Granzyme B Disrupts Central Metabolism and Protein Synthesis in Bacteria to Promote an Immune Cell Death Program.

Human cytotoxic lymphocytes kill intracellular microbes. The cytotoxic granule granzyme proteases released by cytotoxic lymphocytes trigger oxidative bacterial death by disrupting electron transport, generating superoxide anion and inactivating bacterial oxidative defenses. However, they also cause non-oxidative cell death because anaerobic bacteria are also killed. Here, we use differential proteomics to identify granzyme B substrates in three unrelated bacteria: Escherichia coli, Listeria monocytogenes, and Mycobacteria tuberculosis. Granzyme B cleaves a highly conserved set of proteins in all three bacteria, which function in vital biosynthetic and metabolic pathways that are critical for bacterial survival under diverse environmental conditions. Key proteins required for protein synthesis, folding, and degradation are also substrates, including multiple aminoacyl tRNA synthetases, ribosomal proteins, protein chaperones, and the Clp system. Because killer cells use a multipronged strategy to target vital pathways, bacteria may not easily become resistant to killer cell attack.

Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.

Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1ß. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined.

Variation in growth and evaluation of cross-protection in Listeria monocytogenes under salt and bile stress.

AIMS: Exposure of Listeria monocytogenes to osmotic stress can induce increased resistance to subsequent lethal exposure to cell envelope stressors, such as nisin and bile salts. We wanted to determine if similar cross-protection phenotypes could occur when L. monocytogenes strains were treated with osmotic stress and exposed to sublethal levels of the cell envelope stressor, bile. METHOD AND RESULTS: Growth phenotypes were measured for six L. monocytogenes strains exposed to 6% NaCl, 0·3 and 1% bile in BHI. To evaluate cross-protection, cells were pre-exposed to 6% NaCl, followed by exposure to BHI+1% bile for 26 h and vice versa. Significant increases in λ (lag phase) and doubling time were observed under salt and bile stresses compared with BHI alone. Average λ and Nmax (maximum cell density) in 0·3 and 1% bile for all strains were significantly lower than that in 6% NaCl. Pre-exposure to 6% NaCl followed by exposure to 1% bile significantly increased λ (P < 0·05), whereas pre-exposure to 1% bile followed by exposure to 6% NaCl led to formation of filamentous cells, with no changes in cell density over 26 h. CONCLUSIONS: Variation in growth characteristics was observed among strains exposed to bile. Exposure to osmotic stress did not lead to increased resistance to bile. Exposure to bile significantly impacted the ability of L. monocytogenes to adapt to grow under osmotic stress, where cells did not multiply but formed filamentous cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Pre-exposure to a cell envelope stress and subsequent exposure to an osmotic stress appears to pose a significant stress to L. monocytogenes cells.

Characterization of purified antimicrobial peptide produced by Pediococcus pentosaceus LJR1, and its application in preservation of white leg shrimp.

The bacteriocinogenic lactic acid bacterium Pediococcus pentosaceus LJR1 isolated from rumen liquor of goat had strong anti-bacterial activity toward Listeria monocytogenes in vitro. This antibacterial activity was lost on treatment with protease indicating that the bacteriocin is proteinaceous in nature. The bacteriocin LJR1 produced by P. pentosaceus was purified following a three step procedure consisting of ammonium sulphate precipitation, gel filtration chromatography and reverse phase-high performance liquid chromatography. The molecular weight of purified bacteriocin was determined to be 4.6 kDa using Tricine SDS-PAGE. Further, we found that the proteinaceous bacteriocin was stable at 100 °C as well as 121 °C for 30 min and 15 min respectively and also at different pH ranging from 4 to 10 when stored for 15 min at 37 °C. Its minimum inhibitory concentration for S. typhi MTCC134 and L. monocytogenes MTCC 1143 was 7.81 µg/ml and 15.63 µg/ml respectively. Scanning electron microscopy analysis of the surface of S. typhi treated with the bacteriocin showed the presence of craters; while in the case of treated L. monocytogenes blebs were observed. The addition of the bacteriocin to shrimp (white leg shrimp) has led to reduction of about 1 log units of L. monocytogenes on day 1 and maintained for 7 days on storage at 4 °C. It is clear that the purified bacteriocin has good potential as a bio preservative for application in food industry.

Discovery of genes required for lipoteichoic acid glycosylation predicts two distinct mechanisms for wall teichoic acid glycosylation.

The bacterial cell wall is an important and highly complex structure that is essential for bacterial growth because it protects bacteria from cell lysis and environmental insults. A typical Gram-positive bacterial cell wall is composed of peptidoglycan and the secondary cell wall polymers, wall teichoic acid (WTA) and lipoteichoic acid (LTA). In many Gram-positive bacteria, LTA is a polyglycerol-phosphate chain that is decorated with d-alanine and sugar residues. However, the function of and proteins responsible for the glycosylation of LTA are either unknown or not well-characterized. Here, using bioinformatics, genetic, and NMR spectroscopy approaches, we found that the Bacillus subtilis csbB and yfhO genes are essential for LTA glycosylation. Interestingly, the Listeria monocytogenes gene lmo1079, which encodes a YfhO homolog, was not required for LTA glycosylation, but instead was essential for WTA glycosylation. LTA is polymerized on the outside of the cell and hence can only be glycosylated extracellularly. Based on the similarity of the genes coding for YfhO homologs that are required in B. subtilis for LTA glycosylation or in L. monocytogenes for WTA glycosylation, we hypothesize that WTA glycosylation might also occur extracellularly in Listeria species. Finally, we discovered that in L. monocytogenes, lmo0626 (gtlB) was required for LTA glycosylation, indicating that the encoded protein has a function similar to that of YfhO, although the proteins are not homologous. Together, our results enable us to propose an updated model for LTA glycosylation and also indicate that glycosylation of WTA might occur through two different mechanisms in Gram-positive bacteria.

The antimicrobial activities and action-mechanism of tea tree oil against food-borne bacteria in fresh cucumber juice.

BACKGROUND: Foodborne diseases caused by foodborne pathogens have increasingly become a worldwide public health concern. Due to potential harmful effects of synthetic chemicals, there is a pressure for adoption of natural alternatives to obtain microbial safety of food. Tea tree oil (TTO) exhibited a wide range of pharmacological actions attribute to the broad spectrum activities. However, to the best of our knowledge, no systematic research on the mode of antibacterial actions of TTO against Listeria monocytogenes (L. monocytogenes) and Escherichia coli (E. coli) in vitro models have been conducted so far. RESULTS: The present investigation reported on the antimicrobial activities of TTO and examined its possible antimicrobial mode of action against L. monocytogenes and E. coli. Results showed that the susceptibility of L. monocytogenes were excellent with the lower minimal inhibitory concentration (MIC) values and larger inhibition zones. TTO changed the integrity of the membrane, as evidenced by the release of 260 nm absorbing intracellular materials and the alteration of membrane potential. The results of flow cytometry showed that TTO caused bacterial membrane permeabilization in a dose-dependent manner. The remarkable cellular morphological changes in bacteria caused by TTO were observed using the scanning electron microscope, indicating cell damage. In addition, antimicrobial preserving properties of TTO were evaluated by time-kill assay after its incorporation in cucumber juice, the results showed TTO successfully inhibited L. monocytogenes and E. coli development, at room temperature and in refrigerator (25 °C and 4 °C) respectively, demonstrating it had good preservative activities in food system. CONCLUSIONS: These findings suggested that TTO exhibited good antimicrobial effect against food-borne pathogens and could be potentially used in food industries as a food preservative.

Use of DNA aptamer for sandwich type detection of Listeria monocytogenes.

A single stranded (ss) DNA aptamer, specific to members of Listeria genus, was used to develop a two-site binding sandwich assay for capture and detection of L. monocytogenes. Antibody-immobilized immunomagnetic beads were used to capture L. monocytogenes, followed by their exposure to the aptamer detector. Detection was achieved by amplification of cell-bound aptamers by qPCR. The lower limit of detection for the combined assay was 2.5 CFU L. monocytogenes in 500 µl buffer. This is juxtaposed to a detection limit of 2.4 log10 CFU in 500 µl buffer for immunomagnetic separation coupled with qPCR detection of L. monocytogenes targeting the hly gene. When applied to turkey deli meat, subjected to 24 h of non-selective enrichment, the two-site binding sandwich assay showed positive results at initial inoculum levels of 1-2 log10 CFU per 25 g sample. Because of its lower limit of detection, the assay reported here could be useful for detection of L. monocytogenes in foods and environmental samples.

Peyer's Patches as a Portal for DNA Delivery by Lactococcus lactis in Vivo.

Application of food-grade Lactococcus lactis (L. lactis) as a safe delivery tool for DNA vaccines and therapeutic proteins has been well investigated. Although some studies showed that eukaryotic expression plasmids were transferred from L. lactis to enterocytes, the precise mechanism of the DNA transfer remains unknown. In this study, we generated an invasive L. lactis strain that expresses "murinized" Internalin A, an invasin of intracellular bacteria Listeria monocytogenes with two amino acid alterations for invasion into murine cells, and confirmed that this L. lactis strain delivered DNA in an invasin-dependent manner into a monolayer of epithelial cells polarized to mimic the gastrointestinal tract environment. Although invasive L. lactis inoculated orally can deliver DNA into enterocytes in the gastrointestinal tract of mice, the efficiency of DNA transfer was similar to that of non-invasive L. lactis strain, suggesting that the in vivo DNA transfer from L. lactis occurs invasin-independently. A ligated-intestinal loop assay, a method for a short-term culturing of the whole intestine filled with materials to evaluate the interaction of the materials with intestinal cells, demonstrated that both non-invasive and invasive L. lactis strains were present in the Peyer's patches of the small intestine. On the other hand, few L. lactis was detected in the non-Peyer's patch epithelial region. Thus, our observations lead us to speculate that DNA transfer from L. lactis occurs predominantly in the Peyer's patches in an invasin-independent manner.

Structure of the bacterial cell division determinant GpsB and its interaction with penicillin-binding proteins.

Each bacterium has to co-ordinate its growth with division to ensure genetic stability of the population. Consequently, cell division and growth are tightly regulated phenomena, albeit different bacteria utilise one of several alternative regulatory mechanisms to maintain control. Here we consider GpsB, which is linked to cell growth and division in Gram-positive bacteria. ΔgpsB mutants of the human pathogen Listeria monocytogenes show severe lysis, division and growth defects due to distortions of cell wall biosynthesis. Consistent with this premise, GpsB interacts both in vitro and in vivo with the major bi-functional penicillin-binding protein. We solved the crystal structure of GpsB and the interaction interfaces in both proteins are identified and validated. The inactivation of gpsB results in strongly attenuated virulence in animal experiments, comparable in degree to classical listerial virulence factor mutants. Therefore, GpsB is essential for in vitro and in vivo growth of a highly virulent food-borne pathogen, suggesting that GpsB could be a target for the future design of novel antibacterials.

Cyclic di-AMP targets the cystathionine beta-synthase domain of the osmolyte transporter OpuC.

Cellular turgor is of fundamental importance to bacterial growth and survival. Changes in external osmolarity as a consequence of fluctuating environmental conditions and colonization of diverse environments can significantly impact cytoplasmic water content, resulting in cellular lysis or plasmolysis. To ensure maintenance of appropriate cellular turgor, bacteria import ions and small organic osmolytes, deemed compatible solutes, to equilibrate cytoplasmic osmolarity with the extracellular environment. Here, we show that elevated levels of c-di-AMP, a ubiquitous second messenger among bacteria, result in significant susceptibility to elevated osmotic stress in the bacterial pathogen Listeria monocytogenes. We found that levels of import of the compatible solute carnitine show an inverse correlation with intracellular c-di-AMP content and that c-di-AMP directly binds to the CBS domain of the ATPase subunit of the carnitine importer OpuC. Biochemical and structural studies identify conserved residues required for this interaction and transport activity in bacterial cells. Overall, these studies reveal a role for c-di-AMP mediated regulation of compatible solute import and provide new insight into the molecular mechanisms by which this essential second messenger impacts bacterial physiology and adaptation to changing environmental conditions.

Discrete and overlapping functions of peptidoglycan synthases in growth, cell division and virulence of Listeria monocytogenes.

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with ß-lactam antibiotics. These target Listeria's five high molecular weight penicillin-binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi-functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross-linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)-PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod-shape and PBP B2 for cross-wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased ß-lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross-linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.

Assay for Listeria monocytogenes cells in whole blood using isotachophoresis and recombinase polymerase amplification.

We present a new approach which enables lysis, extraction, and detection of inactivated Listeria monocytogenes cells from blood using isotachophoresis (ITP) and recombinase polymerase amplification (RPA). We use an ITP-compatible alkaline and proteinase K approach for rapid and effective lysis. We then perform ITP purification to separate bacterial DNA from whole blood contaminants using a microfluidic device that processes 25 µL sample volume. Lysis, mixing, dispensing, and on-chip ITP purification are completed in a total of less than 50 min. We transfer extracted DNA directly into RPA master mix for isothermal incubation and detection, an additional 25 min. We first validate our assay in the detection of purified genomic DNA spiked into whole blood, and demonstrate a limit of detection of 16.7 fg µL-1 genomic DNA, the equivalent of 5 × 103 cells per mL. We then show detection of chemically-inactivated L. monocytogenes cells spiked into whole blood, and demonstrate a limit of detection of 2 × 104 cells per mL. Lastly, we show preliminary experimental data demonstrating the feasibility of the integration of ITP purification with RPA detection on a microfluidic chip. Our results suggest that ITP purification is compatible with RPA detection, and has potential to extend the applicability of RPA to whole blood.

RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP.

Cyclic di-AMP (cdiA) is a second messenger predicted to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intracellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore, a flow cytometry assay based on this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, we have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.

A function of DivIVA in Listeria monocytogenes division site selection.

The cell division protein DivIVA influences protein transport via the accessory SecA2 secretion route in Listeria monocytogenes. In contrast, DivIVA from the closely related bacterium Bacillus subtilis contributes to division site selection via the MinCDJ system. However, no classical min phenotype, i.e. filamentation and minicell production was observed with a listerial ΔdivIVA mutant. This has prompted the speculation that division site selection is DivIVA-independent in L. monocytogenes. We addressed this question with genetic, cytological and bacterial two-hybrid experiments and the data obtained correct this view. DivIVA not only binds to MinJ but also directly interacts with MinD. Experiments with fluorescently tagged proteins showed that localization of MinC and MinD was clearly DivIVA-dependent, whereas localization of MinJ was not. An impact of DivIVA on cell division was confirmed by careful comparisons of cell size distributions of divIVA and secA2 mutants. Gene deletion studies and epistasis experiments consistently reinforced these findings, and also revealed that MinJ must have a DivIVA-independent function. The frequency of minicell formation is low in L. monocytogenes min mutants. However, since listerial minicells might be useful as carriers for the introduction of therapeutic compounds into eukaryotic cells, we present a strategy how minicell frequency can be increased.

Microfluidic filtration and extraction of pathogens from food samples by hydrodynamic focusing and inertial lateral migration.

Detecting pathogenic bacteria in food or other biological samples with lab-on-a-chip (LOC) devices requires several sample preparation steps prior to analysis which commonly involves cleaning complex sample matrices of large debris. This often underestimated step is important to prevent these larger particles from clogging devices and to preserve initial concentrations when LOC techniques are used to concentrate or isolate smaller target microorganisms for downstream analysis. In this context, we developed a novel microfluidic system for membrane-free cleaning of biological samples from debris particles by combining hydrodynamic focusing and inertial lateral migration effects. The microfluidic device is fabricated using thermoplastic elastomers being compatible with thermoforming fabrication techniques leading to low-cost single-use devices. Microfluidic chip design and pumping protocols are optimized by investigating diffusive losses numerically with coupled Navier-Stokes and convective-diffusion theoretical models. Stability of inertial lateral migration and separation of debris is assessed through fluorescence microscopy measurements with labelled particles serving as a model system. Efficiency of debris cleaning is experimentally investigated by monitoring microchip outlets with in situ optical turbidity sensors, while retention of targeted pathogens (i.e., Listeria monocytogenes) within the sample stream is assessed through bacterial culture techniques. Optimized pumping protocols can remove up to 50 % of debris from ground beef samples while percentage for preserved microorganisms can account for 95 % in relatively clean samples. However, comparison between inoculated turbid and clean samples (i.e., with and without ground beef debris) indicate some degree of interference between debris inertial lateral migration and hydrodynamic focusing of small microorganisms. Although this interference can lead to significant decrease in chip performance through loss of target bacteria, it remains possible to reach 70 % for sample recovery and more than 50 % for debris removal even in the most turbid samples tested. Due to the relatively simple design, the robustness of the inertial migration effect itself, the high operational flow rates and fabrication methods that leverage low-cost materials, the proposed device can have an impact on a wide range of applications where high-throughput separation of particles and biological species is of interest.

Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.

Inactivation of the SecA2 protein export pathway in Listeria monocytogenes promotes cell aggregation, impacts biofilm architecture and induces biofilm formation in environmental condition.

Listeria monocytogenes has a dichotomous lifestyle, existing as an ubiquitous saprophytic species and as an opportunistic intracellular pathogen. Besides its capacity to grow in a wide range of environmental and stressful conditions, L. monocytogenes has the ability to adhere to and colonize surfaces. Morphotype variation to elongated cells forming rough colonies has been reported for different clinical and environmental isolates, including biofilms. This cell differentiation is mainly attributed to the reduced secretion of two SecA2-dependent cell-wall hydrolases, CwhA and MurA. SecA2 is a non-essential SecA paralogue forming an alternative translocase with the primary Sec translocon. Following investigation at temperatures relevant to its ecological niches, i.e. infection (37°C) and environmental (20°C) conditions, inactivation of this SecA2-only protein export pathway led, despite reduced adhesion, to the formation of filamentous biofilm with aerial structures. Compared to the wild type strain, inactivation of the SecA2 pathway promoted extensive cell aggregation and sedimentation. At ambient temperature, this effect was combined with the abrogation of cell motility resulting in elongated sedimented cells, which got knotted and entangled together in the course of filamentous-biofilm development. Such a cell differentiation provides a decisive advantage for listerial surface colonization under environmental condition. As further discussed, this morphotypic conversion has strong implication on listerial physiology and is also of potential significance for asymptomatic human/animal carriage.

Selection and characterization of DNA aptamers specific for Listeria species.

Single-stranded (ss) DNA aptamers with binding affinity to Listeria spp. were selected using a whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Listeria monocytogenes cells were grown at 37°C and harvested at mid-log phase or early stationary phase to serve as the targets in SELEX. A total of 10 unique aptamer sequences were identified, six associated with log phase cells and four with stationary phase cells. Binding affinity of the aptamers was determined using flow cytometry and ranged from 10% to 44%. Four candidates having high binding affinity were further studied and found to show genus-specific binding affinity when screened against five different species within the Listeria genus. Using sequential binding assays combined with flow cytometry, it was determined that three of the aptamers (LM6-2, LM12-6, and LM12-13) bound to one apparent cell surface moiety, while a fourth aptamer (LM6-116) appeared to bind to a different cell surface region. This is the first study in which SELEX targeted bacterial cells at different growth phases. When used together, aptamers that bind to different cell surface moieties could increase the analytical sensitivity of future capture and detection assays.

Tannin-rich fraction from pomegranate rind damages membrane of Listeria monocytogenes.

Pomegranate rind has been reported to inhibit several foodborne pathogens, and its antimicrobial activity has been attributed mainly to its tannin fraction. This study aimed to investigate the antimicrobial activity of the tannin-rich fraction from pomegranate rind (TFPR) against Listeria monocytogenes and its mechanism of action. The tannin-related components of TFPR were analyzed by high-performance liquid chromatography and liquid chromatography-mass spectrometry, and the minimum inhibitory concentration (MIC) of TFPR was determined using the agar dilution method. Extracellular potassium concentration, the release of cell constituents, intra- and extracellular ATP concentrations, membrane potential, and intracellular pH (pHin) were measured to elucidate a possible antibacterial mechanism. Punicalagin (64.2%, g/g) and ellagic acid (3.1%, g/g) were detected in TFPR, and the MICs of TFPR were determined to be 1.25-5.0 mg/mL for different L. monocytogenes strains. Treatment with TFPR induced a decrease of the intracellular ATP concentration, an increase of the extracellular concentrations of potassium and ATP, and the release of cell constituents. A reduction of pHin and cell membrane hyperpolarization were observed after treatment. Electron microscopic observations showed that the cell membrane structures of L. monocytogenes were apparently impaired by TFPR. It is concluded that TFPR could destroy the integrity of the cell membrane of L. monocytogenes, leading to a loss of cell homeostasis. These findings indicate that TFPR has the potential to be used as a food preservative in order to control L. monocytogenes contamination in food and reduce the risk of listeriosis.

Control of actin-based motility through localized actin binding.

A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it, if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disc. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young's modulus of the actin network and can explain several aspects of actin-based motility.