A secretome view of colonisation factors in Shiga toxin-encoding<i>Escherichia coli</i>(STEC): from enterohaemorrhagic<i>E. coli</i>(EHEC) to related enteropathotypes

Monteiro, Ricardo; Ageorges, Valentin; Rojas-López, Maricarmen; Schmidt, Herbert; Weiß, Agnes; Bertin, Yolande; Forano, Evelyne; Jubelin, Grégory; Henderson, Ian R.; Livrelli, Valérie; Gobert, Alain P.; Rosini, Roberto; Soriani, Marco; Desvaux, Mickaël

doi:10.1093/femsle/fnw179

Cited by 28 publications

(28 citation statements)

References 164 publications

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“…Other OMPs act as secretion pores, transporting a variety of macromolecules across the outer membrane, such as, lipopolysaccharide (Dong et al, 2014 ), biofilm matrix components (Hufnagel et al, 2015 ), other proteins (Chagnot et al, 2013 ), or, in the case of autotransporters, parts of the same polypeptide chain (Leo et al, 2012 ). OMPs are further involved in such functions as self-recognition (Aoki et al, 2005 , 2008 ), protein hydrolysis (Haiko et al, 2009 ), and virulence (Monteiro et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

A New Strain Collection for Improved Expression of Outer Membrane Proteins

Meuskens

Michalik

Chauhan

et al. 2017

Front. Cell. Infect. Microbiol.

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Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21(DE3) designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB), both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

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Section: Introductionmentioning

confidence: 99%

A New Strain Collection for Improved Expression of Outer Membrane Proteins

Meuskens

Michalik

Chauhan

et al. 2017

Front. Cell. Infect. Microbiol.

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“…The first indication that ppd/hof genes may encode a functional system has come from studies of enterohaemorrhagic E. coli (EHEC), which produce pili composed of the major subunit PpdD in minimal casein medium (Xicohtencatl-Cortes et al, 2007). EHEC is a major intestinal pathogen causing haemorrhagic colitis and haemolytic uremic syndrome (HUS), associated with significant mortality and morbidity (Monteiro et al, 2016). Sera of patients recovering from HUS contain PpdD-reactive antibodies (Xicohtencatl-Cortes et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Functional reconstitution of the type IVa pilus assembly system from enterohaemorrhagic Escherichia coli

Rico

Zheng

Petiot

et al. 2019

Molecular Microbiology

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Summary Type 4a pili (T4aP) are long, thin and dynamic fibres displayed on the surface of diverse bacteria promoting adherence, motility and transport functions. Genomes of many Enterobacteriaceae contain conserved gene clusters encoding putative T4aP assembly systems. However, their expression has been observed only in few strains including Enterohaemorrhagic Escherichia coli (EHEC) and their inducers remain unknown. Here we used EHEC genomic DNA as a template to amplify and assemble an artificial operon composed of four gene clusters encoding 13 pilus assembly proteins. Controlled expressions of this operon in nonpathogenic E. coli strains led to efficient assembly of T4aP composed of the major pilin PpdD, as shown by shearing assays and immunofluorescence microscopy. When compared with PpdD pili assembled in a heterologous Klebsiella T2SS type 2 secretion system (T2SS) by using cryo‐electron microscopy (cryoEM), these pili showed indistinguishable helical parameters, emphasizing that major pilins are the principal determinants of the fibre structure. Bacterial two‐hybrid analysis identified several interactions of PpdD with T4aP assembly proteins, and with components of the T2SS that allow for heterologous fibre assembly. These studies lay ground for further characterization of the T4aP structure, function and biogenesis in enterobacteria.

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“…They may cause watery diarrhea and serious complications such as bloody diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome in humans [25]. These complications are caused by several toxins, such as Shiga toxin, cytolethal distending toxin V, enterohemolysin, and subtilase cytotoxin, as well as accessory virulence factors [24,34]. Most outbreaks occurred by strains of the serotypes O157:H7 and O157:H − , as well as the non-O157 serotypes O26, O91, O103, O111, O118, O145 and O166 [6,19,20].…”

Section: Introductionmentioning

confidence: 99%

Application of MALDI-TOF mass spectrometry and specific PCR for tracking of E. coli O157:H− strain 431/97 in Batavia lettuce

Weiß

Heinold

Brunisholz

et al. 2019

Chem. Biol. Technol. Agric.

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Background: In this study, lettuce roots and leaves were contaminated with enterohemorrhagic Escherichia coli O157:H − strain 431/97 under greenhouse conditions. Furthermore, the internalization of strain 431/97 in lettuce roots and leaves was examined. To track the inoculated bacteria during the experiments and to differentiate them from the autochthonous microbiota, a combined protocol including matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and PCR was established. Results: At different time points after inoculation of Batavia lettuce with 5.5 × 10 6 cfu/mL (high inoculation level) or 3.0 × 10 1 cfu/mL (low inoculation level) E. coli O157:H − strain 431/97 as well as sterile 0.9% (w/v) sodium chloride solution (negative control), samples from the root and the leaf were taken and surface disinfected with gentamicin. After homogenization, microorganisms were isolated from the samples and analyzed by MALDI-TOF MS. Analysis of the root samples resulted in bacterial counts of 1.0 × 10 2-1.0 × 10 6 cfu/0.25 g depending on the inoculated viable counts and the incubation period. In the leaf samples, strain 431/97 was not detected. The investigation of the viable cell counts of E. coli O157:H − 431/97 following irrigation of the leaves resulted in bacterial counts of 10 2 cfu/0.25 g for the disinfected leaf samples. Conclusions: Thus, the established protocol is suitable for detecting the investigated strain under greenhouse conditions in plant infection experiments. This strain may indeed survive in the soil, but did not enter the plant via the root in detectable numbers. Contrarily, viable counts exceeding the generally accepted infective dose of less than 100 cells for enterohemorrhagic E. coli were determined internalized after irrigation of the leaves. As this may pose a risk for the consumer, the present study provides a valuable set of tools for further research.

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A secretome view of colonisation factors in Shiga toxin-encodingEscherichia coli(STEC): from enterohaemorrhagicE. coli(EHEC) to related enteropathotypes

Cited by 28 publications

References 164 publications

A New Strain Collection for Improved Expression of Outer Membrane Proteins

A New Strain Collection for Improved Expression of Outer Membrane Proteins

Functional reconstitution of the type IVa pilus assembly system from enterohaemorrhagic Escherichia coli

Application of MALDI-TOF mass spectrometry and specific PCR for tracking of E. coli O157:H− strain 431/97 in Batavia lettuce

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