Fimbriae reprogram host gene expression – Divergent effects of P and type 1 fimbriae

Pathogens rely on a complex virulence gene repertoire to successfully attack their hosts. We were therefore surprised to find that a single fimbrial gene reconstitution can return the virulence-attenuated commensal strain Escherichia coli 83972 to virulence, defined by a disease phenotype in human hosts. E. coli 83972pap stably reprogrammed host gene expression, by activating an acute pyelonephritis-associated, IRF7-dependent gene network. The PapG protein was internalized by human kidney cells and served as a transcriptional agonist of IRF-7, IFN-β and MYC, suggesting direct involvement of the fimbrial adhesin in this process. IRF-7 was further identified as a potent upstream regulator (-log (p-value) = 61), consistent with the effects in inoculated patients. In contrast, E. coli 83972fim transiently attenuated overall gene expression in human hosts, enhancing the effects of E. coli 83972. The inhibition of RNA processing and ribosomal assembly indicated a homeostatic rather than a pathogenic end-point. In parallel, the expression of specific ion channels and neuropeptide gene networks was transiently enhanced, in a FimH-dependent manner. The studies were performed to establish protective asymptomatic bacteriuria in human hosts and the reconstituted E. coli 83972 variants were developed to improve bacterial fitness for the human urinary tract. Unexpectedly, P fimbriae were able to drive a disease response, suggesting that like oncogene addiction in cancer, pathogens may be addicted to single super-virulence factors

A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato

Stork I, Gartemann K-H, Burger A, Eichenlaub R. A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato. Molecular Plant Pathology. 2008;9(5):599-608.Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant

Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression

Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II)-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes). Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease–associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT) strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an environment that enhances their persistence

In vitro functional analyses of arrhythmogenic right ventricular cardiomyopathy-associated desmoglein-2-missense variations

Gaertner A, Klauke B, Stork I, Niehaus K, Niemann G. In vitro functional analyses of arrhythmogenic right ventricular cardiomyopathy-associated desmoglein-2-missense variations. PloS one. 2012;7(10): e47097.BACKGROUND: Although numerous sequence variants in desmoglein-2 (DSG2) have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), the functional impact of new sequence variations is difficult to estimate. METHODOLOGY/PRINCIPAL FINDINGS: To test the functional consequences of DSG2-variants, we established an expression system for the extracellular domain and the full-length DSG2 using the human cell line HT1080. We established new tools to investigate ARVC-associated DSG2 variations and compared wild-type proteins and proteins with one of the five selected variations (DSG2-p.R46Q, -p.D154E, -p.D187G, -p.K294E, -p.V392I) with respect to prodomain cleavage, adhesion properties and cellular localisation. CONCLUSIONS/SIGNIFICANCE: The ARVC-associated DSG2-p.R46Q variation was predicted to be probably damaging by bioinformatics tools and to concern a conserved proprotein convertase cleavage site. In this study an impaired prodomain cleavage and an influence on the DSG2-properties could be demonstrated for the R46Q-variant leading to the classification of the variant as a potential gain-of-function mutant. In contrast, the variants DSG2-p.K294E and -p.V392I, which have an arguable impact on ARVC pathogenesis and are predicted to be benign, did not show functional differences to the wild-type protein in our study. Notably, the variants DSG2-p.D154E and -p.D187G, which were predicted to be damaging by bioinformatics tools, had no detectable effects on the DSG2 protein properties in our study

Adhesion properties of rECDs.

<p><b>A+B</b> Flow cytometry-based assay for the binding of 0.8 µM rECD-wt or -variants to HT1080. <b>A</b> Representative histograms of FITC- fluorescence for binding of rECD-wt- and rECD-R46Q (as indicated). Bound rECD was detected with anti-HisFITC. As a negative control, HT1080 cells were incubated with only anti-HisFITC (grey filled area). <b>B</b> Column plots representing the ratio of rECD-binding related to the negative control (ratio_rECD-bound) as detected by flow cytometry. Ratios_rECD-bound are indicated as mean± SEM of 7 independent measurements for rECD-variants and 9 independent measurements for rECD-wt with rECDs from at least 3 different purifications. Statistical analysis was performed by one-way ANOVA with Dunnett’s posttest using rECD-wt as a control (GraphPad Prism 5.01). rECD-R46Q-binding to HT1080 is increased 1.8-fold as compared to rECD-wt. Other ARVC-associated variants have no influence on rECD-binding to HT1080. <b>C</b> Representative Western blot (with anti-DSG2-10G11) of rECDs crosslinked in a 5 mM CaCl₂ containing buffer with BS³ (+) or of controls (-) reveals that rECD wild-type and variants exist in solution as monomers (m), dimers (d), and oligomers (o).</p

Representative images for detecting the localisation of wild-type and variants of full-length-DSG2-EYFP in HT1080 for three independent transfection experiments:

<p><b>DSC2b-HT1080 cells were transfected with full-length(fl)-DSG2-pEYFP; live cells were analysed with a fluorescence miscroscope one day after transfection.</b> R46Q, D154E, D187G, K294E and V392I indicate the sequence variant in fl-DSG2-EYFP, wt fl-DSG2-wt-pEYFP, and C the LFA mock transfected control. Chimeric DSG2-proteins localised preferentially to the cell borders. ARVC-associated variations had no detectable influence on the localisation of fl-DSG2-EYFP in DSC2b-HT1080. Images were acquired through YFP and phase-contrast filters. Scale (red bar) = 10 µm.</p

Investigated DSG2-variants.

<p>All data were obtained from the ARVC database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-vanderZwaag1" target="_blank">[64]</a> and the corresponding references. TFC = task force criteria, EC = extracellular cadherin domain, DCM = dilatative cardiomyopathy.</p>a<p>The prevalence in controls for the DSG2-V392I was adapted to the results in our research group <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Klauke1" target="_blank">[35]</a>.</p>b<p>Grantham, R. (1974). “Amino acid difference formula to help explain protein evolution.” Science 185(4154):862–864.; Li, W. H., C. I. Wu, et al. (1984). “Nonrandomness of point mutation as reflected in nucleotide substitutions in pseudogenes and its evolutionary implications.” J Mol Evol 21(1): 58–71.</p>c<p>Kumar, P., S. Henikoff, et al. (2009). “Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.” Nat Protoc 4(7): 1073–1081.</p>d<p>Ramensky, V., P. Bork, et al. (2002). “Human non-synonymous SNPs: server and survey.” Nucleic Acids Res 30(17): 3894–3900.</p

Evaluation of CD data.

<p>Results of the deconvolution with DichroWeb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Whitmore1" target="_blank">[67]</a> using the CONTINLL-algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Provencher1" target="_blank">[68]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-vanStokkum1" target="_blank">[69]</a> and the CRYST175 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047097#pone.0047097-Evans1" target="_blank">[70]</a> reference data set. The results are presented as means±SEM [%] for three independent measurements. α-helical content (<b>A</b>) was 5.9±0.8 and 5.9±0.5 without Ca²⁺ (-Ca²⁺) and 3.2±1.4 and 3.2±0.2 with 5 mM CaCl₂ for rECD-wt-nat and rECD-wt-denat, respectively. β-strand content (<b>B</b>) was 38.2±0.3 and 36.6±1.3 without Ca²⁺ and 40.5±0.8 and 40.7±0.6 with 5 mM CaCl₂. Analysis of the secondary structure with two-way ANOVA showed that the α-helix content (<b>A</b>) was significantly decreased (p<0.05) while the β-strand content (<b>B</b>) was significantly increased (p<0.01) by the addition of 5 mM CaCl₂ (+Ca²⁺). However, as shown by two-way ANOVA, purification conditions had no significant effect on the rECD secondary structure.</p

Flow cytometry-based assay for rECD-binding.

<p><b>A</b> Representative histograms of FITC-fluorescence for rECD-wt binding with 5 mM CaCl₂ (1) or with 2 mM EGTA (2). HT1080 cells were incubated with (black line) or without (negative control, grey filled area) rECD-wt. Bound rECD-wt was detected with anti-HisFITC. <b>B</b> Column plots representing the ratios of rECD-wt-binding (ratio_rECD-bound; for calculation see Supporting Information) indicated as mean±SEM of 3 independent measurements as detected by flow cytometry. The ratio_rECD-bound was significantly (p<0.01) decreased from 1.21±0.03 for samples incubated in 5 mM CaCl₂ (1) to 1.05±0.03 for samples incubated with 2 mM EGTA (2) showing that rECD-wt has a Ca²⁺-dependent binding to HT1080 cells. Statistical analysis was performed with unpaired student’s t-test (GraphPad Prism 5.01).</p

In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations - A

<p><b>Schematic view of the rECD with analysed ARVC-associated variations.</b> The dotted line shows the predicted PC cleavage site. SS = signal sequence, Pro = prodomain, EC1-EC4 = DSG2 extracellular cadherin subdomains 1-4. <b>B</b> Recombinantly expressed proteins were identified as DSG2-ECD with anti-DSG2-10G11 by Western blot analysis. The calculated apparent molecular weights were 67.5±1.5, 72.5±3.5, 70.0±3.0, 70.0±3.0, 70.5±2.5, and 69.0±4.0 (mean±SEM; n = 2) for the proteins in the traces in 1, 2, 3, 4, 5 and 6, respectively. <b>C</b> Coomassie-R-250 staining revealed the purity of the proteins. 1 = rECD-wt, 2-6 = rECDs as labelled in <b>A</b>.</p