Characterization of a novel plasmid encoding F4-like fimbriae present in a Shiga-toxin producing enterotoxigenic Escherichia coli isolated during the investigation on a case of hemolytic-uremic syndrome.

In February 2017 a case of Hemolytic-Uremic Syndrome (HUS) was reported to the National Registry of HUS in an adult living in Northern Italy. Stool specimens from the patient and his family contacts were collected and the analyses led to the isolation of a Locus of Enterocyte Effacement (LEE)-negative Shiga toxin 2 (Stx2)-producing Escherichia coli. The epidemiological investigations performed brought to collect fecal samples from the animals reared in a farm held by the case's family and a mixture of bovine and swine feces proved positive for Shiga toxin-producing E. coli (STEC) and yielded the isolation of a LEE-negative stx2-positive E. coli strain. Further characterization by whole genome sequencing led to identify the isolates as two identical O2:H27 hybrid Enterotoxigenic Shiga toxin-producing E. coli (ETEC-STEC). Sequencing of a high molecular weight plasmid present in the human isolate disclosed a peculiar plasmid harboring virulence genes characteristic for both pathotypes, including the enterohemolysin-coding gene and sta1, encoding the heat stable enterotoxin. Moreover, a complete fae locus encoding the ETEC F4 fimbriae could be identified, including a novel variant of faeG gene responsible for the production of the main structural subunit of the fimbriae. This novel faeG showed great diversity in the nucleotidic sequence when compared with the reference genes encoding the swine F4 allelic variants, whereas at the amino acid sequence level the predicted protein sequence showed some similarity with FaeG from E. coli strains of bovine origin. Further investigation on the plasmid region harboring the newly identified faeG allelic variant allowed to identify similar plasmids in NCBI sequence database, as part of the genome of other previously uncharacterized ETEC-STEC strains of bovine origin, suggesting that the novel F4-like fimbriae may play a role in bovine host specificity.

Laser microdissection of grapevine leaf phloem infected by stolbur reveals site-specific gene responses associated to sucrose transport and metabolism.

Bois Noir is an emergent disease of grapevine that has been associated to a phytoplasma belonging to the XII-A stolbur group. In plants, phytoplasmas have been found mainly in phloem sieve elements, from where they spread moving through the pores of plates, accumulating especially in source leaves. To examine the expression of grapevine genes involved in sucrose transport and metabolism, phloem tissue, including sieve element/companion cell complexes and some parenchyma cells, was isolated from healthy and infected leaves by means of laser microdissection pressure catapulting (LMPC). Site-specific expression analysis dramatically increased sensitivity, allowing us to identify specific process components almost completely masked in whole-leaf analysis. Our findings showed decreased phloem loading through inhibition of sucrose transport and increased sucrose cleavage activity, which are metabolic changes strongly suggesting the establishment of a phytoplasma-induced switch from carbohydrate source to sink. The analysis focused at the infection site also showed a differential regulation and specificity of two pathogenesis-related thaumatin-like genes (TL4 and TL5) of the PR-5 family.

Neutral invertases in grapevine and comparative analysis with Arabidopsis, poplar and rice.

Neutral invertases (NIs, EC 3.2.1.26) cleave sucrose to glucose and fructose. They are encoded by a small gene family of 9 members in the Arabidopsis genome, 8 in rice, 16 in poplar and 9 in Vitis vinifera (L.). The grapevine NIs were identified in the 8.4X genome assembly of the quasi-homozygous line PN40024. In addition, alleles of three NIs were sequenced in the heterozygous cultivar 'Cabernet Sauvignon'. Analyses of sequence variation between alleles, homoeologous and paralogous copies in grapevine and their orthologues in Arabidopsis, poplar and rice are provided. In grapevine, NIs were classified into four alpha NIs and five beta NIs and subsequently grouped into hierarchical clades using a combination of evidence including amino acid identity, exon/intron structure, rate of synonymous substitutions (K (s)) and chromosomal distribution. Estimation of K (s) proved the ancient origin of all NIs and the lack of expansion by gene duplication past the event of polyploidisation. We then focused on transcription analysis of five NIs for which evidence of expression was available from expressed sequence tag databases. Among these, four NIs consisted of pairs of homoeologous copies, each pair lying on a pair of chromosomes duplicated by polyploidy. Unequal expression of homoeologous genes was observed by quantitative RT-PCR in leaf, flower, seed and root tissues. Since NIs might play significant roles in fruit and wine quality, NIs expression was monitored in flesh and skin of 'Merlot' berries and shown in parallel with the suite of changes that accompany fruit ripening, including glucose and fructose accumulation.