Fluorescent Protein-expressing Bacteria Research Articles

RNA fluorescence in situ hybridization (FISH) as a method to visualize bacterial colonization in the C. elegans gut.

Caenorhabditis elegans is an excellent model to study host-microbe interactions as it is easy to visualize bacterial presence in their intestine. However, previous studies have shown that utilizing transgenic, fluorescent protein-expressing bacteria is not a reliable method to distinguish living bacteria from dead bacteria in the lumen of C. elegans . In this study, we compared methods for visualizing bacterial presence within the C. elegans intestine and found that RNA f luorescent i n s itu h ybridization (RNA FISH) could distinguish the difference between intact and dead bacteria. Thus, we propose RNA FISH as the preferred method to visualize live bacterial presence in the intestines of C. elegans prior to fixation.

Murine Endometrial Organoids to Model Chlamydia Infection

The obligate intracellular bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections. Once internalized in host cells, C. trachomatis undergoes a biphasic developmental cycle within a membrane-bound compartment, known as the inclusion. Successful establishment of the intracellular niche relies on bacterial Type III effector proteins, such as Inc proteins. In vitro and in vivo systems have contributed to elucidating the intracellular lifestyle of C. trachomatis, but additional models combining the archetypal environment of infection with the advantages of in vitro systems are needed. Organoids are three-dimensional structures that recapitulate the microanatomy of an organ's epithelial layer, bridging the gap between in vitro and in vivo systems. Organoids are emerging as relevant model systems to study interactions between bacterial pathogens and their hosts. Here, we took advantage of recently developed murine endometrial organoids (EMOs) and present a C. trachomatis-murine EMO infection model system. Confocal microscopy of EMOs infected with fluorescent protein-expressing bacteria revealed that inclusions are formed within the cytosol of epithelial cells. Moreover, infection with a C. trachomatis strain that allows for the tracking of RB to EB transition indicated that the bacteria undergo a full developmental cycle, which was confirmed by harvesting infectious bacteria from infected EMOs. Finally, the inducible gene expression and cellular localization of a Chlamydia Inc protein within infected EMOs further demonstrated that this model is compatible with the study of Type III secreted effectors. Altogether, we describe a novel and relevant system for the study of Chlamydia-host interactions.

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology.

Background & AimsThe human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host–microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling.MethodsA microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe.ResultsCVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein– or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity.ConclusionsHigh-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.

Optical tracking of phagocytosis with an activatable profluorophore metabolically incorporated into bacterial peptidoglycan.

Phagocytosis is critical for immunity against pathogens. Prior imaging using dye-labeled synthetic beads or green fluorescent protein-expressing bacteria is limited by "always-on" signals which compromise discerning phagocytosed particles from adherent particles. Targeting cellular internalization of pathogens into acidic phagolysosomes, we herein report "turn-on" fluorescence imaging of phagocytosis with viable bacteria featuring peptidoglycans covalently modified with rhodamine-lactam responsive to acidic pH. Culturing of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) with d-lysine conjugated rhodamine-lactam and fluorescein isocyanate (FITC) leads to efficient metabolic incorporation of FITC and rhodamine-lactam into bacterial peptidoglycan. E. coli and S. aureus become red-emissive upon phagocytosis into Raw 264.7 macrophages. With FITC as the reference signal, the mono- and dual-color emission allow efficient in situ distinction of ingested bacteria from extracellular bacteria. Given the ease of optical peptidoglycan labeling, the prevalence of microbial peptidoglycan and preservation of microbial surface landscape, this approach would be of use for investigation on microbial pathogenesis and high-throughput screening of immunomodulators of phagocytosis.

Bacterial imaging with photostable upconversion fluorescent nanoparticles

Autofluorescence, photodamage and photobleaching are often encountered when using downconverting fluorophores and fluorescent proteins for bacteria labeling. These caveats represent a serious limitation when trying to map bacteria dissemination for prolonged periods. Upconversion nanoparticles (UCNs), which are able to convert low energy near-infrared (NIR) excitation light into higher energy visible or NIR light, can address these limitations. These particles' unique optical properties translate into attractive advantages of minimal autofluorescence, reduced photodamage, deeper tissue penetration and prolonged photostability. Here, we report a UCN-based bacteria labeling strategy using Escherichia coli as prototypic bacteria. A comparative analysis highlighted the superior photostability of UCN-labeled bacteria over green fluorescent protein-expressing bacteria. Infection study of UCN-labeled bacteria in dendritic cells indicated co-localization of the UCN signal with bacterial position for up to 6 h post-infection. Furthermore, long-term monitoring of the same infected cells demonstrated the potential to utilize photostable UCN-based imaging for bacterial trafficking purposes.

Elaboration of antibiofilm materials by chemical grafting of an antimicrobial peptide

A peptide antibiotic, gramicidin A, was covalently bound to cystamine self-assembled monolayers on gold surfaces. Each step of the surface functionalization was characterized by polarization modulation infrared reflection absorption spectroscopy and X-ray photoelectron spectroscopy. The antimicrobial activity of the anchored gramicidin was tested against three Gram-positive bacteria (Listeria ivanovii, Enterococcus faecalis, and Staphylococcus aureus), the Gram-negative bacterium Escherichia coli and the yeast Candida albicans. The results revealed that the adsorbed gramicidin reduced, from 60% for E. coli to 90% for C. albicans, the number of culturable microorganisms attached to the surface. The activity was proven to be persistent overtime, up to 6 months after the first use. The bacteria attached to the functionalized surfaces were permeabilized as shown by confocal microscopy. Taken together, these results indicate a bacteriostatic mode of action of the immobilized peptide. Finally, using green fluorescent protein-expressing bacteria, it was shown that the development of a bacterial biofilm was delayed on peptide-grafted surfaces for at least 24 h.

Development of a Fluorescent Multiwell Assay for Evaluating the Capacity of the Ciliated Protozoan Tetrahymena for Bacterivory in Water Samples

Abstract Bacterivory by ciliates in various water ecosystems, both natural and artificial, plays a significant role on the microbial population composition and consequently affects water quality. A convenient, rapid and inexpensive methodology to evaluate the capacity of the ciliate protozoan Tetrahymena thermophila for bacterivory was developed utilizing fluorescent protein expressing bacteria (FPEB) in a microtitre plate fluorimeter. Bacterivory was correlated with a loss in fluorescence measured in the fluorimeter and confirmed by fluorescence microscopy showing that the FPEB were engulfed during the assay and subsequently lost their fluorescence, whereas Cytochalasin B, a known inhibitor of phagocytosis, prevented a decrease in relative fluorescence units (RFUs). The ciliate bacterivory (CB) assay has a great dynamic range allowing the assay to be performed with a variety of predator:prey concentrations. A model toxicant, CuCl2, known to have a toxicological impact on protozoa and often present in different types of wastewater, resulted in measurable decreases in bacterivory. As well, starvation of Tetrahymena for 24 h resulted in reduced bacterivory. In the future, the CB assay could be developed for water monitoring purposes to rapidly assess water samples for the capacity to support bacterivory as an indicator of ecosystem health.

The Global Regulator Ler Is Necessary for Enteropathogenic Escherichia coli Colonization of Caenorhabditis elegans

Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries and is useful for general investigations of the bacterial infection process. However, the study of the molecular pathogenesis of EPEC has been hampered by the lack of genetically tractable, convenient animal models. We have therefore developed the use of the nematode Caenorhabditis elegans as a small animal model of infection for this diarrheal pathogen. We found that nematodes died faster on nematode growth medium in the presence of EPEC pathogens than in the presence of the laboratory control strain MG1655. Increased numbers of pathogens in the gut, determined by standard plate count assays and fluorescence microscopy using green fluorescent protein-expressing bacteria, correlated with killing. Deletion of the gene encoding the global regulator Ler severely reduced the ability of EPEC to colonize the nematode gut and could be complemented by providing the ler gene on a multicopy plasmid in trans. Neither the type III secretion system nor the type IV bundle-forming pilus was required for colonization. Combined, the similarities and distinct differences between EPEC infection of nematodes and that of humans offer a unique opportunity to study several stages of the infection process, namely, attachment, colonization, and persistence, in a genetically tractable, inexpensive, and convenient in vivo system.

Genes of Helicobacter pylori regulated by attachment to AGS cells.

Reciprocal interactions between Helicobacter pylori and cells of the gastric epithelium to which it adheres may affect colonization. Changes in gene expression of H. pylori induced by adhesion to AGS gastric cancer cells by coculture were compared to changes in gene expression of H. pylori cultured without AGS cells by using cDNA filter macroarrays. Adhesion was quantitatively verified by confocal microscopy of green fluorescent protein-expressing bacteria. Four experiments showed that 22 and 21 H. pylori genes were consistently up- and down-regulated, respectively. The up-regulated genes included pathogenicity island, motility, outer membrane protein, and translational genes. The sigma(28) factor antagonist flgM, flgG, the stress response gene, flaA, omp11, and the superoxide dismutase gene (sodB) were down-regulated. The up-regulation of cag3, flgB, tonB, rho, and deaD was confirmed by quantitative PCR, and the up-regulation of lpxD, omp6, secG, fabH, HP1285, HP0222, and HP0836 was confirmed by reverse transcription (RT)-PCR. The down-regulation of flaA, sodB, and HP0874 was confirmed by quantitative PCR, and the down-regulation of omp11 was confirmed by RT-PCR. The alteration of gene expression in H. pylori after adhesion to gastric cells in vitro suggests that changes in motility, outer membrane composition, and stress responses, among other changes, may be involved in gastric colonization.

Bacterial persistence within erythrocytes: A unique pathogenic strategy of Bartonella spp.

Abstract The genus Bartonella comprises human-specific and zoonotic pathogens responsible for a wide range of clinical manifestations, including Carrion's disease, trench fever, cat scratch disease, bacillary angiomatosis and peliosis, endocarditis and bacteremia. These arthropod-borne pathogens typically parasitise erythrocytes in their mammalian reservoir host(s), resulting in a long-lasting haemotropic infection. We have studied the process of Bartonella erythrocyte parasitism by tracking green fluorescent protein-expressing bacteria in the blood of experimentally infected animals. Following intravenous infection, bacteria colonise a yet enigmatic primary niche, from where they are seeded into the blood stream in regular intervals of ∼five days. Bacteria invade mature erythrocytes, replicate temporarily and persist in this unique intracellular niche for the remaining life span of the infected erythrocytes. A triggered antibody response typically results in an abrogation of bacteremia within 3 months of infection, likely by blocking new waves of bacterial invasion into erythrocytes. The recent establishment of genetic tools for Bartonella spp. permitted us to identify several putative pathogenicity determinants. Application of differential fluorescence induction technology resulted in the isolation of bacterial genes differentially expressed during infection in vitro and in vivo, including an unknown family of autotransporter proteins as well as a novel type IV secretion system homologous to the conjugation system of E. coli plasmid R388. Mutational analysis of a previously described type IV secretion system displaying homology to the virB locus of Agrobacterium tumefaciens provided the first example of an essential pathogenicity locus in Bartonella. Though required for establishing haemotropic infection, it remains to be demonstrated if this type IV secretion system is necessary for colonisation of the primary niche or for the subsequent colonisation of erythrocytes.