Identification of the <i>Y. pestis</i> quorum-sensing molecules.

<p>(A) HPLC fractionation profiles of ¹⁴C-labeled AHL produced by R88 <i>Y. pestis</i> (solid circles) compared to R115 QS^-<i>Y. pestis</i> (empty circles). The peaks absent from organic extracts of R115 <i>Y. pestis</i> supernatants correspond to C8-, C6-, and oxo-C6-AHL. (B) AI-2 production during the growth of R88 <i>Y. pestis</i> (solid circles) and R115 (empty circles) was monitored by adding the cell-free supernatants at the indicated time points to a <i>V. harveyii</i> reporter strain that is bioluminescent (RLU) in response to AI-2. Data are representative of at least three independent studies. (C and D) The production of AI-2 (C) and AHL (D) signals as a function of growth.</p

Selected genes significantly up-regulated by QS.

*<p>FC = fold change.</p

AHLs upregulate the maltose operon and enhance growth on maltose.

<p>(A) Growth of wild-type (WT) (black), and AHL mutant (dashed) <i>Y. pestis</i> in minimal maltose medium (light grey) was monitored in Bioscreen C microplate reader incubating at 28°C with agitation. (B) Complementation of AHL mutant bacteria with p<i>ypeIR</i> (black, dashed) or p<i>yspIR</i> (grey, dashed) restores growth on maltose, whereas control plasmid (black, solid) does not.</p

AHL quorum sensing upregulates glyoxylate bypass and enhances growth on acetate.

<p>(A) Growth of R88 (black), and an R115 AHL null mutant strain (dashed) of <i>Y. pestis</i> in minimal acetate medium (light grey is medium only control) was monitored in a Bioscreen C microplate reader incubating at 28°C with agitation. (B) Complementation of AHL mutant bacteria with p<i>ypeIR</i> (black, dashed) or p<i>yspIR</i> (grey, dashed) restores growth on acetate, whereas control plasmid (black, solid) does not.</p

Quorum-sensing regulates fermentation of sugars.

<p>(A) Colony phenotype after 96 hrs growth on LB plates containing Congo red and 0.2% maltose and incubated at the temperature indicated. <i>Y. pestis</i> ferments maltose to acid, converting the Congo red to black, whereas a <i>yspIR ypeIR</i> mutant does not. This fermentation does not occur at temperatures above 30°C, or on other sugars tested (data not shown.) (B) Duplicate growth stabs of <i>Y. pestis</i> grown on solid medium under anaerobic conditions for 96 h at 28°C. An indicator dye, Congo Red, turns dark upon production of fermentative end products. Deletion of <i>yspIR</i> and <i>ypeIR</i> in R114 and R115 results in a lag in fermentation of maltose under anaerobic conditions. 1 = <i>Y. pseudotuberculosis</i>; 2 = R88; 3 = R114; 4 = R115.</p

Transcriptome Analysis of Acetyl-Homoserine Lactone-Based Quorum Sensing Regulation in <i>Yersinia pestis</i>

<div><p>The etiologic agent of bubonic plague, <i>Yersinia pestis,</i> senses self-produced, secreted chemical signals in a process named quorum sensing. Though the closely related enteric pathogen <i>Y. pseudotuberculosis</i> uses quorum sensing system to regulate motility, the role of quorum sensing in <i>Y. pestis</i> has been unclear. In this study we performed transcriptional profiling experiments to identify <i>Y. pestis</i> quorum sensing regulated functions. Our analysis revealed that acyl-homoserine lactone-based quorum sensing controls the expression of several metabolic functions. Maltose fermentation and the glyoxylate bypass are induced by acyl-homoserine lactone signaling. This effect was observed at 30°C, indicating a potential role for quorum sensing regulation of metabolism at temperatures below the normal mammalian temperature. It is proposed that utilization of alternative carbon sources may enhance growth and/or survival during prolonged periods in natural habitats with limited nutrient sources, contributing to maintenance of plague in nature.</p></div

AHL-based QS regulates secondary metabolism.

<p>RNA was isolated from the indicated strains of <i>Y. pestis</i>, and levels of mRNA were measured by qRT-PCR as outlined in Material and Methods. Data represents the mean of triplicate measurements of the transcript differences between each mutant and that of strain R88 normalized to the 16 S rRNA of each sample, and are representative of at least three independent experiments. AHL^– = strain R114; QS^– = strain R115; AI-2^– = strain ISM1980.</p

P159 from <i>Mycoplasma hyopneumoniae</i> Binds Porcine Cilia and Heparin and Is Cleaved in a Manner Akin to Ectodomain Shedding

<i>Mycoplasma hyopneumoniae</i> colonizes the ciliated epithelial lining of the upper respiratory tract of swine and results in chronic infection. Previously, we have observed that members of P97 and P102 paralog families of cilium adhesins undergo endoproteolytic processing on the surface of <i>M. hyopneumoniae</i>. We show that P159 (MHJ_0494), an epithelial cell adhesin unrelated to P97 and P102 paralog families, is a cilium adhesin that undergoes dominant cleavage events at S/T-X-F↓X-D/E-like motifs located at positions ²³³F↓Q²³⁴ and ⁹⁸¹F↓Q⁹⁸², generating P27, P110, and P52. An unrelated cleavage site ⁷³⁸L-K-V↓G-A-A⁷⁴³ in P110 shows sequence identity with a cleavage site (L-N-V↓A-V-S) identified in the P97 paralog, Mhp385, and generates 76 (P76) and 35 kDa (P35) fragments. LC–MS/MS analysis of biotinylated surface proteins identified six peptides with a biotin moiety on their N-terminus indicating novel, low abundance neo-N-termini. LC–MS/MS of proteins separated by 2D-PAGE, 2D immunoblotting using monospecific antiserum raised against recombinant fragments spanning P159 (F1_P159-F4_P159), and proteins that bound to heparin-agarose were all used to map P159 cleavage fragments. P159 is the first cilium adhesin not belonging to the P97/P102 paralog families and is extensively processed in a manner akin to ectodomain shedding in eukaryotes

<i>Mycoplasma hyopneumoniae</i> Surface Proteins Mhp385 and Mhp384 Bind Host Cilia and Glycosaminoglycans and Are Endoproteolytically Processed by Proteases That Recognize Different Cleavage Motifs

P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of <i>Mycoplasma hyopneumoniae</i> that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F↓X-D/E-like site, creating P60₃₈₄ and P50₃₈₄. The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115₃₈₅) and 88 kDa (P88₃₈₅) and 27 kDa (P27₃₈₅) cleavage fragments identified by LC–MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide ⁷⁵²IQFELEPISLNV⁷⁶³ denotes the C-terminus of P88₃₈₅ and defines the novel cleavage site ⁷⁶¹L-N-V↓A-V-S⁷⁶⁶ in Mhp385. P115₃₈₅, P88₃₈₅, P27₃₈₅, P60₃₈₄, and P50₃₈₄ were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60₃₈₄, P50₃₈₄, and P88₃₈₅. No primary function was attributed to P27₃₈₅; however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of <i>M. hyopneumoniae</i>