Glycomimicry: Display of the GM3 sugar epitope on Escherichia coli and Salmonella enterica sv Typhimurium

Oligosaccharides present on the surface of pathogenic bacteria play an important role in their interaction with their host. Bacteria with altered cell surface structures can be used to study these interactions, and glycoengineering represents a tool to display a glycoepitope on a different bacterium. Here, we present non-pathogenic Escherichia coli and Salmonella enterica serovar Typhimurium expressing the sialyllactose oligosaccharide epitope of the ganglioside GM3. By expression of the galactosyltransferase LgtE and the sialic acid transferase Lst as well as the CMP-sialic acid synthetase SiaB from Neisseria gonorrhoeae and Neisseria meningitidis in engineered strains devoid of the sialic acid catabolism, the GM3 sugar epitope was displayed on these bacteria as demonstrated by live cell immunostaining and a detailed analysis of their lipooligosaccharides. These strains offer the possibility to investigate the role of sialic acid in the recognition of bacteria by the immune system in a non-pathogenic backgroun

Glycomimicry: display of fucosylation on the lipo-oligosaccharide of recombinant Escherichia coli K12

We recently described the design of Escherichia coli K12 and Salmonella enterica sv Typhimurium to display the gangliomannoside 3 (GM3) antigen on the cell surface [1]. We report here the fucosylation of modified lipooligosaccharide in a recombinant E.coli strain with a truncated lipid A core due to deletion of the core glycosyltransferases genes waaO and waaB. This truncated structure was used as a scaffold to assemble the Lewis Y motif by consequent action of the heterologously expressed β-1,4 galactosyltransferase LgtE (Neisseria gonorrheae), the β-1,3 N-acetylglucosaminyltransferase LgtA and the β-1,3 galactosyltransferase LgtB from Neisseria meningitidis, as well as the α-1,2 and α-1,3 fucosyltransferases FutC and FutA from Helicobacter pylori. We show the display of the Lewis Y structure by immunological and chemical analysi

SiRNA inhibits replication of Langat virus, a member of the tick-borne encephalitis virus complex in organotypic rat brain slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000-3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3' untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro

A tick-borne encephalitis model in infant rats infected with langat virus

Tick-borne encephalitis virus (TBEV) is the causative agent of human TBE, a severe infection that can cause long-lasting neurologic sequelae. Langat virus (LGTV), which is closely related to TBEV, has a low virulence for human hosts and has been used as a live vaccine against TBEV. Tick-borne encephalitis by natural infection of LGTV in humans has not been described, but one of 18,500 LGTV vaccinees developed encephalitis. The pathogenetic mechanisms of TBEV are poorly understood and, currently, no effective therapy is available. We developed an infant rat model of TBE using LGTV as infective agent. Infant Wistar rats were inoculated intracisternally with 10 focus-forming units of LGTV and assessed for clinical disease and neuropathologic findings at Days 2, 4, 7, and 9 after infection. Infection with LGTV led to gait disturbance, hypokinesia, and reduced weight gain or weight loss. Cerebrospinal fluid concentrations of RANTES, interferon-γ, interferon-β, interleukin-6, and monocyte chemotactic protein-1 were increased in infected animals. The brains of animals with LGTV encephalitis exhibited characteristic perivascular inflammatory cuffs and glial nodules; immunohistochemistry documented the presence of LGTV in the thalamus, hippocampus, midbrain, frontal pole, and cerebellum. Thus, LGTV meningoencephalitis in infant rats mimics important clinical and histopathologic features of human TBE. This new model provides a tool to investigate disease mechanisms and to evaluate new therapeutic strategies against encephalitogenic flaviviruses

Glycomimicry: display of fucosylation on the lipo-oligosaccharide of recombinant Escherichia coli K12

ISSN:0282-0080ISSN:1573-498

Glycomimicry: Display of the GM3 sugar epitope on Escherichia coli and Salmonella enterica sv Typhimurium

ISSN:0959-665

Rapid Increase of CTX-M-Producing Shigella sonnei Isolates in Switzerland: Spread of Common Plasmids and International Clones

The Swiss Centre for Antibiotic Resistance (ANRESIS) has recently noted an increase of extended-spectrum cephalosporin-resistant (ESC-R) S. sonnei isolates nationwide (3.8% in 2016 vs. 37.5% in 2019). To understand this phenomenon, we analyzed 25 representative isolates (of which 14 ESC-R) collected in Switzerland during 2016-2019. Whole-genome sequencing was achieved using both Illumina and Nanopore platforms. Both ESC-R and susceptible isolates belonged to ST152. The ESC-R isolates carried blaCTX-M-3 in IncI1-pST57 (n=5), blaCTX-M-15 in IncFII (F2:A-:B-) (n=5), blaCTX-M-15 in IncI1-pST16, and blaCTX-M-27, blaCTX-M-55, or blaCTX-M-134 in other IncFII plasmids (n=1 each). Plasmids having the same bla and Inc group exhibited high genetic identity to each other, but also to plasmids previously reported in other Enterobacterales. Core-genome analysis showed that there were 4 main clusters, each of which included strains that differed by <58 SNVs, both blaCTX-M-positive and blaCTX-M-negative isolates. Moreover, most isolates belonging to the same cluster shared an identical cgST. For instance, cluster-1 included 4 isolates of cgST113036, of which only 3 harbored the IncI1-pST57 blaCTX-M-3-positive plasmid. The 25 S. sonnei isolates were also subjected to phylogenetic comparison with deposited international strains. As a result, matching isolates (same cgST and differing by <8 SNVs) have been reported in the UK, USA, France, and the Netherlands. Overall, our results suggest that some common S. sonnei clusters can spread between continents and can be imported into other nations after international trips. Such clusters include, in part, isolates that do not possess blaESBL-harboring plasmids, indicating their tendency to acquire them from other Enterobacterales

Antiviral effect of siRNA D3 in relation to the infectious dose of Langat.

<p>HeLa cells were infected with Langat virus at different infectious dose (MOI of 10, 1, 0.1 or 0.01) and one hour after infection, cells were treated with 200 nM siRNA sequence D3 or with nonsense siRNA. Virus titer was assessed by quantitative real-time RT-PCR 6 days after infection in the cell culture supernatant. The reduction of viral RNA in siRNA D3 treated cells is statistically significant compared to nonsense siRNA treated cells for all four MOI (p<0.05). The data are presented as mean ± SD of three independent experiments and significance calculated using an unpaired T-test.</p

Inhibitory effects of siRNA D3 on viral replication on HeLa cells over time.

<p>The replication profile for Langat virus was determined for the time period of 6 days. Indicated are the numbers of RNA copies (A) and the number of infectious particles (B) for cell cultures treated with nonsense siRNA or specific siRNA D3.</p

LGTV-specific siRNA sequences inhibit production of viral RNA in HeLa cells.

<p>Nineteen siRNA sequences (Q1–Q6 and D1–D13) targeting genes within the whole open reading frame of LGTV genome were analyzed for their antiviral potential on HeLa cells (A). After transfection with siRNA, cells were infected with Langat virus (MOI = 10) and six days later virus replication was assessed by quantitative real-time RT-PCR (B). Results are shown as a percentage of virus inhibition compared to the control cells transfected with the non-coding siRNA. Data are presented as mean ± SD of three independent experiments. Fourteen (Q2, Q3, Q4, Q5, Q6, D3, D4, D5, D6, D8, D9 D11 D12 and D13) out of 19 were significantly reduced (p<0.05) compared to a theoretical mean of 100% expressed by cells treated with nonsense siRNA; measured by One sample t test.</p