Disruption of the NlpD lipoprotein of the plague pathogen Yersinia pestis affects iron acquisition and the activity of the twin-arginine translocation system

Abstract

We have previously shown that the cell morphogenesis NlpD lipoprotein is essential for virulence of the plague bacteria, Yersinia pestis. To elucidate the role of NlpD in Y. pestis pathogenicity, we conducted a whole-genome comparative transcriptome analysis of the wild-type Y. pestis strain and an nlpD mutant under conditions mimicking early stages of infection. The analysis suggested that NlpD is involved in three phenomena: (i) Envelope stability/integrity evidenced by compensatory up-regulation of the Cpx and Psp membrane stress-response systems in the mutant; (ii) iron acquisition, supported by modulation of iron metabolism genes and by limited growth in iron-deprived medium; (iii) activity of the twin-arginine (Tat) system, which translocates folded proteins across the cytoplasmic membrane. Virulence studies of Y. pestis strains mutated in individual Tat components clearly indicated that the Tat system is central in Y. pestis pathogenicity and substantiated the assumption that NlpD essentiality in iron utilization involves the activity of the Tat system. This study reveals a new role for NlpD in Tat system activity and iron assimilation suggesting a modality by which this lipoprotein is involved in Y. pestis pathogenesis.

Fig 1. Role of iron in nlpD mutant growth and pathogenesis.

A. Growth of Y. pestis strains under iron-limiting conditions. Kim53 (upper panel), Kim53ΔnlpD (lower panel). The displayed data is one representative experiment. B. Survival curves of iron-treated mice infected with attenuated Y. pestis strains. Two groups of 12 mice were infected with the attenuated Y. pestis EV76 (circles) or Kim53ΔnlpD (square) strains (s.c. infection with 10⁷ cfu/mouse). In each group, six mice were treated with iron dextran (filled symbols), and six mice served as control (open symbols). Survival curve of mice infected with the Y. pestis Kim53 strain (s.c. infection with 10⁶ cfu/mouse, triangles).

Fig 2. Intracellular localization of the TorA_signal-GFP Tat-reporter protein in Y. pestis strains.

A. Y. pestis strains Kim53:pTorA_signal-GFP, Kim53ΔnlpD:pTorA_signal-GFP Kim53ΔtatA:pTorA_signal-GFP, and Kim53ΔtatC:pTorA_signal-GFP, were inspected under a fluorescence microscope for identification of TorA_signal-GFP localization. The scale bar represents 1 μm. B. Y. pestis cells overproducing TorA_signal-GFP were fractionated into Whole cell (W), spheroplast (S) and periplasm (P), and were subject to immunoblotting with antibodies against GFP or against the cytoplasmic protein Pcm [20]. The blots were derived from the same experiment and were processed in parallel. The data presented are representative of at least two independent experiments and the displayed data is one representative experiment.

Fig 3. Expression of TatC in the Y. pestis strains.

A. Quantitative RT-PCR analysis of tatC mRNA levels. mRNA from Kim53 (white histogram) and the nlpD mutant (gray histogram) was subjected to qRT-PCR analysis of tatC gene expression. The relative mRNA level was determined by calculating the threshold cycle (ΔCt) of target genes via the classic ΔCt method [90]. The results presented are an average of three independent experiments. B. Western blot analysis of TatC protein levels in total cell lysates of the wild-type Kim53 strain and the nlpD mutant. Whole cell lysates (10⁶ cfu/lane grown at 37°C), were subjected to Western blot analysis using anti-NlpD and anti-TatC antibodies. The Coomassie blue stained gel and the blots were derived from the same experiment and were processed in parallel. C. Distribution of TatC protein on the bacterial membrane of Y. pestis strains. Fluorescence microscopy images of wild-type Kim53 (top panel) and the nlpD mutant (lower panel) are presented after TatC staining alone (right panel) or with DAPI staining (left panel). Images (100×) were captured with a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany). Scale bar = 1 μm. The inset shows a magnification (×3) of stained bacterial cells. The data presented are representative of at least two independent experiments and the displayed data is one representative experiment.

Fig 4. The Sec translocon is operational in the ΔnlpD mutant.

A. The indicated bacterial cells were labeled with FITC-conjugated α-F1 antibodies (left), DAPI (right) or both α-F1 and DAPI (middle). B. Fluorescence microscopy of Y. pestis strains expressing the Sec-substrates BtuC and GadC fused to GFP. Scale bar = 1 μm. C. Relative fluorescence units (RFUs) of the ΔnlpD mutant expressing BtuC-GFP or GadC-GFP, compared to the wild Kim53 strain expressing the reporter proteins, according to [40]. Ns, non-significant (Unpaired t test). The data presented are representative of at least two independent experiments and the displayed data in A and B is one representative experiment.

Fig 5. Phenotypic characterization of Y. pestis tat mutants.

A. Gram staining of Y. pestis strains wild-type Kim53, ΔnlpD, ΔtatA, ΔtatC, ΔtatA+ptatA and, ΔtatC+ptatC was performed, and bacilli were observed by light microscopy at a magnification of ×1000. Scale bar = 10 μm. B. Growth of Y. pestis strains under iron-limiting conditions. Y. pestis strains (see description in the lower panel, iv), were grown under iron-limiting conditions (see Materials and Methods). The medium included: 1% agarose, 1× PMH2, 20 μM MgCl₂ and 80 μM DIP (i), 100 μM DIP (ii) or 100 μM DIP with addition of iron dextran (0.5mg/ml, iii). The displayed data is one representative experiment.