Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes

Andersen, Jens Bo; Roldgaard, Bent; Lindner, Ariel B.; Christensen, Bjarke Bak; Licht, Tine Rask

doi:10.1186/1471-2180-6-86

Cited by 40 publications

(18 citation statements)

References 30 publications

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“…For this purpose, we created Kpn I/ Not I DNA fragments where the mCherry or cfp [42] ORFs were flanked by the promoter and terminator regions of the incDEFG operon. The E. coli-C. trachomatis shuttle plasmids, p2TK2-SW2 IncDProm-mCherry-IncDTerm and p2TK2-SW2 IncDProm-CFP-IncDTerm were respectively obtained by ligation of the IncDProm-mCherry-IncDTerm or IncDProm-CFP-IncDTerm DNA fragments into the Kpn I and Not I restriction sites of p2TK2-SW2 (Figure 3B–3C (map), Figure S5–S6 (sequence) and Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

A C. trachomatis Cloning Vector and the Generation of C. trachomatis Strains Expressing Fluorescent Proteins under the Control of a C. trachomatis Promoter

2013

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Here we describe a versatile cloning vector for conducting genetic experiments in C. trachomatis. We successfully expressed various fluorescent proteins (i.e. GFP, mCherry and CFP) from C. trachomatis regulatory elements (i.e. the promoter and terminator of the incDEFG operon) and showed that the transformed strains produced wild type amounts of infectious particles and recapitulated major features of the C. trachomatis developmental cycle. C. trachomatis strains expressing fluorescent proteins are valuable tools for studying the C. trachomatis developmental cycle. For instance, we show the feasibility of investigating the dynamics of inclusion fusion and interaction with host proteins and organelles by time-lapse video microscopy.

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Section: Resultsmentioning

confidence: 99%

A C. trachomatis Cloning Vector and the Generation of C. trachomatis Strains Expressing Fluorescent Proteins under the Control of a C. trachomatis Promoter

2013

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“…pJEBAN3 plasmid encoding yellow fluorescent protein (YFP) and pJEBAN6 plasmid encoding red fluorescence protein (DsRedEx), was transferred into L. monocytogenes by electroporation to give yellow and red fluorescent strain, respectively30. L. monocytogenes harboring pJEBAN3 or pJEBAN6 plasmid was cultured in tryptic soy broth supplemented with 5 μg/ml erythromycin (Wako Pure Chemical Industries, Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

Passive immunization with anti-ActA and anti-listeriolysin O antibodies protects against Listeria monocytogenes infection in mice

Asano

Sashinami

Osanai

et al. 2016

Sci Rep

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Listeria monocytogenes is an intracellular pathogen that causes listeriosis. Due to its intracellular niche, L. monocytogenes has evolved to limit immune recognition and response to infection. Antibodies that are slightly induced by listerial infection are completely unable to protect re-infection of L. monocytogenes. Thus, a role of antibody on the protective effect against L. monocytogenes infection has been neglected for a long time. In the present study, we reported that passive immunization with an excessive amount of antibodies against ActA and listeriolysin O (LLO) attenuates severity of L. monocytogenes infection. Combination of these antibodies improved survival of L. monocytogenes infected mice. Bacterial load in spleen and liver of listerial infected mice and infected RAW264.7 cells were significantly reduced by administration of anti-ActA and anti-LLO antibodies. In addition, anti-LLO antibody neutralized LLO activity and inhibited the bacterial escape from the lysosomal compartments. Moreover, anti-ActA antibody neutralized ActA activity and suppressed actin tail formation and cell-to-cell spread. Thus, our studies reveal that passive immunization with the excessive amount of anti-ActA and -LLO antibodies has potential to provide the protective effect against listerial infection.

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“…L. monocytogenes 10403S wild type (WT) and its isogenic ⌬flaA (HEL-304) mutant (24) were used to evaluate the role of flagella in biofilm architecture. For real-time confocal observation, autofluorescent variants (25) harboring the pNF8 plasmid encoding GFPmut1 (26) or pJEBAN6 encoding DsRedExpress (27) were used. All strains were stored at Ϫ80°C in tryptone soya broth (TSB) (Oxoid, France) containing 20% (vol/vol) glycerol.…”

Section: Methodsmentioning

confidence: 99%

Exploring the Diversity of Listeria monocytogenes Biofilm Architecture by High-Throughput Confocal Laser Scanning Microscopy and the Predominance of the Honeycomb-Like Morphotype

Guilbaud

Piveteau

Desvaux

et al. 2015

Appl Environ Microbiol

123

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Listeria monocytogenes is involved in food-borne illness with a high mortality rate. The persistence of the pathogen along the food chain can be associated with its ability to form biofilms on inert surfaces. While most of the phenotypes associated with biofilms are related to their spatial organization, most published data comparing biofilm formation by L. monocytogenes isolates are based on the quantitative crystal violet assay, which does not give access to structural information. Using a high-throughput confocal-imaging approach, the aim of this work was to decipher the structural diversity of biofilms formed by 96 L. monocytogenes strains isolated from various environments. Prior to large-scale analysis, an experimental design was created to improve L. monocytogenes biofilm formation in microscopic-grade microplates, with special emphasis on the growth medium composition. Microscopic analysis of biofilms formed under the selected conditions by the 96 isolates revealed only weak correlation between the genetic lineages of the isolates and the structural properties of the biofilms. However, a gradient in their geometric descriptors (biovolume, mean thickness, and roughness), ranging from flat multilayers to complex honeycomb-like structures, was shown. The dominant honeycomb-like morphotype was characterized by hollow voids hosting free-swimming cells and localized pockets containing mixtures of dead cells and extracellular DNA (eDNA). Listeria monocytogenes still represents an important risk for public health; 1,740 listeriosis cases were reported in the European Union (EU) in 2011 with a mortality rate of 12.7% (1). Listeriosis is particularly dangerous for pregnant women and elderly or immunocompromised people. Persistence of L. monocytogenes strains on food plant surfaces can occur due to maladapted design of equipment and biofilm formation (2, 3). L. monocytogenes is able to attach to and colonize various surfaces, such as stainless steel, glass, and polystyrene, and to contaminate food products during processing (4-6). Biofilms of L. monocytogenes are associated with important ecological advantages, such as protection against biocide action (7). Several molecular determinants, such as flagella, biofilm-associated proteins (Bap), SecA2, and cell-cell communication systems, have been shown to be involved in biofilm construction within the species (8, 9). While no exopolysaccharidic components have been evidenced in the L. monocytogenes biofilm matrix (8), extracellular DNA (eDNA) has been shown to participate in initial cellular adhesion and biofilm organization under specific growth conditions (10). Biofilm formation by the species is highly dependent on environmental conditions, such as variations in temperature, pH, and nutrients (11, 12). L. monocytogenes is structured into four major phylogenetic lineages, each of which is genetically heterogeneous and substructured into highly recognizable clonal complexes as defined by multilocus sequence typing (MLST) (13,14). Attempts to relate biofilm formation ...

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Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes

Cited by 40 publications

References 30 publications

A C. trachomatis Cloning Vector and the Generation of C. trachomatis Strains Expressing Fluorescent Proteins under the Control of a C. trachomatis Promoter

A C. trachomatis Cloning Vector and the Generation of C. trachomatis Strains Expressing Fluorescent Proteins under the Control of a C. trachomatis Promoter

Passive immunization with anti-ActA and anti-listeriolysin O antibodies protects against Listeria monocytogenes infection in mice

Exploring the Diversity of Listeria monocytogenes Biofilm Architecture by High-Throughput Confocal Laser Scanning Microscopy and the Predominance of the Honeycomb-Like Morphotype

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