Draft genome sequences of Pseudomonas strains zfem001-005 isolated from the intestine of larval zebrafish Danio rerio.

Here, we report the draft genome sequences of Pseudomonas strains zfem001-005, five isolates from the intestinal microbiota of healthy larval zebrafish Danio rerio at a developmental age of 7 days post fertilization. The isolates have been identified as Pseudomonas sediminis, Pseudomonas japonica, Pseudomonas otitidis, Pseudomonas sichuanensis, and Pseudomonas tohonis, respectively.

Colonization of larval zebrafish (Danio rerio) with adherent-invasive Escherichia coli prevents recovery of the intestinal mucosa from drug-induced enterocolitis.

IMPORTANCE: Although inflammatory bowel diseases are on the rise, what factors influence IBD risk and severity, and the underlying mechanisms remain to be fully understood. Although host genetics, microbiome, and environmental factors have all been shown to correlate with the development of IBD, cause and effect are difficult to disentangle in this context. For example, AIEC is a known pathobiont found in IBD patients, but it remains unclear if gut inflammation during IBD facilitates colonization with AIEC, or if AIEC colonization makes the host more susceptible to pro-inflammatory stimuli. It is critical to understand the mechanisms that contribute to AIEC infections in a susceptible host in order to develop successful therapeutics. Here, we show that the larval zebrafish model recapitulates key features of AIEC infections in other animal models and can be utilized to address these gaps in knowledge.

Bacterial outer membrane vesicles provide an alternative pathway for trafficking of Escherichia coli O157 type III secreted effectors to epithelial cells.

IMPORTANCE: Bacteria can package protein cargo into nanosized membrane blebs that are shed from the bacterial membrane and released into the environment. Here, we report that a type of pathogenic bacteria called enterohemorrhagic Escherichia coli O157 (EHEC) uses their membrane blebs (outer membrane vesicles) to package components of their type 3 secretion system and send them into host cells, where they can manipulate host signaling pathways including those involved in infection response, such as immunity. Usually, EHEC use a needle-like apparatus to inject these components into host cells, but packaging them into membrane blebs that get taken up by host cells is another way of delivery that can bypass the need for a functioning injection system.

Systematic analysis of proximal midgut- and anorectal-originating contractions in larval zebrafish using event feature detection and supervised machine learning algorithms.

BACKGROUND: Zebrafish larvae are translucent, allowing in vivo analysis of gut development and physiology, including gut motility. While recent progress has been made in measuring gut motility in larvae, challenges remain which can influence results, such as how data are interpreted, opportunities for technical user error, and inconsistencies in methods. METHODS: To overcome these challenges, we noninvasively introduced Nile Red fluorescent dye to fill the intraluminal gut space in zebrafish larvae and collected serial confocal microscopic images of gut fluorescence. We automated the detection of fluorescent-contrasted contraction events against the median-subtracted signal and compared it to manually annotated gut contraction events across anatomically defined gut regions. Supervised machine learning (multiple logistic regression) was then used to discriminate between true contraction events and noise. To demonstrate, we analyzed motility in larvae under control and reserpine-treated conditions. We also used automated event detection analysis to compare unfed and fed larvae. KEY RESULTS: Automated analysis retained event features for proximal midgut-originating retrograde and anterograde contractions and anorectal-originating retrograde contractions. While manual annotation showed reserpine disrupted gut motility, machine learning only achieved equivalent contraction discrimination in controls and failed to accurately identify contractions after reserpine due to insufficient intraluminal fluorescence. Automated analysis also showed feeding had no effect on the frequency of anorectal-originating contractions. CONCLUSIONS & INFERENCES: Automated event detection analysis rapidly and accurately annotated contraction events, including the previously neglected phenomenon of anorectal contractions. However, challenges remain to discriminate contraction events based on intraluminal fluorescence under treatment conditions that disrupt functional motility.

Defective phagocyte association during infection of Galleria mellonella with Yersinia pseudotuberculosis is detrimental to both insect host and microbe.

Adhesins facilitate bacterial colonization and invasion of host tissues and are considered virulence factors, but their impact on immune-mediated damage as a driver of pathogenesis remains unclear. Yersinia pseudotuberculosis encodes for a multivalent adhesion molecule (MAM), a mammalian cell entry (MCE) family protein and adhesin. MAMs are widespread in Gram-negative bacteria and enable enteric bacteria to colonize epithelial tissues. Their role in bacterial interactions with the host innate immune system and contribution to pathogenicity remains unclear. Here, we investigated howY. pseudotuberculosis MAM contributes to pathogenesis during infection of the Galleria mellonella insect model. We show that Y. pseudotuberculosis MAM is required for efficient bacterial binding and uptake by hemocytes, the host phagocytes. Y. pseudotuberculosis interactions with insect and mammalian phagocytes are determined by bacterial and host factors. Loss of MAM, and deficient microbe-phagocyte interaction, increased pathogenesis in G. mellonella. Diminished phagocyte association also led to increased bacterial clearance. Furthermore, Y. pseudotuberculosis that failed to engage phagocytes hyperactivated humoral immune responses, most notably melanin production. Despite clearing the pathogen, excessive melanization also increased phagocyte death and host mortality. Our findings provide a basis for further studies investigating how microbe- and host-factors integrate to drive pathogenesis in a tractable experimental system.

The E. coli transcription factor GrlA is regulated by subcellular compartmentalization and activated in response to mechanical stimuli.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that colonizes the gastrointestinal tract and has evolved intricate mechanisms to sense and respond to the host environment. Upon the sensation of chemical and physical cues specific to the host's intestinal environment, locus of enterocyte effacement (LEE)-encoded virulence genes are activated and promote intestinal colonization. The LEE transcriptional activator GrlA mediates EHEC's response to mechanical cues characteristic of the intestinal niche, including adhesive force that results from bacterial adherence to epithelial cells and fluid shear that results from intestinal motility and transit. GrlA expression and release from its inhibitor GrlR was not sufficient to induce virulence gene transcription; mechanical stimuli were required for GrlA activation. The exact mechanism of GrlA activation, however, remained unknown. We isolated GrlA mutants that activate LEE transcription, independent of applied mechanical stimuli. In nonstimulated EHEC, wild-type GrlA associates with cardiolipin membrane domains via a patch of basic C-terminal residues, and this membrane sequestration is disrupted in EHEC that expresses constitutively active GrlA mutants. GrlA transitions from an inactive, membrane-associated state and relocalizes to the cytoplasm in response to mechanical stimuli, allowing GrlA to bind and activate the LEE1 promoter. GrlA expression and its relocalization in response to mechanical stimuli are required for optimal virulence regulation and colonization of the host intestinal tract during infection. These data suggest a posttranslational regulatory mechanism of the mechanosensor GrlA, whereby virulence gene expression can be rapidly fine-tuned in response to the highly dynamic spatiotemporal mechanical profile of the gastrointestinal tract.

The zebrafish as a model for gastrointestinal tract-microbe interactions.

The zebrafish (Danio rerio) has become a widely used vertebrate model for bacterial, fungal, viral, and protozoan infections. Due to its genetic tractability, large clutch sizes, ease of manipulation, and optical transparency during early life stages, it is a particularly useful model to address questions about the cellular microbiology of host-microbe interactions. Although its use as a model for systemic infections, as well as infections localised to the hindbrain and swimbladder having been thoroughly reviewed, studies focusing on host-microbe interactions in the zebrafish gastrointestinal tract have been neglected. Here, we summarise recent findings regarding the developmental and immune biology of the gastrointestinal tract, drawing parallels to mammalian systems. We discuss the use of adult and larval zebrafish as models for gastrointestinal infections, and more generally, for studies of host-microbe interactions in the gut.

Mathematical model predicts anti-adhesion-antibiotic-debridement combination therapies can clear an antibiotic resistant infection.

As antimicrobial resistance increases, it is crucial to develop new treatment strategies to counter the emerging threat. In this paper, we consider combination therapies involving conventional antibiotics and debridement, coupled with a novel anti-adhesion therapy, and their use in the treatment of antimicrobial resistant burn wound infections. Our models predict that anti-adhesion-antibiotic-debridement combination therapies can eliminate a bacterial infection in cases where each treatment in isolation would fail. Antibiotics are assumed to have a bactericidal mode of action, killing bacteria, while debridement involves physically cleaning a wound (e.g. with a cloth); removing free bacteria. Anti-adhesion therapy can take a number of forms. Here we consider adhesion inhibitors consisting of polystyrene microbeads chemically coupled to a protein known as multivalent adhesion molecule 7, an adhesin which mediates the initial stages of attachment of many bacterial species to host cells. Adhesion inhibitors competitively inhibit bacteria from binding to host cells, thus rendering them susceptible to removal through debridement. An ordinary differential equation model is developed and the antibiotic-related parameters are fitted against new in vitro data gathered for the present study. The model is used to predict treatment outcomes and to suggest optimal treatment strategies. Our model predicts that anti-adhesion and antibiotic therapies will combine synergistically, producing a combined effect which is often greater than the sum of their individual effects, and that anti-adhesion-antibiotic-debridement combination therapy will be more effective than any of the treatment strategies used in isolation. Further, the use of inhibitors significantly reduces the minimum dose of antibiotics required to eliminate an infection, reducing the chances that bacteria will develop increased resistance. Lastly, we use our model to suggest treatment regimens capable of eliminating bacterial infections within clinically relevant timescales.

Optimal translational fidelity is critical for Salmonella virulence and host interactions.

Translational fidelity is required for accurate flow of genetic information, but is frequently altered by genetic changes and environmental stresses. To date, little is known about how translational fidelity affects the virulence and host interactions of bacterial pathogens. Here we show that surprisingly, either decreasing or increasing translational fidelity impairs the interactions of the enteric pathogen Salmonella Typhimurium with host cells and its fitness in zebrafish. Host interactions are mediated by Salmonella pathogenicity island 1 (SPI-1). Our RNA sequencing and quantitative RT-PCR results demonstrate that SPI-1 genes are among the most down-regulated when translational fidelity is either increased or decreased. Further, this down-regulation of SPI-1 genes depends on the master regulator HilD, and altering translational fidelity destabilizes HilD protein via enhanced degradation by Lon protease. Our work thus reveals that optimal translational fidelity is pivotal for adaptation of Salmonella to the host environment, and provides important mechanistic insights into this process.

Outer Membrane Vesicle-Host Cell Interactions.

Outer membrane vesicles (OMVs) are nanosized proteoliposomes derived from the outer membrane of Gram-negative bacteria. They are ubiquitously produced both in culture and during infection and are now recognized to play crucial roles during host-microbe interactions. OMVs can transport a broad range of chemically diverse cargoes, including lipids and lipopolysaccharides, membrane-embedded and associated proteins and small molecules, peptidoglycan, and nucleic acids. Particularly, virulence factors such as adhesins and toxins are often enriched in OMVs. Here we discuss a variety of ways in which OMVs facilitate host-microbe interactions, including their contributions to biofilm formation, nutrient scavenging, and modulation of host cell function. We particularly examine recent findings regarding OMV-host cell interactions in the oral cavity and the gastrointestinal tract.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae.

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Correction: Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation in Vibrio cholerae and downregulate the expression of virulence genes.

[This corrects the article DOI: 10.1039/C7SC00615B.].

Structural Determinants of the Stability of Enzyme-Responsive Polyion Complex Nanoparticles Targeting Pseudomonas aeruginosa's Elastase.

Here, we report how the stability of polyion complex (PIC) particles containing Pseudomonas aeruginosa's elastase (LasB) degradable peptides and antimicrobial poly(ethylene imine) is significantly improved by careful design of the peptide component. Three LasB-degradable peptides are reported herein, all of them carrying the LasB-degradable sequence -GLA- and for which the number of anionic amino acids and cysteine units per peptide were systematically varied. Our results suggest that while net charge and potential to cross-link via disulfide bond formation do not have a predictable effect on the ability of LasB to degrade these peptides, a significant effect of these two parameters on particle preparation and stability is observed. A range of techniques has been used to characterize these new materials and demonstrates that increasing the charge and cross-linking potential of the peptides results in PIC particles with better stability in physiological conditions and upon storage. These results highlight the importance of molecular design for the preparation of PIC particles and should underpin the future development of these materials for responsive drug delivery.

Aggregation of Vibrio cholerae by Cationic Polymers Enhances Quorum Sensing but Overrides Biofilm Dissipation in Response to Autoinduction.

Vibrio cholerae is a Gram-negative bacterium found in aquatic environments and a human pathogen of global significance. Its transition between host-associated and environmental lifestyles involves the tight regulation of niche-specific phenotypes such as motility, biofilm formation, and virulence. V. cholerae's transition from the host to environmental dispersal usually involves suppression of virulence and dispersion of biofilm communities. In contrast to this naturally occurring transition, bacterial aggregation by cationic polymers triggers a unique response, which is to suppress virulence gene expression while also triggering biofilm formation by V. cholerae, an artificial combination of traits that is potentially very useful to bind and neutralize the pathogen from contaminated water. Here, we set out to uncover the mechanistic basis of this polymer-triggered bacterial behavior. We found that bacteria-polymer aggregates undergo rapid autoinduction and achieve quorum sensing at bacterial densities far below those required for autoinduction in the absence of polymers. We demonstrate this induction of quorum sensing is due both to a rapid formation of autoinducer gradients and local enhancement of autoinducer concentrations within bacterial clusters as well as the stimulation of CAI-1 and AI-2 production by aggregated bacteria. We further found that polymers cause an induction of the biofilm-specific regulator VpsR and the biofilm structural protein RbmA, bypassing the usual suppression of biofilm during autoinduction. Overall, this study highlights that synthetic materials can be used to cross-wire natural bacterial responses to achieve a combination of phenotypes with potentially useful applications.

cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization.

The bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes.IMPORTANCE Cholera is an infectious disease that is caused by the bacterium Vibrio cholerae Best known for causing disease in humans, the bacterium is most commonly found in aquatic ecosystems. Hence, humans acquire cholera following ingestion of food or water contaminated with V. cholerae Transition between an aquatic environment and a human host triggers a lifestyle switch that involves reprogramming of V. cholerae gene expression patterns. This process is controlled by a network of transcription factors. In this paper, we show that the cAMP receptor protein (CRP) is a key regulator of V. cholerae gene expression in response to lifestyle changes.

Predictive modelling of a novel anti-adhesion therapy to combat bacterial colonisation of burn wounds.

As the development of new classes of antibiotics slows, bacterial resistance to existing antibiotics is becoming an increasing problem. A potential solution is to develop treatment strategies with an alternative mode of action. We consider one such strategy: anti-adhesion therapy. Whereas antibiotics act directly upon bacteria, either killing them or inhibiting their growth, anti-adhesion therapy impedes the binding of bacteria to host cells. This prevents bacteria from deploying their arsenal of virulence mechanisms, while simultaneously rendering them more susceptible to natural and artificial clearance. In this paper, we consider a particular form of anti-adhesion therapy, involving biomimetic multivalent adhesion molecule 7 coupled polystyrene microbeads, which competitively inhibit the binding of bacteria to host cells. We develop a mathematical model, formulated as a system of ordinary differential equations, to describe inhibitor treatment of a Pseudomonas aeruginosa burn wound infection in the rat. Benchmarking our model against in vivo data from an ongoing experimental programme, we use the model to explain bacteria population dynamics and to predict the efficacy of a range of treatment strategies, with the aim of improving treatment outcome. The model consists of two physical compartments: the host cells and the exudate. It is found that, when effective in reducing the bacterial burden, inhibitor treatment operates both by preventing bacteria from binding to the host cells and by reducing the flux of daughter cells from the host cells into the exudate. Our model predicts that inhibitor treatment cannot eliminate the bacterial burden when used in isolation; however, when combined with regular or continuous debridement of the exudate, elimination is theoretically possible. Lastly, we present ways to improve therapeutic efficacy, as predicted by our mathematical model.

Mathematical modelling of the antibiotic-induced morphological transition of Pseudomonas aeruginosa.

Here we formulate a mechanistic mathematical model to describe the growth dynamics of P. aeruginosa in the presence of the ß-lactam antibiotic meropenem. The model is mechanistic in the sense that carrying capacity is taken into account through the dynamics of nutrient availability rather than via logistic growth. In accordance with our experimental results we incorporate a sub-population of cells, differing in morphology from the normal bacillary shape of P. aeruginosa bacteria, which we assume have immunity from direct antibiotic action. By fitting this model to experimental data we obtain parameter values that give insight into the growth of a bacterial population that includes different cell morphologies. The analysis of two parameters sets, that produce different long term behaviour, allows us to manipulate the system theoretically in order to explore the advantages of a shape transition that may potentially be a mechanism that allows P. aeruginosa to withstand antibiotic effects. Our results suggest that inhibition of this shape transition may be detrimental to bacterial growth and thus suggest that the transition may be a defensive mechanism implemented by bacterial machinery. In addition to this we provide strong theoretical evidence for the potential therapeutic strategy of using antimicrobial peptides (AMPs) in combination with meropenem. This proposed combination therapy exploits the shape transition as AMPs induce cell lysis by forming pores in the cytoplasmic membrane, which becomes exposed in the spherical cells.

Lipopolysaccharide structure impacts the entry kinetics of bacterial outer membrane vesicles into host cells.

Outer membrane vesicles are nano-sized microvesicles shed from the outer membrane of Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle-host cell interactions is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery to host cells. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded, vesicle-targeted probe. We found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of lipopolysaccharide O antigen enables vesicles to bypass clathrin-mediated endocytosis, which enhances both their entry rate and efficiency into host cells. Collectively, our findings highlight the composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections.

Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation in Vibrio cholerae and downregulate the expression of virulence genes.

Here we report the first application of non-bactericidal synthetic polymers to modulate the physiology of a bacterial pathogen. Poly(N-[3-(dimethylamino)propyl] methacrylamide) (P1) and poly(N-(3-aminopropyl)methacrylamide) (P2), cationic polymers that bind to the surface of V. cholerae, the infectious agent causing cholera disease, can sequester the pathogen into clusters. Upon clustering, V. cholerae transitions to a sessile lifestyle, characterised by increased biofilm production and the repression of key virulence factors such as the cholera toxin (CTX). Moreover, clustering the pathogen results in the minimisation of adherence and toxicity to intestinal epithelial cells. Our results suggest that the reduction in toxicity is associated with the reduction to the number of free bacteria, but also the downregulation of toxin production. Finally we demonstrate that these polymers can reduce colonisation of zebrafish larvae upon ingestion of water contaminated with V. cholerae. Overall, our results suggest that the physiology of this pathogen can be modulated without the need to genetically manipulate the microorganism and that this modulation is an off-target effect that results from the intrinsic ability of the pathogen to sense and adapt to its environment. We believe these findings pave the way towards a better understanding of the interactions between pathogenic bacteria and polymeric materials and will underpin the development of novel antimicrobial polymers.

3-Sulfogalactosyl-dependent adhesion of Escherichia coli HS multivalent adhesion molecule is attenuated by sulfatase activity.

Bacterial adhesion to host receptors is an early and essential step in bacterial colonization, and the nature of adhesin-receptor interactions determines bacterial localization and thus the outcome of these interactions. Here, we determined the host receptors for the multivalent adhesion molecule (MAM) from the gut commensal Escherichia coli HS (MAMHS), which contains an array of seven mammalian cell entry domains. The MAMHS adhesin interacted with a range of host receptors, through recognition of a shared 3-O-sulfogalactosyl moiety. This functional group is also found in mucin, a component of the intestinal mucus layer and thus one of the prime adherence targets for commensal E. coli Mucin gels impeded the motility of E. coli by acting as a physical barrier, and the barrier effect was enhanced by specific interactions between mucin and MAMHS in a sulfation-dependent manner. Desulfation of mucin by pure sulfatase or the sulfatase-producing commensal Bacteroides thetaiotaomicron decreased binding of E. coli to mucin and increased the attachment of bacteria to the epithelial surface via interactions with surface-localized sulfated lipid and protein receptors. Together, our results demonstrate that the E. coli adhesin MAMHS facilitates retention of a gut commensal by attachment to mucin. They further suggest that the amount of sulfatase secreted by mucin-foraging bacteria such as B. thetaiotaomicron, inhabiting the same niche, may affect the capacity of the mucus barrier to retain commensal E. coli.