Quantitative contribution of efflux to multi-drug resistance of clinical Escherichia coli and Pseudomonas aeruginosa strains

Cunrath, Olivier; Meinel, Dominik M.; Maturana, Pauline; Fanous, Joseph; Buyck, Julien; Auguste, Pamela Saint; Seth-Smith, Helena M. B.; Körner, Jonas; Dehio, Christoph; Trebosc, Vincent; Kemmer, Christian; Neher, Richard A.; Egli, Adrian; Bumann, Dirk

doi:10.1016/j.ebiom.2019.02.061

Cited by 45 publications

(49 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To limit toxic exposure to volatile dimethyl telluride, we used kanamycin for suicide vector generation and used tellurite only for the positive selection of ex-conjugants. We confirmed the utility of pFOKT by deleting tolC with high efficiency in a multi-drug resistant clinical Escherichia coli isolate [29] (Fig. 2c).…”

Section: Resultssupporting

confidence: 72%

“…tpm codes for a thiopurine-S-methyltranferase conferring resistance to tellurite [33]. This plasmid can be used for multi-drug resistant (MDR) bacteria for which the choice of positive selection markers is limited [29]. To limit toxic exposure to volatile dimethyl telluride, we used kanamycin for suicide vector generation and used tellurite only for the positive selection of ex-conjugants.…”

Section: Resultsmentioning

confidence: 99%

“…Salmonella enterica serovar Typhimurium SL1344 was cultured in LB in the presence of 90 µg/ml streptomycin (Sigma-Aldrich S9137-100G ). E. coli EC01 [29] and P. aeruginosa PA14 were cultured in LB. For preparing chemical competent cells, fresh LB medium was inoculated at OD 600nm 0.01 with an overnight culture of JKe201 and grown until OD 600nm 0.4-0.6.…”

Section: Methodsmentioning

confidence: 99%

“…We employed an E. coli donor strain that simplifies donor removal after conjugation and avoids common problems with contaminating phages [28]. We used different positive selection markers that enable selection in many bacterial species, including MDR pathogens [29]. Combination of these elements yielded a reliable and fast method for genetic engineering of multiple bacterial species that, in concert with a simplified screening procedure, minimized hands-on time and significantly accelerated genome editing in our lab.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Efficient Dual-Negative Selection for Bacterial Genome Editing

Cianfanelli

Cunrath

Bumann

2020

Preprint

Self Cite

View full text Add to dashboard Cite

11We describe a versatile method for chromosomal gene editing based on classical consecutive 12 single-crossovers. The method exploits rapid plasmid construction using Gibson assembly, a 13 convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector 14 resolution. We used this method to generate in frame deletions, insertions and point mutations in 15Salmonella enterica with limited hands-on time. Similar strategies allowed efficient gene editing 16 also in Pseudomonas aeruginosa and multi-drug-resistant (MDR) Escherichia coli clinical isolates. 17 18 [19][20][21], but this strategy is more cumbersome for bacteria with limited recombination activities 34 [22][23][24]. 35Here, we combined several improvements for establishing a time-efficient versatile 36 method for consecutive single cross-overs in multiple bacterial species. We used rapid Gibson 37 assembly of PCR products [25] to generate suicide vectors with dual negative selection with I-38SceI and SacB [26, 27] (Fig 1a), which increased counter-selection efficiency to 100% for nearly 39 all tested deletions, insertions and point mutations. We employed an E. coli donor strain that 40 simplifies donor removal after conjugation and avoids common problems with contaminating 100 mM (Sigma Aldrich C1016-500G) containing 15% of glycerol (AppliChem, A1123,1000). 57 Bacteria were resuspended in 5 ml ice-cold CaCl2 100 mM containing 15% of glycerol and 200 µl 58 aliquots were frozen and stored at -80°C. Super-Optimal broth with Catabolite repression (SOC) 59 (tryptone 20 g/L, yeast extract 5 g/L, NaCl 0.5 g/L, KCl 0.186 g/L, MgSO4 4.8 g/L and glucose 3.6 60 g/L) medium was used after heat shock. Kanamycin (Roth T832.4) at a final concentration of 50 61 µg/ml and gentamicin (Gibco 15750-037) at a final concentration of 15 µg/ml were used to select 62 E. coli transformants. For positive selection, kanamycin (Roth T832.4) at a final concentration of 63 50 µg/ml, gentamicin (Gibco 15750-037) at a final concentration of 30 µg/ml, or potassium tellurite 64 (Sigma P0677) at a final concentration of 10 µg/ml, were used to select for S. typhimurium, P.

show abstract

Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Efficient Dual-Negative Selection for Bacterial Genome Editing

Cianfanelli

Cunrath

Bumann

2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Interestingly, the unexpected finding that a FIC domain exerts a biological function unrelated to a catalytic activity is unique and only remotely reminiscent of the Fic protein AvrB, which lacks all residues required for enzymatic activity [40]. Thus, it opens new perspectives for the function of the many Beps, and other Fic proteins, carrying a non-canonical FIC motif and possibly also lacking enzymatic activity [16, 20, 41].…”

Section: Discussionmentioning

confidence: 99%

Bartonellaeffector protein C mediates actin stress fiber formation via recruitment of GEF-H1 to the plasma membrane

Marlaire

Dehio

2020

Preprint

Self Cite

View full text Add to dashboard Cite

16Bartonellae are Gram-negative facultative-intracellular pathogens that use a type-IV- 17 secretion system (T4SS) to translocate a cocktail of Bartonella effector proteins (Beps) 18 into host cells to modulate diverse cellular functions. BepC was initially reported to act 19 in concert with BepF in triggering major actin cytoskeletal rearrangements that result 20 in the internalization of a large bacterial aggregate by the so-called 'invasome'. Later, 21 infection studies with bepC deletion mutants and ectopic expression of BepC have 22 implicated this effector in triggering an actin-dependent cell contractility phenotype 23 characterized by fragmentation of migrating cells due to deficient rear detachment at 24 the trailing edge, and BepE was shown to counterbalance this remarkable phenotype. 25However, the molecular mechanism of how BepC triggers cytoskeletal changes and 26 the host factors involved remained elusive. Using infection assays, we show here that 27 T4SS-mediated transfer of BepC is sufficient to trigger stress fiber formation in non-28 migrating epithelial cells and additionally cell fragmentation in migrating endothelial 29 cells. Interactomic analysis revealed binding of BepC to a complex of the Rho guanine 30 nucleotide exchange factor GEF-H1 and the serine/threonine-protein kinase MRCKα. 31Knock-out cell lines revealed that only GEF-H1 is required for mediating BepC-32 triggered stress fiber formation and inhibitor studies implicated activation of the 33 RhoA/ROCK pathway downstream of GEF-H1. Ectopic co-expression of tagged 34 versions of GEF-H1 and BepC truncations revealed that the C-terminal 'Bep 35 intracellular delivery' (BID) domain facilitated anchorage of BepC to the plasma 36 membrane, whereas the N-terminal 'filamentation induced by cAMP' (FIC) domain 37 facilitated binding of GEF-H1. While FIC domains typically mediate post-translational 38 modifications, most prominently AMPylation, a mutant with quadruple amino acid 39 exchanges in the putative active site indicated that the BepC FIC domain acts in a non-40 catalytic manner to activate GEF-H1. Our data support a model in which BepC 41 activates the RhoA/ROCK pathway by re-localization of GEF-H1 from microtubules to 42 the plasma membrane. 43 4 Author Summary 44 A wide variety of bacterial pathogens evolved numerous virulence factors to subvert 45 cellular processes in support of a successful infection process. Likewise, bacteria of 46 the genus Bartonella translocate a cocktail of effector proteins (Beps) via a type-IV-47 secretion system into infected cells in order to interfere with host signaling processes 48 involved in cytoskeletal dynamics, apoptosis control, and innate immune responses. In 49 this study, we demonstrate that BepC triggers actin stress fiber formation and a linked 50 cell fragmentation phenotype resulting from distortion of rear-end retraction during cell 51 migration. The ability of BepC to induce actin stress fiber formation is directly 52 associated with its ability to bind GEF-H1, an activato...

show abstract