Induction Of Lysis Research Articles

The LuxO-OpaR quorum-sensing cascade differentially controls Vibriophage VP882 lysis-lysogeny decision making in liquid and on surfaces.

Quorum sensing (QS) is a process of cell-to-cell communication that bacteria use to synchronize collective behaviors. QS relies on the production, release, and group-wide detection of extracellular signaling molecules called autoinducers. Vibrios use two QS systems: the LuxO-OpaR circuit and the VqmA-VqmR circuit. Both QS circuits control group behaviors including biofilm formation and surface motility. The Vibrio parahaemolyticus temperate phage φVP882 encodes a VqmA homolog (called VqmAφ). When VqmAφ is produced by φVP882 lysogens, it binds to the host-produced autoinducer called DPO and launches the φVP882 lytic cascade. This activity times induction of lysis with high host cell density and presumably promotes maximal phage transmission to new cells. Here, we explore whether, in addition to induction from lysogeny, QS controls the initial establishment of lysogeny by φVP882 in naïve host cells. Using mutagenesis, phage infection assays, and phenotypic analyses, we show that φVP882 connects its initial lysis-lysogeny decision to both host cell density and whether the host resides in liquid or on a surface. Host cells in the low-cell-density QS state primarily undergo lysogenic conversion. The QS regulator LuxO~P promotes φVP882 lysogenic conversion of low-cell-density planktonic host cells. By contrast, the ScrABC surface-sensing system regulates lysogenic conversion of low-cell-density surface-associated host cells. ScrABC controls the abundance of the second messenger molecule cyclic diguanylate, which in turn, modulates motility. The scrABC operon is only expressed when its QS repressor, OpaR, is absent. Thus, at low cell density, QS-dependent derepression of scrABC drives lysogenic conversion in surface-associated host cells. These results demonstrate that φVP882 integrates cues from multiple sensory pathways into its lifestyle decision making upon infection of a new host cell.

Comparison of two viscoelastic testing devices in a porcine model of surgery, hemorrhage and resuscitation.

Viscoelastic hemostatic assays (VHA) are integral in contemporary hemostatic resuscitation, offering insights into clot formation, firmness, and lysis for rapid diagnosis and targeted therapy. Large animal models, particularly swine, provide anatomical and physiological analogies for coagulation research. Despite the growing use of VHAs, the ClotPro® device's applicability in porcine models remains unexplored. This study investigates ClotPro® in a porcine model of abdominal surgery, severe hemorrhage, and resuscitation, comparing it with the established ROTEM® delta system. Twenty-seven healthy pigs underwent abdominal surgery, hemorrhage and resuscitation. ClotPro® and ROTEM® were used to assess viscoelastic hemostatic properties at baseline, after surgery, 60min after shock induction, 60 and 120min after resuscitation. Clotting times in extrinsically and intrinsically stimulated assays exhibited fair to moderate correlation. Clot firmness in extrinsically stimulated tests could be used interchangeably while fibrin polymerization assays revealed significant differences between the devices. Fibrin polymerization assays in ClotPro® consistently yielded higher values than ROTEM®. Furthermore, the study evaluated the ClotPro® TPA-test's applicability in porcine blood, revealing failure of lysis induction in porcine blood samples. This research contributes valuable insights into the use of ClotPro® in porcine models of hemorrhage and coagulopathy, highlighting both its applicability and limitations in comparison to ROTEM® delta. The observed differences, especially in fibrin polymerization assays, emphasize the importance of understanding device-specific characteristics when interpreting results. Due to its inapplicability, TPA-test should not be used in porcine blood to evaluate fibrinolytic potential. The study provides a foundation for future investigations into the use of different viscoelastic hemostatic assays in porcine animal models.

Pfs230 Domain 7 is targeted by a potent malaria transmission-blocking monoclonal antibody

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that block Plasmodium parasite development in the mosquito midgut, thus preventing mosquitoes from becoming infectious. While the Pro-domain and first of fourteen 6-Cysteine domains (Pro-D1) of the Plasmodium gamete surface protein Pfs230 are known targets of transmission-blocking antibodies, no studies to date have discovered other Pfs230 domains that are functional targets. Here, we show that a murine monoclonal antibody (mAb), 18F25.1, targets Pfs230 Domain 7. We generated a subclass-switched complement-fixing variant, mAb 18F25.2a, using a CRISPR/Cas9-based hybridoma engineering method. This subclass-switched mAb 18F25.2a induced lysis of female gametes in vitro. Importantly, mAb 18F25.2a potently reduced P. falciparum infection of Anopheles stephensi mosquitoes in a complement-dependent manner, as assessed by standard membrane feeding assays. Together, our data identify Pfs230 Domain 7 as target for transmission-blocking antibodies and provide a strong incentive to study domains outside Pfs230Pro-D1 as TBV candidates.

The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy

Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of the encephalomyocarditis virus (EMCV) Leader protein, a ‘viral security protein’ that subverts the host antiviral response. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of a non-structural viral protein in the non-lytic release of picornaviruses via packaging in EVs.

THCz: Small molecules with antimicrobial activity that block cell wall lipid intermediates

Emerging antibiotic resistance demands identification of novel antibacterial compound classes. A bacterial whole-cell screen based on pneumococcal autolysin-mediated lysis induction was developed to identify potential bacterial cell wall synthesis inhibitors. A hit class comprising a 1-amino substituted tetrahydrocarbazole (THCz) scaffold, containing two essential amine groups, displayed bactericidal activity against a broad range of gram-positive and selected gram-negative pathogens in the low micromolar range. Mode of action studies revealed that THCz inhibit cell envelope synthesis by targeting undecaprenyl pyrophosphate-containing lipid intermediates and thus simultaneously inhibit peptidoglycan, teichoic acid, and polysaccharide capsule biosynthesis. Resistance did not readily develop invitro, and the ease of synthesizing and modifying these small molecules, as compared to natural lipid II-binding antibiotics, makes THCz promising scaffolds for development of cell wall-targeting antimicrobials.

The Phage T4 Antiholin RI Has a Cleavable Signal Peptide, Not a SAR Domain.

Holin/endolysin-mediated lysis of phage T4 of Escherichia coli is tightly regulated by the antiholins RI and RIII. While regulation by the cytoplasmic RIII plays a minor role, the periplasmic antiholin RI binds tightly to the holin T and is believed to directly sense periplasmic phage DNA from superinfections as a trigger for the inhibition of lysis. RI has been reported to contain a non-cleavable signal peptide that anchors the protein to the membrane. Lysis is believed to be induced at some stage by a membrane depolarization that causes a release of RI into the periplasm without cleavage of the signal anchor. For the current model of phage lysis induction, it is thus a fundamental assumption that the N-terminal trans-membrane domain (TMD) of RI is such a signal anchor release (SAR) domain. Here we show that, in contrast to previous reports, this domain of RI is a cleavable signal peptide. RI is processed and released into the periplasm as a mature protein, and inactivation of its signal peptidase cleavage site blocks processing and membrane release. The signal peptide of RI can also mediate the normal translocation of a well-characterized Sec substrate, PhoA, into the periplasm. This simplifies the current view of phage lysis regulation and suggests a fundamentally different interpretation of the recently published structure of the soluble domains of the RI–T complex.

Bacteriophage therapy for challenging bacterial infections: achievements, limitations and prospects for future clinical use by veterinary dermatologists.

Bacteriophages were discovered just over 100 years ago and have been used to treat bacterial infections in animals since the 1920s. The antimicrobial resistance crisis has led to a new surge of interest in the use of bacteriophage therapy as an alternative or supplement to antimicrobial therapy in humans and other animals. To describe the nature of bacteriophages and provide a critical review and update on the clinical use of bacteriophages in the treatment of challenging bacterial infections, with an emphasis on companion animal veterinary applications. The scientific literature on the subject was critically evaluated. Findings from the most pertinent papers have been presented in summary form and critiqued. Over the last 20 years there has been a considerable increase in the volume and quality of publications dealing with bacteriophage therapy. Some recent papers build on excellent work published in the 1980s and describe promising veterinary applications. Challenges related particularly to the registration and approval of phage remedies will need to be overcome before phage therapy can become a mainstream tool for use in veterinary settings. Considerably more research, particularly controlled clinical trials, needs to be done. Bacteriophage therapy is one of the most promising approaches to tackling the looming antimicrobial resistance crisis, yet substantial regulatory challenges will need to be overcome before it enters widespread use. Phage therapy also may, in the future, improve the management of challenging bacterial infections that are not necessarily multidrug-resistant.

Growth suppression and apoptosis-like cell death in Microcystis aeruginosa by H2O2: A new insight into extracellular and intracellular damage pathways

H2O2 has been suggested and applied as effective algaecide for harmful cyanobacterial bloom control, however, the transport of exogenous H2O2 into microalgal cells, the subsequent intracellular damage pathway and dose-response variations were little studied. We addressed these questions in a bloom-forming cyanobacterium Microcystis aeruginosa with H2O2 at 0.1–1.5 mM. The results showed that H2O2 at 0.4 mM and above significantly suppressed M. aeruginosa growth for over two weeks, and induced apoptosis-like death in terms of membrane potential dissipation, caspase-3 activation, chromatin condensation, and lysis induction. However, the dose-response effects were not monotonic. H2O2 at 0.7 mM resulted in the severest growth suppression among 0.1–1.5 mM treatments, including the lowest biomass for 74% loss, the highest cell lysis ratio for 79%, and the highest utilization rate of H2O2 for 0.101 mM d−1. Moreover, several evidence point to severer apoptosis-like cell death in 0.7 mM treatments, involving fastest and severest cell lysis, smallest cell size and wrinkled surface and lowest membrane potential. Therefore, the apoptosis-like cell death induced by H2O2 at moderate dosages should be a crucial cause for the non-monotonic dose-response effects on growth suppression. Additionally, intracellular H2O2 level increased rapidly within 20 min after exposure at 0.4 mM and above, directly confirming the transport of exogenous H2O2 into M. aeruginosa cells and the intracellular damages due to subsequent elevation in intracellular oxidative stress. The study demonstrates that H2O2 at moderate dosages could be a promising method for the biomass control, in a fast and efficient way, on M. aeruginosa blooms.

Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute to Campylobacter jejuni Biofilm Formation.

Campylobacter jejuni is a microaerophilic bacterium and is believed to persist in a biofilm to antagonize environmental stress. This study investigated the influence of environmental conditions on the formation of C. jejuni biofilm. We report an extracellular DNA (eDNA)-mediated mechanism of biofilm formation in response to aerobic and starvation stress. The eDNA was determined to represent a major form of constitutional material of C. jejuni biofilms and to be closely associated with bacterial lysis. Deletion mutation of the stress response genes spoT and recA enhanced the aerobic influence by stimulating lysis and increasing eDNA release. Flagella were also involved in biofilm formation but mainly contributed to attachment rather than induction of lysis. The addition of genomic DNA from either Campylobacter or Salmonella resulted in a concentration-dependent stimulation effect on biofilm formation, but the effect was not due to forming a precoating DNA layer. Enzymatic degradation of DNA by DNase I disrupted C. jejuni biofilm. In a dual-species biofilm, eDNA allocated Campylobacter and Salmonella at distinct spatial locations that protect Campylobacter from oxygen stress. Our findings demonstrated an essential role and multiple functions of eDNA in biofilm formation of C. jejuni, including facilitating initial attachment, establishing and maintaining biofilm, and allocating bacterial cells.IMPORTANCECampylobacter jejuni is a major cause of foodborne illness worldwide. In the natural environment, the growth of C. jejuni is greatly inhibited by various forms of environmental stress, such as aerobic stress and starvation stress. Biofilm formation can facilitate the distribution of C. jejuni by enabling the survival of this fragile microorganism under unfavorable conditions. However, the mechanism of C. jejuni biofilm formation in response to environmental stress has been investigated only partially. The significance of our research is in identifying extracellular DNA released by bacterial lysis as a major form of constitution material that mediates the formation of C. jejuni biofilm in response to environmental stress, which enhances our understanding of the formation mechanism of C. jejuni biofilm. This knowledge can aid the development of intervention strategies to limit the distribution of C. jejuni.

The induction of lysis in lysogenic strains of Escherichia coli by a new antitumor transplatin derivative and its DNA interactions.

The toxicity of the new derivative of transplatin, namely trans-[PtCl2(DEA)(NH3)] (DEA = diethylamine), in which only one NH3 group was replaced by a small, non-bulky DEA ligand, in the cisplatin sensitive and resistant tumor cell lines was examined. The results indicate that this very small modification of the transplatin molecule results in a considerable enhancement of toxicity in the cancer cells. Thus, this finding is consistent with the thesis that the trans geometry in Pt(II)-dichlorido compounds can also be effectively activated by the replacement of only one NH3 ligand in transplatin by the non-bulky ligand, such as the short aliphatic amine, (C2H5)2NH. We also demonstrate that trans-[PtCl2(DEA)(NH3)], in contrast to transplatin, can be grouped with the coordination compounds exhibiting antitumor activity and capable of inducing lysis in lysogenic bacteria. Thus, these results afford, for the first time, experimental support for the view that DNA is the potential cellular target also for antitumor derivatives of transplatin. DNA binding mode of trans-[PtCl2(DEA)(NH3)] in cell-free media was examined as well. The results show that the small aliphatic DEA ligand has significant consequences for the DNA conformational changes. Unlike 'classical' transplatin, modification of DNA with trans-[PtCl2(DEA)(NH3)] leads to mainly bifunctional cross-links. The extent and destabilization of the double-helical structure of DNA by this new trans-platinum complex are similar to those induced by cisplatin. As a consequence the lesions effectively inhibit transcription of template DNA similarly to the lesions of cisplatin, but markedly more than the lesions of transplatin. The stronger inhibition of DNA transcription by the adducts of trans-[PtCl2(DEA)(NH3)] in comparison with the adducts of transplatin adds a new dimension to the impact of the activated trans geometry in platinum compounds on biological processes, possibly including DNA transcription.

Characterization of Lysis of the Multicellular CyanobacteriumLimnothrix/Pseudanabaenasp. Strain ABRG5-3

The cyanobacterium semi-filamentous multicellular strain ABRG5-3 undergoes cell lysis as a unique feature that occurs due to growth condition changes from normal cultivation with shaking to static cultivation without shaking in liquid culture (Nishizawa et al., 2010). Microscopic observation and energy dispersive X-ray spectrometer (EDX) analysis have revealed that lysis is involved in the accumulation of polyphosphate compounds and the disintegration of thylakoid membranes in cells. Static cultivation, dark or red light exposure, and temperature (22 to 42 °C) conditions were found to be effective factors for the induction of lysis. Moreover, stress induced by salts, osmotic pressure with sucrose, and the depletion of nitrogen or phosphate in cultures also induced ABRG5-3 cell lysis. Based on these results, we discuss lysis and its utilization in the biotechnology industry.

A bacteriophage detection tool for viability assessment of Salmonella cells

Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods.

Mechanisms of tumor immune escape in triple-negative breast cancers (TNBC) with and without mutated BRCA 1

Triple negative breast cancers (TNBC) are associated with an adverse outcome, although these tumors are sensitive to chemotherapy. In part, this phenomenon could be caused by tumor immune escape. The current study investigates immunogenicity of TNBC cells in vitro and the presence of immunosuppressive factors in the tumor microenvironment (pAKT and B7H1 expression, infiltration with regulatory T cells, [Tregs]). Natural killer (NK)-cell induced lysis was evaluated in estrogen receptor (ER) positive MCF 7 breast cancers, in MDA-MB231 and MDA-MB468 and in HCC-1937 (BRCA 1 mutated) and HCC-1806 TNBC cells. Expression of pAKT, B7H1 and infiltration with Tregs were determined by immunohistochemistry in human specimens of benign and malignant breast disease. NK-cell induced lysis was significantly increased (p < 0.05) in four TNBC cell lines compared to ER + MCF 7 cells. Fibroadenomas and mastectomy samples were not infiltrated with Tregs. Infiltration with Tregs was 0.92 ± 0.21 in ER/PR + breast cancers and significantly higher in TNBC without (2.30 ± 0.34) and also significantly higher with mutation of BRCA 1 (2.10 ± 0.34). Expression of pAKT was absent in benign controls and 1.23 ± 0.36 in ER/PR + breast cancers, 1.78 ± 0.40 in TNBC without and 2.40 ± 0.30 with mutated BRCA 1. No significant differences of B7H1 expression occurred among the breast cancer subgroups. TNBC cell stimulate the NK-cell immune response significantly stronger than ER positive breast cancer cells. This could explain why infiltration with immunosuppressive Tregs is increased in human specimens of TNBC with and without mutated BRCA 1. Accordingly, immunomodulatory treatment strategies should be further explored in TNBC.

Membrane stabilization as a mechanism of the anti-inflammatory activity of methanol extract of garden egg (Solanum aethiopicum)

BackgroundSome observations and reports show that people with high consumption of Solanum aethiopicum (African garden egg) have relief in arthritic pains and swelling. We aimed at assessing the effect of methanol extract of Solanum aethiopicum in experimentally induced inflammation using leukocyte mobilization and vascular permeability tests in rats and human red blood cell (HRBC) membrane stabilization as studies.MethodsTwenty five (25) adult Wistar rats of either sex (120 g – 200 g) divided into five groups of five rats each were used for each of the animal models. Groups 2, 3 and 4 were administered varied doses of the extract (100, 200 and 400 mg/kg), while groups 1 (vehicle control) and 5 (treatment control) received normal saline and indomethacin (50 mg/kg) respectively. Vascular permeability was induced by the intra-peritoneal injection of 1 ml of acetic acid and monitored using 0.5 ml intravenous injection of 1% Evans blue solution. Leukocyte mobilization was induced by the intra-peritoneal injection of 0.5 ml of 3% agar suspension in normal saline. Heat and hypotonicity induced heamolysis of HRBC membrane was used to assess membrane stabilization.ResultsThe methanol extracts of garden egg significantly and dose dependently reduced (p≤0.05) the acetic acid induced vascular permeability and agar induced leukocyte mobilization in rats. The percentage inhibitions of induced vascular permeability were 21 ± 3.39, 25 ±1.92 and 60 ± 3.81 for the 100, 200 and 400 mg/kg of the extract while the inhibitions of the agar induced leucocyte migration were 23 ± 2.17, 26 ± 1.58 and 32 ± 1.58 for the 100, 200 and 400 mg/kg of the extract respectively. The extract also, at doses of 100, 200, 400, 600 and 800 μg/ml significantly inhibited heat induced lysis of the human red cell membrane with values of 66.46 ± 2.89, 65.14 ± 4.58, 46.53 ± 2.52, 61.88 ± 4.51and 86.67 ± 3.06 respectively.ConclusionsThese results show that methanol extract of Solanum aethiopicum has anti-inflammatory properties and can reduce inflammatory injury and tissue damage.

Global analysis of host response to induction of a latent bacteriophage

BackgroundThe transition from viral latency to lytic growth involves complex interactions among host and viral factors, and the extent to which host physiology is buffered from the virus during induction of lysis is not known. A reasonable hypothesis is that the virus should be evolutionarily selected to ensure host health throughout induction to minimize its chance of reproductive failure. To address this question, we collected transcriptional profiles of Escherichia coli and bacteriophage lambda throughout lysogenic induction by UV light.ResultsWe observed a temporally coordinated program of phage gene expression, with distinct early, middle and late transcriptional classes. Our study confirmed known host-phage interactions of induction of the heat shock regulon, escape replication, and suppression of genes involved in cell division and initiation of replication. We identified 728 E. coli genes responsive to prophage induction, which included pleiotropic stress response pathways, the Arc and Cpx regulons, and global regulators crp and lrp. Several hundred genes involved in central metabolism, energy metabolism, translation and transport were down-regulated late in induction. Though statistically significant, most of the changes in these genes were mild, with only 140 genes showing greater than two-fold change.ConclusionOverall, we observe that prophage induction has a surprisingly low impact on host physiology. This study provides the first global dynamic picture of how host processes respond to lambda phage induction.

Control of piglet coccidiosis by chemical disinfection with a cresol-based product (Neopredisan 135-1®)

Isospora suis is a common pathogen in piggeries and one of the main causative agents of scours in suckling piglets. Besides specific treatment, optimised hygiene including chemical disinfection is considered essential in the control of isosporosis. The suitability of the cresol-based product Neopredisan 135-1(R) (NP) to inactivate oocysts in vitro and to reduce infection pressure in commercial piggeries was evaluated. Under in vitro conditions, NP at a final concentration of 2 or 4% induced lysis of more than 95% of sporulated oocysts at a contact time of 30 min and destroyed all oocysts after a contact time of 90 min or more. A total of six trials (T1-T6) were performed on two farms (I and II). T5 was split into two parts, T5/1 and T5/2. Two groups of litters kept in farrowing crates either disinfected conventionally before farrowing (controls, group C) or disinfected with 4% dilution of NP before farrowing and with 2% NP one to three times thereafter (group NP) were compared in each trial. Altogether, 81 litters were randomly allocated to group NP and 77 litters to group C (comprising a total of 1,465 piglets). Piglet faeces were collected individually 5 days after birth and six times thereafter in intervals of 2 or 3 days from four piglets per litter and microscopically examined for oocysts of I. suis. Diarrhoea scores, other clinical data (skin turgidity, coat length etc.), weights and loss of piglets until weaning were recorded. One trial (T3) could not be analysed because of insufficient cleaning before disinfection. In group C, litter prevalence of I. suis ranged between 40 and 80%. The proportion of positive litters was considerably reduced by approximately 50% in disinfected crates except for one trial, and the number of affected piglets decreased by up to 80%. Diarrhoea and oocyst excretion were significantly associated. Diarrhoea was less frequently observed in disinfected crates. In general, isosporosis appeared mild to subclinical, and no significant effects of disinfection on other clinical data, weight gain and number of weaned piglets were noted. It is concluded that NP efficiently inactivates oocysts of I. suis, and that additional disinfection after farrowing is suited to reduce infection pressure. No clear relation of infection prevalence to the frequency of intermediate disinfection (one, two or three times) was seen, and thus, single intermediate disinfection 1 week after farrowing is considered sufficient.

Lytr, a phage-derived amidase is most effective in induced lysis of Lactococcus lactis compared with other lactococcal amidases and glucosaminidases

In the genome of Lactococcus lactis IL1403 five genes encoding peptidoglycan hydrolases are present: four glucosaminidases ( acmA, acmB, acmC and acmD) and an endopeptidase ( yjgB). Genes for six prophage lysins have also been identified. The genes acmB, acmC, acmD, yjgB and the lysin lytR of prophage r1t and the lytic gene of the prophage bIL309 were cloned downstream of the nisin-inducible promoter in L. lactis NZ9000. Upon nisin induction, increased lysis of the expressing strain was obtained in all cases. Most lysis was obtained with AcmC and the two prophage lysins. Moreover, lysis by AcmB, AcmD and YjgB depends on the presence of the autolysin AcmA. The genes lytR, acmD and acmD in an operon-like structure with acmA were expressed in a foodgrade manner in a L. lactis strain that was used in conjunction with a starter to make Gouda-type cheese. Overexpression of lytR or the combination acmA/acmD resulted in increased cellular lysis in the cheese compared to the reference culture. LytR induced lysis was delayed but eventually resulted in increased lysis in trans of the acidifying L. lactis strain 13 M.

Altering Renneting pH Changes Microstructure, Cell Distribution, and Lysis of Lactococcus lactis AM2 in Cheese Made from Ultrafiltered Milk

The objective of this study was to investigate the lysis of a highly autolytic strain of Lactococcus lactis ssp. cremoris AM2 in a model cheese made from concentrated ultrafiltered milk. From the same initial ultrafiltered retentate inoculated with L. lactis AM2, 5 cheeses were made by the addition of rennet at different pH values (6.6, 6.2, 5.8, 5.4, and 5.2). Lysis was monitored by measurement of the release of lactate dehydrogenase, an intracellular marker enzyme, and by immunodetection of intracellular proteins with species-specific antibodies. Confocal scanning laser microscopy (CSLM) was used to investigate the cheese microstructure by staining for protein and fat. Dual staining with a bacterial viability kit with CSLM was performed to reveal the integrity and localization of the bacterial cells. Levels of soluble calcium significantly increased when the pH at which the rennet was added decreased. In cheese renneted at pH 6.6, CSLM revealed an open porous structure containing a dense protein network with fat globules of different sizes distributed in the aqueous phase. In cheese renneted at pH 5.2, the protein network was homogeneous, with a less dense protein network, and an even distribution of fat globules. On d 1, bacterial cells were organized into colonies in cheese renneted at pH 6.6, whereas in cheeses renneted at pH 5.2, bacteria were evenly dispersed as single cells throughout the protein network. Lysis was detected on d 1 in cheeses renneted at high pH values and continued to increase throughout ripening, whereas induction of lysis was delayed in cheeses renneted at lower pH values until the end of ripening. This study demonstrates that alterations in the microstructure of the cheese and the distribution of cells play a role in lysis induction of L. lactis AM2.

59. Development of Conditionally Replicative Adenoviruses as Platforms for the Expression of shRNAs or Therapeutic Genes

Conditionally replicative adenoviruses (CRAds) represent a novel class of anticancer agents, designed to selectively replicate in tumor cells and to destroy these cells by the induction of lysis. We aim to enhance CRAd oncolytic potency by suppressing apoptosis inhibitors or by expressing toxic proteins in CRAd-infected cancer cells. Towards the first goal, we explored the utility of RNA interference (RNAi) in the context of CRAd replication in cancer cells. RNAi is the sequence-specific gene-silencing induced by double-stranded RNA (dsRNA) and is now widely used to study the function of individual genes. By virtue of its excellent target specificity, RNAi also holds great promise as a new anticancer modality. The success of this approach largely depends on efficient delivery of dsRNA to tumor cells. As a proof of principle, we constructed CRAds encoding short hairpin RNAs (shRNAs) targeted against firefly luciferase and assessed their silencing efficiency. These replicating viruses silenced firefly luciferase expression relative to control virus in the four human cancer cell lines tested. In a time course experiment, the expression inhibition was especially evident during later stages of infection, reaching a maximum of approximately 70% silencing at the time when CPE became apparent. The CRAd-shRNA induced silencing was not observed in an experiment using a reporter plasmid with point mutations in the target site of the shRNA, confirming the sequence-specificity of the silencing effect. Thus, successful RNAi was accomplished.

Saturated free fatty acid release and intracellular ceramide generation during apoptosis induction are closely related processes

Apoptosis induced by cells from the immune system is frequently associated with an increase in the ceramide content of target cells, due to the activation of sphingomyelinases (SMase). Some studies have also reported the release of saturated and monounsaturated free fatty acids (FFA) from apoptotic cells. However, the possible relationship between these lipid biochemistry events has not been characterized. We have analysed for the first time the release of FFA triggered by tumor necrosis factor-α (TNF-α), Fas/CD95 or the perforin/granzyme system of cytotoxic T lymphocytes (CTL) and their relationship to intracellular ceramide generation. TNF-α- and Fas-induced apoptosis are associated with both intracellular ceramide generation from sphingomyelin (SM) and release of palmitic-derived FFA, with similar kinetics. Intracellular SMase activation and FFA release from target cells during Fas-induced apoptosis are much more rapid and efficient if Fas-based cytotoxicity is exerted by alloantigenic CTL. In the case of perforin/granzyme-based cytotoxicity exerted by CTL, intracellular ceramide generation and FFA release from target cells seem to depend on the type of lysis induction used. Importantly, the correlation between intracellular SMase activation and the release of palmitic acid-derived FFA from target cells has been observed in all types of cytotoxicity assayed. In addition, exogenous natural ceramide induces the rapid release of the same FFA, well before any apoptotic sign is detected, and FFA release during Fas-induced apoptosis is inhibited in SM-depleted cells by chronic fumonisin-B 1 treatment. These results demonstrate a novel connection between the release of palmitic acid-derived FFA and intracellular ceramide accumulation during apoptosis induction.