractions. Five characterized Y. pestis iron/siderophore acquisition systems (Ybt, Yfe, Yfu, Yiu and Hmu) and a putative iron/chelate outer membrane receptor (Y0850) were increased in abundance in iron-starved cells. The iron-sulfur (Fe-S) cluster assembly system Suf, adapted to oxidative stress and iron starvation in E. coli, was also more abundant, suggesting functional activity of Suf in Y. pestis under iron-limiting conditions. Metabolic and reactive oxygen-deactivating enzymes dependent on Fe-S clusters or other iron cofactors were decreased in abundance in iron-depleted cells. This data was consistent with lower activities of aconitase and catalase in iron-starved vs. iron-rich cells. In contrast, pyruvate oxidase B which metabolizes pyruvate via electron transfer to ubiquinone-8 for direct utilization in the respiratory chain was strongly increased in abundance and activity in iron-depleted cells.

Conclusions: Many protein abundance differences were indicative of the important regulatory role of the ferric uptake regulator Fur. Iron deficiency seems to result in a coordinated shift from iron-utilizing to iron-independent biochemical pathways in the cytoplasm of Y. pestis. With growth temperature as an additional variable in proteomic comparisons of the Y. pestis fractions (26 degrees C and 37 degrees C), there was little evidence for temperature-specific adaptation processes to iron starvation.

Figure 1

Protein display in 2D gels of Y. pestis KIM6+ periplasmic fractions in the pI range 4-7 (-Fe vs. +Fe conditions). Proteins were derived from cell growth in the presence of 10 μM FeCl₃ at 26°C (top) or the absence of FeCl₃ at 26°C (bottom). Gels (20 × 25 cm) were stained with Coomassie Brilliant Blue G250 (CBB), with five gel replicates representing each group, and subjected to differential display analysis using the software Proteomweaver v.4.0. Protein assignment to a spot required validation by MS data from at least two representative gels. The denoted spot numbers are equivalent to those listed in Table 1 with their '-Fe vs. +Fe' protein abundance ratios and other data.

Figure 2

Protein display in 2D gels of Y. pestis KIM6+ periplasmic fractions in the pI range 6.5-9 (-Fe vs. +Fe conditions). Proteins were derived from cell growth in the presence of 10 μM FeCl₃ at 26°C (top) or absence of FeCl₃ at 26°C (bottom). Gels (20 × 25 cm) were stained with CBB, with three gel replicates representing each group, and subjected to differential display analysis using the software Proteomweaver v.4.0. Protein assignment to a spot required validation by MS data from at least two representative gels. The denoted spot numbers are equivalent to those listed in Table 1 with their '-Fe vs. +Fe' protein abundance ratios and other data.

Figure 3

Protein display in 2D gels of Y. pestis KIM6+ membrane fractions in the pI range 4-7 (-Fe vs. +Fe conditions). Proteins were derived from cell growth in the presence of 10 μM FeCl₃ at 26°C (top) or absence of FeCl₃ at 26°C (bottom). Gels (20 × 25 cm) were stained with CBB, with five gel replicates representing each of the groups, and subjected to differential display analysis using the software Proteomweaver v.4.0. Protein assignments to a spot (or a spot train) required validation by MS data from at least two representative gels. The denoted spots and spot trains are equivalent to those listed in Table 2 with their '-Fe vs. +Fe' protein abundance ratios and other data.

Figure 4

Protein display in 2D gels of Y. pestis KIM6+ cytoplasmic fractions in the pI range 4-7 (-Fe vs. +Fe conditions). Proteins were derived from cell growth in the presence of 10 μM FeCl₃ at 26°C (top) or the absence of FeCl₃ at 26°C (bottom). Gels (20 × 25 cm) were stained with CBB, with four gel replicates representing each group, and subjected to differential display analysis using the software Proteomweaver v.4.0. Protein assignment to a spot required validation by MS data from at least two representative gels. The denoted spot numbers are equivalent to those listed in Table 3 with their '-Fe vs. +Fe' protein abundance ratios and other data.

Figure 5

Iron homeostasis in Y. pestis. The center of the schematic depicts a network of regulators (orange color), known or potentially involved in iron homeostasis. Details are provided in the text. CRP (carbon metabolism); OxyR (oxidative stress); Fur and small RNAs like RyhB (iron homeostasis). Red lines/arrows show which genes (or mRNAs) are controlled by these regulators. Additional arrows symbolize enzymatic reactions (blue line) or small molecule transport processes (dotted green line). The lower/left side of the schematic depicts components of the energy metabolism. It includes glycolytic steps from dihydroxyacetone phosphate (DHAP) to pyruvate, the TCA/glyoxalate bypass cycle and on the left side alternative pyruvate metabolism branches generating acetate or acetyl-CoA. Subunits of electron transport systems (NuoCD, FrdAB and CybC) are also displayed. The top/left side of the schematic pertains to quorum sensing. LuxS converts S-ribosylhomocysteine (SRH) to 4,5-dihydroxy-2,3-pentanedione (DPD) which is a precursor of autoinducer-2 (yellow pentagon). In E. coli, the autoinducer-2 is exported and imported via periplasmic LsrB into different cells followed by activation of LuxR via small RNA regulators. The precise functional role of YebC in quorum sensing is not known. LuxR influences the expression of virulence factors in pathogenic E. coli strains. The role of LuxR in the regulation of the type VI secretion system is speculative, but both iron starvation [73] and the T6SS [74] have been linked to quorum sensing in other organisms. In the upper part of the schematic, iron transporter subunits are placed according their predicted or known subcellular localizations. Transporters with a blue color background are known to be functional in Y. pestis. On the center/right side, iron storage proteins, the Suf Fe-S cluster assembly system and putative sulfur-mobilizing enzymes (TauD and CysIJ) are displayed. The bottom/right part of the schematic features oxidative stress response proteins. Finally, the top/right part of the schematic displays the flea survival factor Ymt and its fragments, as well as the protein YqhD. These proteins may be implicated in the enzymatic modifications of IM phospholipids. Proteins with a red and green background harbor iron/heme and Fe-S cluster cofactors, respectively.