Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions¹. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.     

Elevated Shear Stress Protects Escherichia coli Cells Adhering to Surfaces via Catch Bonds from Detachment by Soluble Inhibitors

Soluble inhibitors find widespread applications as therapeutic drugs to reduce the ability of eukaryotic cells, bacteria, or viruses to adhere to surfaces and host tissues. Mechanical forces resulting from fluid flow are often present under in vivo conditions, and it is commonly presumed that fluid flow will further add to the inhibitive effect seen under static conditions. In striking contrast, we discover that when surface adhesion is mediated by catch bonds, whose bond life increases with increased applied force, shear stress may dramatically increase the ability of bacteria to withstand detachment by soluble competitive inhibitors. This shear stress-induced protection against inhibitor-mediated detachment is shown here for the fimbrial FimH-mannose-mediated surface adhesion of Escherichia coli. Shear stress-enhanced reduction of bacterial detachment has major physiological and therapeutic implications and needs to be considered when developing and screening drugs.     

En route from artificial to natural: Evaluation of inhibitors of mannose-specific adhesion of E. coli under flow

We investigated the properties of six Escherichia coli adhesion inhibitors under static and under flow conditions. On mannan-covered model substrates and under static conditions, all inhibitors were able to almost completely abolish lectin-mediated E. coli adhesion. On a monolayer of living human microvascular endothelial cells (HMEC-1), the inhibitors reduced adhesion under static conditions as well, but a large fraction of bacteria still managed to adhere even at highest inhibitor concentrations. In contrast, under flow conditions E. coli did not exhibit any adhesion to HMEC-1 not even at inhibitor concentrations where significant adhesion was detected under static conditions. This indicates that the presence of shear stress strongly affects inhibitor properties and must be taken into account when evaluating the potency of bacterial adhesion inhibitors.     

Expression of dengue virus NS3 protein in Drosophila alters its susceptibility to infection

We developed a Drosophila model in which the dengue virus NS3 protein is expressed in a tissue specific and inducible manner. Dengue virus NS3 is a multifunctional protein playing a major role during viral replication. Both protease and helicase domains of NS3 are interacting with human and insect host proteins including innate immune components of the host machinery. We characterized the NS3 transgenic flies showing that NS3 expression did not affect fly development. To further study the links between NS3 and the innate immune response, we challenge the flies with gram-positive and gram-negative bacteria. Interestingly, the Drosophila transgenic flies expressing NS3 were more susceptible to bacterial infections than control flies. However ubiquitous or immune-specific NS3 expression affected neither the life span nor the response to a non-infectious stress of the flies. In conclusion, we generated a new in vivo system to study the functional impact of DENV NS3 protein on the innate immune response.     

Extracellular Bacterial Pathogen Induces Host Cell Surface Reorganization to Resist Shear Stress

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow–generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.     

Biofilm formation by Pseudomonas aeruginosa in solid murine tumors - a novel model system

The ability of opportunistic bacterial pathogens to grow in biofilms is decisive in the pathogenesis of chronic infectious diseases. Growth within biofilms does not only protect the bacteria against the host immune system but also from the killing by antimicrobial agents. Here, we introduce a mouse model in which intravenously administered planktonic Pseudomonas aeruginosa bacteria are enriched in transplantable subcutaneous mouse tumors. Electron microscopy images provide evidence that such bacteria reside in the tumor tissue within biofilm structures. Immunohistology furthermore demonstrated that infection of the tumor tissue elicits a host response characterized by strong neutrophilic influx. Interestingly, the biofilm defective PA14 pqsA transposon mutant formed less biofilm in vivo and was more susceptible to clearance by intravenous ciprofloxacin treatment as compared to the wild-type control. In conclusion, we have established an experimentally tractable model that may serve to identify novel bacterial and host factors important for in vivo biofilm formation and to re-evaluate bactericidal and anti-biofilm effects of currently used and novel antibacterial compounds.     

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.     

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside ‘septin cages’ and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.     

Impact of the exopolysaccharide layer on biofilms,adhesion and resistance to stress in Lactobacillus johnsonii FI9785

Background

The bacterial cell surface is a crucial factor in cell-cell and cell-host interactions. Lactobacillus johnsonii FI9785 produces an exopolysaccharide (EPS) layer whose quantity and composition is altered in mutants that harbour genetic changes in their eps gene clusters. We have assessed the effect of changes in EPS production on cell surface characteristics that may affect the ability of L. johnsonii to colonise the poultry host and exclude pathogens.

Results

Analysis of physicochemical cell surface characteristics reflected by Zeta potential and adhesion to hexadecane showed that an increase in EPS gave a less negative, more hydrophilic surface and reduced autoaggregation. Autoaggregation was significantly higher in mutants that have reduced EPS, indicating that EPS can mask surface structures responsible for cell-cell interactions. EPS also affected biofilm formation, but here the quantity of EPS produced was not the only determinant. A reduction in EPS production increased bacterial adhesion to chicken gut explants, but made the bacteria less able to survive some stresses.

Conclusions

This study showed that manipulation of EPS production in L. johnsonii FI9785 can affect properties which may improve its performance as a competitive exclusion agent, but that positive changes in adhesion may be compromised by a reduction in the ability to survive stress.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0347-2) contains supplementary material, which is available to authorized users.     

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.     

Unraveling the Role of Surface Mucus-Binding Protein and Pili in Muco-Adhesion of Lactococcus lactis

Adhesion of bacteria to mucus may favor their persistence within the gut and their beneficial effects to the host. Interactions between pig gastric mucin (PGM) and a natural isolate of Lactococcus lactis (TIL448) were measured at the single-cell scale and under static conditions, using atomic force microscopy (AFM). In parallel, these interactions were monitored at the bacterial population level and under shear flow. AFM experiments with a L. lactis cell-probe and a PGM-coated surface revealed a high proportion of specific adhesive events (60%) and a low level of non-adhesive ones (2%). The strain muco-adhesive properties were confirmed by the weak detachment of bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were detected at short (100−200 nm) and long distances (up to 600−800 nm). AFM measurements on pili and mucus-binding protein defective mutants demonstrated the comparable role played by these two surface proteinaceous components in adhesion to PGM under static conditions. Under shear flow, a more important contribution of the mucus-binding protein than the pili one was observed. Both methods differ by the way of probing the adhesion force, i.e. negative force contact vs. sedimentation and normal-to-substratum retraction vs. tangential detachment conditions, using AFM and flow chamber, respectively. AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM. This study dissects L. lactis muco-adhesive phenotype, in relation with the nature of the bacterial surface determinants.     

The three-dimensional structure of CFA/I adhesion pili: traveler's diarrhea bacteria hang on by a spring

To survive the harsh environment of a churning intestinal tract, bacteria attach to the host epithelium via thin fibers called pili (or fimbriae). Enterotoxigenic Escherichia coli bacteria expressing colonization factor antigen I (CFA/I) pili and related pili are the most common known bacterial cause of diarrheal disease, including traveler's diarrhea. CFA/I pili, assembled via the alternate chaperone pathway, are essential for binding and colonization of the small bowel by these pathogenic bacteria. Herein, we elucidate unique structural features of CFA/I pili that appear to optimize their function as bacterial tethers in the intestinal tract. Using transmission electron microscopy of negatively stained samples in combination with iterative three-dimensional helical reconstruction methods for image processing, we determined the structure of the CFA/I pilus filament. Our results indicate that strong end-to-end protein interactions and weak interactions between the coils of a sturdy spring-like helix provide the combination of strength, stability, and flexibility required to sustain bacterial adhesion and incite intestinal disease. We propose that CFA/I pili behave like a spring to maintain attachment to the gut lining during vortex mixing and downward flow of the intestinal contents, thereby persisting long enough for these bacteria to colonize the host epithelium and cause enteric disease.     

A MAM7 Peptide-Based Inhibitor of Staphylococcus aureus Adhesion Does Not Interfere with In Vitro Host Cell Function

Adhesion inhibitors that block the attachment of pathogens to host tissues may be used synergistically with or as an alternative to antibiotics. The wide-spread bacterial adhesin Multivalent Adhesion Molecule (MAM) 7 has recently emerged as a candidate molecule for a broad-spectrum adhesion inhibitor which may be used to prevent bacterial colonization of wounds. Here we have tested if the antibacterial properties of a MAM-based inhibitor could be used to competitively inhibit adhesion of methicillin-resistant Staphylococcus aureus (MRSA) to host cells. Additionally, we analyzed its effect on host cellular functions linked to the host receptor fibronectin, such as migration, adhesion and matrix formation in vitro, to evaluate potential side effects prior to advancing our studies to in vivo infection models. As controls, we used inhibitors based on well-characterized bacterial adhesin-derived peptides from F1 and FnBPA, which are known to affect host cellular functions. Inhibitors based on F1 or FnBPA blocked MRSA attachment but at the same time abrogated important cellular functions. A MAM7-based inhibitor did not interfere with host cell function while showing good efficacy against MRSA adhesion in a tissue culture model. These observations provide a possible candidate for a bacterial adhesion inhibitor that does not cause adverse effects on host cells while preventing bacterial infection.     

The Shaft of the Type 1 Fimbriae Regulates an External Force to Match the FimH Catch Bond

Type 1 fimbriae mediate adhesion of uropathogenic Escherichia coli to host cells. It has been hypothesized that due to their ability to uncoil under exposure to force, fimbriae can reduce fluid shear stress on the adhesin-receptor interaction by which the bacterium adheres to the surface. In this work, we develop a model that describes how the force on the adhesin-receptor interaction of a type 1 fimbria varies as a bacterium is affected by a time-dependent fluid flow mimicking in vivo conditions. The model combines in vivo hydrodynamic conditions with previously assessed biomechanical properties of the fimbriae. Numerical methods are used to solve for the motion and adhesion force under the presence of time-dependent fluid profiles. It is found that a bacterium tethered with a type 1 pilus will experience significantly reduced shear stress for moderate to high flow velocities and that the maximum stress the adhesin will experience is limited to ∼120 pN, which is sufficient to activate the conformational change of the FimH adhesin into its stronger state but also lower than the force required for breaking it under rapid loading. Our model thus supports the assumption that the type 1 fimbria shaft and the FimH adhesin-receptor interaction are optimized to each other, and that they give piliated bacteria significant advantages in rapidly changing fluidic environments.     

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.     

DRAM1 promotes the targeting of mycobacteria to selective autophagy

Autophagy provides an important defense mechanism against intracellular bacteria, such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease (TB). We recently reported that pathogen recognition and antibacterial autophagy are connected by the induction of the DNA damage-regulated autophagy modulator DRAM1 via the toll-like receptor (TLR)-MYD88-NFKB innate immunity signaling pathway. Having shown that DRAM1 colocalizes with Mtb in human macrophages, we took advantage of a zebrafish model for TB to investigate the function of DRAM1 in autophagic host defense in vivo. We found that DRAM1 protects the zebrafish host from infection with Mycobacterium marinum (Mm), a close relative of Mtb. Overexpression of DRAM1 increases autophagosome formation and promotes autophagic flux by a mechanism dependent on the cytosolic DNA sensor TMEM173/STING and the ubiquitin receptor SQSTM1/p62. Here we summarize and discuss the implications of these findings.     

Modifying TIMER to generate a slow-folding DsRed derivative for optimal use in quickly-dividing bacteria

It is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, named DsRed₄₂, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however DsRed₄₂ accumulates red fluorescence in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show DsRed₄₂ signal also accumulates during exposure to sources of nitric oxide (NO), suggesting DsRed₄₂ signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, DsRed₄₂ signal was detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was no significant overlap between DsRed₄₂ signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel DsRed₄₂ variant represents a tool that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.     

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important applications in the quest for the development of novel therapeutics. Assays using static conditions fail to capture the dependence of adhesion on shear, limiting their correlation with in vivo environment. Parallel plate flow chambers that quantify adhesion under physiological fluid flow need multiple experiments for the generation of a shear adhesion map. In addition, they do not represent the in vivo scale and morphology and require large volumes (~ml) of reagents for experiments. In this study, we demonstrate the generation of shear adhesion map from a single experiment using a microvascular network based microfluidic device, SynVivo-SMN. This device recreates the complex in vivo vasculature including geometric scale, morphological elements, flow features and cellular interactions in an in vitro format, thereby providing a biologically realistic environment for basic and applied research in cellular behavior, drug delivery, and drug discovery. The assay was demonstrated by studying the interaction of the 2 µm biotin-coated particles with avidin-coated surfaces of the microchip. The entire range of shear observed in the microvasculature is obtained in a single assay enabling adhesion vs. shear map for the particles under physiological conditions.