Solid-Phase Capture of Proteins, Spores, and Bacteria

Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti-Escherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 μg) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and anti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 and B. globigii was 1 spore/cell independent of the sample size. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25°C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.     

Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae

Quorum sensing, bacterial cell-to-cell communication, has been linked to the virulence of pathogenic bacteria. Indeed, in vitro experiments have shown that many bacterial pathogens regulate the expression of virulence genes by this cell-to-cell communication process. Moreover, signal molecules have been detected in samples retrieved from infected hosts and quorum sensing disruption has been reported to result in reduced virulence in different host–pathogen systems. However, data on in vivo quorum sensing activity of pathogens during infection of a host are currently lacking. We previously reported that quorum sensing regulates the virulence of Vibrio harveyi in a standardised model system with gnotobiotic brine shrimp (Artemia franciscana) larvae. Here, we monitored quorum sensing activity in Vibrio harveyi during infection of the shrimp, using bioluminescence as a read-out. We found that wild-type Vibrio harveyi shows a strong increase in quorum sensing activity early during infection. In this respect, the bacteria behave remarkably similar in different larvae, despite the fact that only half of them survive the infection. Interestingly, when expressed per bacterial cell, Vibrio harveyi showed around 200-fold higher maximal quorum sensing-regulated bioluminescence when associated with larvae than in the culture water. Finally, the in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2- and CAI-1-deficient mutants. These results indicate that AI-2 and CAI-1 are the dominant signals during infection of brine shrimp.     

Genome-Scale Co-Expression Network Comparison across Escherichia coli and Salmonella enterica Serovar Typhimurium Reveals Significant Conservation at the Regulon Level of Local Regulators Despite Their Dissimilar Lifestyles

Availability of genome-wide gene expression datasets provides the opportunity to study gene expression across different organisms under a plethora of experimental conditions. In our previous work, we developed an algorithm called COMODO (COnserved MODules across Organisms) that identifies conserved expression modules between two species. In the present study, we expanded COMODO to detect the co-expression conservation across three organisms by adapting the statistics behind it. We applied COMODO to study expression conservation/divergence between Escherichia coli, Salmonella enterica, and Bacillus subtilis. We observed that some parts of the regulatory interaction networks were conserved between E. coli and S. enterica especially in the regulon of local regulators. However, such conservation was not observed between the regulatory interaction networks of B. subtilis and the two other species. We found co-expression conservation on a number of genes involved in quorum sensing, but almost no conservation for genes involved in pathogenicity across E. coli and S. enterica which could partially explain their different lifestyles. We concluded that despite their different lifestyles, no significant rewiring have occurred at the level of local regulons involved for instance, and notable conservation can be detected in signaling pathways and stress sensing in the phylogenetically close species S. enterica and E. coli. Moreover, conservation of local regulons seems to depend on the evolutionary time of divergence across species disappearing at larger distances as shown by the comparison with B. subtilis. Global regulons follow a different trend and show major rewiring even at the limited evolutionary distance that separates E. coli and S. enterica.     

Differential sensitivity to 2'-deoxyguanosine of antigen-specific and nonspecific suppressor T cells in delayed hypersensitivity

The cell wall components involved in the adherence of Bacillus globigii to lymphocytes were studied. Lipoteichoic acid inhibited B. globigii-lymphocyte interaction. The interaction was not affected by Escherichia coli outer membranes or lipopolysaccharides.     

Pertussis Toxin Exploits Specific Host Cell Signaling Pathways for Promoting Invasion and Translocation of Escherichia coli K1 RS218 in Human Brain-derived Microvascular Endothelial Cells

Pertussis toxin (PTx), an AB5 toxin and major virulence factor of the whooping cough-causing pathogen Bordetella pertussis, has been shown to affect the blood-brain barrier. Dysfunction of the blood-brain barrier may facilitate penetration of bacterial pathogens into the brain, such as Escherichia coli K1 (RS218). In this study, we investigated the influence of PTx on blood-brain barrier permissiveness to E. coli infection using human brain-derived endothelial HBMEC and TY10 cells as in vitro models. Our results indicate that PTx acts at several key points of host cell intracellular signaling pathways, which are also affected by E. coli K1 RS218 infection. Application of PTx increased the expression of the pathogen binding receptor gp96. Further, we found an activation of STAT3 and of the small GTPase Rac1, which have been described as being essential for bacterial invasion involving host cell actin cytoskeleton rearrangements at the bacterial entry site. In addition, we showed that PTx induces a remarkable relocation of VE-cadherin and β-catenin from intercellular junctions. The observed changes in host cell signaling molecules were accompanied by differences in intracellular calcium levels, which might act as a second messenger system for PTx. In summary, PTx not only facilitates invasion of E. coli K1 RS218 by activating essential signaling cascades; it also affects intercellular barriers to increase paracellular translocation.     

Inhibitory effect of aminoglycosides and tetracyclines on quorum sensing in <Emphasis Type="Italic">Chromobacterium violaceum</Emphasis>

Effect of aminoglycosides (kanamycin, gentamycin, and amikacin) and tetracyclines (tetracycline and doxycycline) on the CviI/CviR two-component quorum sensing mediated with C₆-AHL autoinducer in Chromobacterium violaceum ATCC 31532 was studied. All antibiotics showed growth-inhibitory activity against the test strain and violated its density-dependent communication system in sub-inhibitory concentrations, which could be seen as suppression of C₆-AHL-dependent violacein biosynthesis. The tested antibiotics exhibited an alternative balance of these activities: a direct antibacterial effect prevailed in tetracyclines, while aminoglycosides inhibited quorum sensing system of C. violaceum ATCC 31532 within a broad concentration range. Sensor strains capable of quantitative detection of C₆-AHL and bearing the genes of resistance to aminoglycosides (C. violaceum 026) or tetracyclines (Escherichia coli pAL103) were used to analyze the effect of antibiotics on autoinducer accumulation in the cultivation medium. Aminoglycosides were found to suppress C₆-AHL production within a broad concentration range; and therefore quorum sensing in C. violaceum ATCC 31532 did not function because of C₆-AHL limitation. The obtained results were analyzed in relation to their biological role in natural microbiocenoses, as well as in relation to antibiotic treatment of bacterial infections caused by organisms using quorum sensing for induction of their pathogenic potential.     

A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its antitoxin and are found on the chromosomes of many bacteria including pathogens. Here we report how each of five such TA systems in E. coli affect bacterial cell death differently in liquid media and during biofilm formation. Of all these systems, only the TA system mazEF mediated cell death both in liquid media and during biofilm formation. At the other extreme, as our results have revealed here, the TA system dinJ-YafQ is unique in that it is involved only in the death process during biofilm formation. Cell death governed by mazEF and dinJ-YafQ seems to participate in biofilm formation through a novel mechanism.     

Engineering Escherichia coli for enhanced sensitivity to the autoinducer-2 quorum sensing signal

The autoinducer-2 (AI-2) quorum sensing system is involved in a range of population-based bacterial behaviors and has been engineered for cell–cell communication in synthetic biology systems. Investigation into the cellular mechanisms of AI-2 processing has determined that overexpression of uptake genes increases AI-2 uptake rate, and genomic deletions of degradation genes lowers the AI-2 level required for activation of reporter genes. Here, we combine these two strategies to engineer an Escherichia coli strain with enhanced ability to detect and respond to AI-2. In an E. coli strain that does not produce AI-2, we monitored AI-2 uptake and reporter protein expression in a strain that overproduced the AI-2 uptake or phosphorylation units LsrACDB or LsrK, a strain with the deletion of AI-2 degradation units LsrF and LsrG, and an “enhanced” strain with both overproduction of AI-2 uptake and deletion of AI-2 degradation elements. By adding up to 40 μM AI-2 to growing cell cultures, we determine that this “enhanced” AI-2 sensitive strain both uptakes AI-2 more rapidly and responds with increased reporter protein expression than the others. This work expands the toolbox for manipulating AI-2 quorum sensing processes both in native environments and for synthetic biology applications.     

Directed assembly of a bacterial quorum

Many reports have elucidated the mechanisms and consequences of bacterial quorum sensing (QS), a molecular communication system by which bacterial cells enumerate their cell density and organize collective behavior. In few cases, however, the numbers of bacteria exhibiting this collective behavior have been reported, either as a number concentration or a fraction of the whole. Not all cells in the population, for example, take on the collective phenotype. Thus, the specific attribution of the postulated benefit can remain obscure. This is partly due to our inability to independently assemble a defined quorum, for natural and most artificial systems the quorum itself is a consequence of the biological context (niche and signaling mechanisms). Here, we describe the intentional assembly of quantized quorums. These are made possible by independently engineering the autoinducer signal transduction cascade of Escherichia coli (E. coli) and the sensitivity of detector cells so that upon encountering a particular autoinducer level, a discretized sub-population of cells emerges with the desired phenotype. In our case, the emergent cells all express an equivalent amount of marker protein, DsRed, as an indicator of a specific QS-mediated activity. The process is robust, as detector cells are engineered to target both large and small quorums. The process takes about 6 h, irrespective of quorum level. We demonstrate sensitive detection of autoinducer-2 (AI-2) as an application stemming from quantized quorums. We then demonstrate sub-population partitioning in that AI-2-secreting cells can ‘call'' groups neighboring cells that ‘travel'' and establish a QS-mediated phenotype upon reaching the new locale.     

The Role of the QseC Quorum-Sensing Sensor Kinase in Epinephrine-Enhanced Motility and Biofilm Formation by Escherichia coli

Biofilms play a pivotal role in infections related to devices. Biofilm formation in Escherichia coli is mediated by the quorum-sensing E. coli regulator C (QseC), the histidine sensor kinase that can sense epinephrine (EPI)/norepinephrine (NE). In this study, we evaluate the role of the QseC quorum-sensing sensor kinase in epinephrine-enhanced motility and biofilm formation by E. coli. An E. coli MC1000 qseC mutant was constructed. We investigated the role of the QseC in the formation of biofilms on the surface of medical-grade polyvinyl chloride using the E. coli K-12 MC1000 strain as well as a corresponding qseC mutant. Addition of EPI/NE increased biofilm formation by wild-type K-12 MC1000 but not by the isogenic qseC mutant. Scanning confocal laser microscopy corroborated these results by showing that EPI/NE addition significantly increased biofilm’s thickness. As expected, the addition of EPI/NE to the qseC mutant, which lacks the ability to sense the hormones, failed to stimulate biofilm formation. Since EPI/NE addition increased bacterial motility, we proposed that their stimulatory effects on biofilm formation occur by enhancing bacterial motility and altering biofilm architecture. We also found that EPI/NE regulate motility and the biofilm phenotype via QseC, as motility was diminished and biofilm formation was significantly decreased in a qseC deletion mutant. These results indicate that EPI/NE induce E. coli biofilm formation on the surface of polyvinyl chloride through QseC. Cross-talk between E. coli (quorum sensing) and host hormones may explain the pathogen-caused opportunistic infections that occur in patients with prosthetic devices used during hormone level fluctuations in the host.     

Control of Escherichia coli growth rate through cell density

The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability. This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration. In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase. This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E. coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators. Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.     

In Vivo Programmed Gene Expression Based on Artificial Quorum Networks

The quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of the Vibrio fischeri luxI-luxR quorum sensing system. In order to achieve in vivo programmed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter P_araBAD, into the QS system. In vitro expression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density. In vivo expression assays confirmed that the araQS system presented an in vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applications in vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuated Edwardsiella tarda strain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expression in vivo and might have potential uses, including, but not limited to, bacterial vector vaccines.     

Bicyclic brominated furanones: A new class of quorum sensing modulators that inhibit bacterial biofilm formation

Both natural and synthetic brominated furanones are known to inhibit biofilm formation by bacteria, but their toxicity to mammalian cells is often not reported. Here, we designed and synthesized a new class of brominated furanones (BBFs) that contained a bicyclic structure having one bromide group with well-defined regiochemistry. This class of molecules exhibited reduction in the toxicity to mammalian cells (human neuroblastoma SK-N-SH) and did not inhibit bacteria (Pseudomonas aeruginosa and Escherichia coli) growth, but retained the inhibitory activity towards biofilm formation of bacteria. In addition, all the BBFs inhibited the production of virulence factor elastase B in P. aeruginosa. To explore the effect of BBFs on quorum sensing, we used a reporter gene assay and found that 6-BBF and 7-BBF exhibited antagonistic activities for LasR protein in the lasI quorum sensing circuit, while 5-BBF showed agonistic activity for the rhlI quorum sensing circuit. This study suggests that structural variation of brominated furanones can be designed for targeted functions to control biofilm formation.     

Interference of phosphane copper (I) complexes of β-carboline with quorum sensing regulated virulence functions and biofilm in foodborne pathogenic bacteria: A first report

Foodborne pathogens are one of the major cause of food-related diseases and food poisoning. Bacterial biofilms and quorum sensing (QS) mechanism of cell–cell communication have also been found to be associated with several outbreaks of foodborne diseases and are great threat to food safety. Therefore, In the present study, we investigated the activity of three tetrahedrally coordinated copper(I) complexes against quorum sensing and biofilms of foodborne bacteria. All the three complexes demonstrated similar antimicrobial properties against the selected pathogens. Concentration below the MIC i.e. at sub-MICs all the three complexes interfered significantly with the quorum sensing regulated functions in C. violaceum (violacein), P. aeruginosa (elastase, pyocyanin and alginate production) and S. marcescens (prodigiosin). The complexes demonstrated potent broad-spectrum biofilm inhibition in Pseudomonas aeruginosa, E. coli, Chromobacterium violaceum, Serratia marcescens, Klebsiella pneumoniae and Listeria monocytogenes. Biofilm inhibition was visualized using SEM and CLSM images. Action of the copper(I) complexes on two key QS regulated functions contributing to biofilm formation i.e. EPS production and swarming motility was also studied and statistically significant reduction was recorded. These results could form the basis for development of safe anti-QS and anti-biofilm agents that can be utilized in the food industry as well as healthcare sector to prevent food-associated diseases.     

Autonomous bacterial localization and gene expression based on nearby cell receptor density

Escherichia coli were genetically modified to enable programmed motility, sensing, and actuation based on the density of features on nearby surfaces. Then, based on calculated feature density, these cells expressed marker proteins to indicate phenotypic response. Specifically, site‐specific synthesis of bacterial quorum sensing autoinducer‐2 (AI‐2) is used to initiate and recruit motile cells. In our model system, we rewired E. coli's AI‐2 signaling pathway to direct bacteria to a squamous cancer cell line of head and neck (SCCHN), where they initiate synthesis of a reporter (drug surrogate) based on a threshold density of epidermal growth factor receptor (EGFR). This represents a new type of controller for targeted drug delivery as actuation (synthesis and delivery) depends on a receptor density marking the diseased cell. The ability to survey local surfaces and initiate gene expression based on feature density represents a new area‐based switch in synthetic biology that will find use beyond the proposed cancer model here.     

Interspecific Quorum Sensing Mediates the Resuscitation of Viable but Nonculturable Vibrios

Entry and exit from dormancy are essential survival mechanisms utilized by microorganisms to cope with harsh environments. Many bacteria, including the opportunistic human pathogen Vibrio vulnificus, enter a form of dormancy known as the viable but nonculturable (VBNC) state. VBNC cells can resuscitate when suitable conditions arise, yet the molecular mechanisms facilitating resuscitation in most bacteria are not well understood. We discovered that bacterial cell-free supernatants (CFS) can awaken preexisting dormant vibrio populations within oysters and seawater, while CFS from a quorum sensing mutant was unable to produce the same resuscitative effect. Furthermore, the quorum sensing autoinducer AI-2 could induce resuscitation of VBNC V. vulnificus in vitro, and VBNC cells of a mutant unable to produce AI-2 were unable to resuscitate unless the cultures were supplemented with exogenous AI-2. The quorum sensing inhibitor cinnamaldehyde delayed the resuscitation of wild-type VBNC cells, confirming the importance of quorum sensing in resuscitation. By monitoring AI-2 production by VBNC cultures over time, we found quorum sensing signaling to be critical for the natural resuscitation process. This study provides new insights into the molecular mechanisms stimulating VBNC cell exit from dormancy, which has significant implications for microbial ecology and public health.     

Identification of boronic acids as antagonists of bacterial quorum sensing in Vibrio harveyi

Bacterial quorum sensing plays a very important role in the regulation of biofilm formation, virulence, conjugation, sporulation, and swarming mobility. Inhibitors of bacterial quorum sensing are important research tools and potential therapeutic agents. In this paper, we describe for the first time the discovery of several boronic acids as single digit micromolar inhibitors of bacterial quorum sensing in Vibrio harveyi.