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Understanding the relationship between population genetic structure and phenotypic diversity is a fundamental question in evolutionary biology. Yeasts display wide genetic diversity and exhibit remarkably diverse heterotrophic metabolisms that allow a variety of niche occupations. However, little is known about how intra-species genetic population structure is related to trait diversity in yeasts. In this study, we investigated the link between intra-species genetic population structure and trait diversity in the floral nectar-inhabiting yeast Metschnikowia reukaufii (Ascomycota). A total of 73 strains obtained from 11 plant species were genotyped by whole genome sequencing, followed by single nucleotide polymorphism (SNP) calling, and phenotyped using a robot-assisted high-throughput screening platform. Analysis of the population structure estimated the number of ancestral populations to be K = 5, each one including strains from different locations and host plants, and 26% of strains showed significant genetic admixture (<80% ancestry from a single population). These mosaic strains were scattered throughout a maximum-likelihood phylogenetic tree built from SNP data, and differed widely in their ancestry. While yeast strains varied in nutrient assimilation and tolerance to inhibitors, trait differentiation among genetic lineages was in most cases negligible. Notably, outlier phenotypes largely corresponded to the mosaic strains, and removal of these from the data had a dramatic effect on the intra-species phylogenetic signal of studied phenotypes and patterns of trait evolution. Overall, these results suggest that genetic mosaicism broadens the phenotypic landscape explored by M. reukaufii and may allow adaptation to highly variable nectar environments. [ABSTRACT FROM AUTHOR]

The global increase in antibiotic resistances demands for additional efforts to identify novel antimicrobials such as bacteriocins. These antimicrobial peptides of bacterial origin are already used widely in food preservation and promising alternatives for antibiotics in animal feed and some clinical setting. Identification of novel antimicrobials is facilitated by appropriate high throughput screening (HTS) methods. Previously, we have described a rapid, simple and cost-efficient assay based on live biosensor bacteria for detection of antimicrobial compounds that act on membrane integrity using the ratiometric pH-dependent fluorescent protein pHluorin2 (pHin2). Here, we use these biosensors to develop an integrated pipeline for high-throughput identification of bacteriocin producers and their biosynthetic gene clusters. We extend the existing portfolio of biosensors by generating pHin2 expressing strains of Escherichia coli , Bacillus cereus , Staphylococcus epidermidis , and methicillin-resistant Staphylococcus aureus . These strains were characterized, and control experiments were performed to assess heterogeneity of these biosensors in response to known bacteriocins and develop a robust HTS system. To allow detection of compounds that inhibit target bacteria by inhibiting growth without disturbing membrane integrity, the HTS system was extended with a growth-dependent readout. Using this HTS system, we screened supernatants of a total of 395 strains of a collection of lactic acid bacteria. After two rounds of screening 19 strains of the collection were identified that produced antimicrobial activity against Listeria innocua and Listeria monocytogenes . Genomes of confirmed hits were sequenced and annotated. In silico analysis revealed that the identified strains encode between one and six biosynthetic gene clusters (BGCs) for bacteriocins. Our results suggest that pHin2 biosensors provides a flexible, cheap, fast, robust and easy to handle HTS system for identification of potential bacteriocins and their BGCs in large strain collections., Competing Interests: Authors LT and TN were employed by NovaTaste Production GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Otto, Teichmann, Fante, Crauwels, Grünberger, Neddermann and Riedel.)

The global increase in antibiotic resistance of pathogenic microorganisms requires the identification and characterization of novel antimicrobials. Bacterial biosensors expressing fluorescent proteins such as pHluorin variants are suitable for high-throughput screenings. Here, we present Listeria spp. pH-sensitive biosensors with improved fluorescence for single-cell analysis of antimicrobials by flow cytometry., (© 2022 The Authors. Microbiology Open published by John Wiley & Sons Ltd.)

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACD Cgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

TLR5 ligand flagellin-containing fusion proteins are potential vaccine candidates for many diseases. A recombinant fusion protein of flagellin A and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) modulates immune responses in vitro and in vivo. We studied the effects of rFlaA:Betv1 on bone marrow-derived macrophages (BMDMs). BMDMs differentiated from BALB/c, C57BL/6, TLR5 -/- , or MyD88 -/- mice were pre-treated with inhibitors, stimulated with rFlaA:Betv1 or respective controls, and analyzed for activation, cytokine secretion, metabolic state, RNA transcriptome, and modulation of allergen-specific Th2 responses. Stimulation of BMDMs with rFlaA:Betv1 resulted in MyD88-dependent production of IL-1β, IL-6, TNF-α, IL-10, CD69 upregulation, and a pronounced shift towards glycolysis paralleled by activation of MAPK, NF κ B, and mTOR signaling. Inhibition of either mTOR (rapamycin) or SAP/JNK-MAPK signaling (SP600125) resulted in dose-dependent metabolic suppression. In BMDM and T cell co-cultures, rFlaA:Betv1 stimulation suppressed rBet v 1-induced IL-5 and IL-13 secretion while inducing IFN-γ production. mRNA-Seq analyses showed HIF-1a, JAK, STAT, phagosome, NLR, NF κ B, TNF, TLR, and chemokine signaling to participate in the interplay of cell activation, glycolysis, and immune response. rFlaA:Betv1 strongly activated BMDMs, resulting in MyD88-, MAPK-, and mTOR-dependent enhancement of glucose metabolism. Our results suggest macrophages are important target cells to consider during restauration of allergen tolerance during AIT.

Listeria monocytogenes is an important food-borne pathogen and a serious concern to food industries. Bacteriocins are antimicrobial peptides produced naturally by a wide range of bacteria mostly belonging to the group of lactic acid bacteria (LAB), which also comprises many strains used as starter cultures or probiotic supplements. Consequently, multifunctional strains that produce bacteriocins are an attractive approach to combine a green-label approach for food preservation with an important probiotic trait. Here, a collection of bacterial isolates from raw cow's milk was typed by 16S rRNA gene sequencing and MALDI-Biotyping and supernatants were screened for the production of antimicrobial compounds. Screening was performed with live Listeria monocytogenes biosensors using a growth-dependent assay and pHluorin, a pH-dependent protein reporting membrane damage. Purification by cation exchange chromatography and further investigation of the active compounds in supernatants of two isolates belonging to the species Pediococcus acidilactici and Lactococcus garvieae suggest that their antimicrobial activity is related to heat-stable proteins/peptides that presumably belong to the class IIa bacteriocins. In conclusion, we present a pipeline of methods for high-throughput screening of strain libraries for potential starter cultures and probiotics producing antimicrobial compounds and their identification and analysis.

The effects of environmental stresses on microorganisms have been well-studied, and cellular responses to stresses such as heat, cold, acids, and salts have been extensively discussed. Although high pressure processing (HPP) is becoming more popular as a preservation method in the food industry, the characteristics of the cellular damage caused by high pressure are unclear, and the microbial response to this stress has not yet been well-explored. We exposed the pathogen Listeria monocytogenes to HPP (400 MPa, 8 min, 8°C) and found that the high pressure created plasma membrane pores. Using a common staining technique involving propidium iodide (PI) combined with high-frequency fluorescence microscopy, we monitored the rate of diffusion of PI molecules into hundreds of bacterial cells through these pores on days 0, 1, 2, 3, and 4 after pressurization. We also developed a mathematical dynamic model based on mass transfer and passive diffusion laws, calibrated using our microscopy experiments, to evaluate the response of bacteria to HPP. We found that the rate of diffusion of PI into the cells decreased over the 4 consecutive days after exposure to HPP, indicating repair of the pressure-created membrane pores. The model suggested a temporal change in the size of pores until closure. To the best of our knowledge, this is the first time that pressure-created membrane pores have been quantitatively described and shown to diminish with time. In addition, we found that the membrane repair rate in response to HPP was linear, and growth was temporarily arrested at the population level during the repair period. These results support the existence of a progressive repair process in some of the cells that take up PI, which can therefore be considered as being sub-lethally injured rather than dead. Hence, we showed that a subgroup of bacteria survived HPP and actively repaired their membrane pores., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nikparvar, Subires, Capellas, Hernandez-Herrero, Crauwels, Riedel and Bar.)

In cutaneous Leishmaniasis the parasitic control in human host macrophages is still poorly understood. We found an increased expression of the human cathelicidin CAMP in skin lesions of Ethiopian patients with cutaneous leishmaniasis. Vitamin D driven, Cathelicidin-type antimicrobial peptides (CAMP) play an important role in the elimination of invading microorganisms. Recombinant cathelicidin was able to induce cell-death characteristics in Leishmania in a dose dependent manner. Using human primary macrophages, we demonstrated pro-inflammatory macrophages (hMDM1) to express a higher level of human cathelicidin, both on gene and protein level, compared to anti-inflammatory macrophages (hMDM2). Activating the CAMP pathway using Vitamin D in hMDM1 resulted in a cathelicidin-mediated- Leishmania restriction. Finally, a reduction of cathelicidin in hMDM1, using a RNA interference (RNAi) approach, increased Leishmania parasite survival. In all, these data show the human cathelicidin to contribute to the innate immune response against Leishmaniasis in a human primary cell model., (Copyright © 2019 Crauwels, Bank, Walber, Wenzel, Agerberth, Chanyalew, Abebe, König, Ritter, Reiling and van Zandbergen.)

Phagocytosis is a cellular process crucial for recognition and removal of apoptotic cells and foreign particles, subsequently initiating appropriate immune responses. The process of phagocytosis is highly complex and involves major rearrangements of the cytoskeleton. Due to its complexity and importance for tissue homoeostasis and immune responses, it is tightly regulated. Over the last decade, microRNAs (miRNAs) have emerged as important regulators of biological pathways including the immune response by fine-tuning expression of gene regulatory networks. In order to identify miRNAs implicated in the regulation of phagocytosis, a systematic screening of all currently known, human miRNAs was performed using THP-1 macrophage-like cells and serum-opsonized latex beads. Of the total of 2,566 miRNAs analyzed, several led to significant changes in phagocytosis. Among these, we validated miR-124-5p as a novel regulator of phagocytosis. Transfection with miR-124-5p mimics reduced the number of phagocytic cells as well as the phagocytic activity of phorbol-12-myristate-13-acetate (PMA)-activated THP-1 cells and ex vivo differentiated primary human macrophages. In silico analysis suggested that miR-124-5p targets genes involved in regulation of the actin cytoskeleton. Transcriptional analyses revealed that expression of genes encoding for several subunits of the ARP2/3 complex, a crucial regulator of actin polymerization, is reduced upon transfection of cells with miR-124-5p. Further in silico analyses identified potential binding motifs for miR-124-5p in the mRNAs of these genes. Luciferase reporter assays using these binding motifs indicate that at least two of the genes ( ARPC3 and ARPC4 ) are direct targets of miR-124-5p. Moreover, ARPC3 and ARPC4 protein levels were significantly reduced following miR-124-5p transfection. Collectively, the presented results suggest that miR-124-5p regulates phagocytosis in human macrophages by directly targeting expression of components of the ARP2/3 complex., (Copyright © 2019 Herdoiza Padilla, Crauwels, Bergner, Wiederspohn, Förstner, Rinas, Ruf, Kleemann, Handrick, Tuckermann, Otte, Walther and Riedel.)

Listeria monocytogenes is a food-borne human pathogen and a serious concern in food production and preservation. Previous studies have shown that biofilm formation of L. monocytogenes and presence of extracellular DNA (eDNA) in the biofilm matrix varies with environmental conditions and may involve agr peptide sensing. Experiments in normal and diluted (hypoosmotic) complex media at different temperatures revealed reduced biofilm formation of L. monocytogenes EGD-e ΔagrD, a mutant deficient in agr peptide sensing, specifically in diluted Brain Heart Infusion at 25°C. This defect was not related to reduced sensitivity to DNase treatment suggesting sufficient levels of eDNA. Re-analysis of a previously published transcriptional profiling indicated that a total of 132 stress-related genes, that is 78.6% of the SigB-dependent stress regulon, are differentially expressed in the ΔagrD mutant. Additionally, a number of genes involved in flagellar motility and a large number of other surface proteins including internalins, peptidoglycan binding and cell wall modifying proteins showed agr-dependent gene expression. However, survival of the ΔagrD mutant in hypoosmotic conditions or following exposure to high hydrostatic pressure was comparable to the wild type. Also, flagellar motility and surface hydrophobicity were not affected. However, the ΔagrD mutant displayed a significantly reduced viability upon challenge with lysozyme. These results suggest that the biofilm phenotype of the ΔagrD mutant is not a consequence of reduced resistance to hypoosmotic or high pressure stress, motility or surface hydrophobicity. Instead, agr peptide sensing seems to be required for proper regulation of biosynthesis, structure and function of the cell envelope, adhesion to the substratum, and/or interaction of bacteria within a biofilm., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Bacteriocins are antimicrobial peptides naturally produced by many bacteria and were shown to be effective against various pathogens including Listeria monocytogenes . L. monocytogenes is a food-borne pathogen that frequently causes disease outbreaks around the world with fatal outcomes in at-risk individuals. Thus, bacteriocins are a promising solution to prevent contaminations with L. monocytogenes and other microorganisms during food production and preservation. In the present study, we constructed L. monocytogenes EGD-e/pNZ-P help -pHluorin, a strain that constitutively expresses the pH-sensitive fluorescent protein pHluorin, as a sensor strain to detect disruption of the pH gradient by the membrane-damaging activity of bacteriocins. The ratiometric fluorescence properties of pHluorin were validated both in crude extracts and permeabilized cells of this sensor strain. L. monocytogenes EGD-e/pNZ-P help -pHluorin was used to assess membrane damaging activity of the bacteriocins nisin A and pediocin PA-1 and to determine the minimal concentrations required for full disruption of the pH gradient across the membrane. Moreover, the sensor strain proved useful to analyze the presence of compounds affecting membrane integrity in supernatants of a nisin Z-producing Lactococcus lactis strain at different timepoints during growth. Supernatants of this strain that were active in disrupting the pH gradient across the membrane were also shown to inhibit growth of L. monocytogenes . In summary, the presented results suggest that the generated sensor strain is a convenient, fast and reliable tool to identify and characterize novel bacteriocins and other compounds that target membrane integrity.

Listeria monocytogenes is a human food-borne facultative intracellular pathogen that is resistant to a wide range of stress conditions. As a consequence, L. monocytogenes is extremely difficult to control along the entire food chain from production to storage and consumption. Frequent and recent outbreaks of L. monocytogene s infections illustrate that current measures of decontamination and preservation are suboptimal to control L. monocytogenes in food. In order to develop efficient measures to prevent contamination during processing and control growth during storage of food it is crucial to understand the mechanisms utilized by L. monocytogenes to tolerate the stress conditions in food matrices and food processing environments. Food-related stress conditions encountered by L. monocytogenes along the food chain are acidity, oxidative and osmotic stress, low or high temperatures, presence of bacteriocins and other preserving additives, and stresses as a consequence of applying alternative decontamination and preservation technologies such high hydrostatic pressure, pulsed and continuous UV light, pulsed electric fields (PEF). This review is aimed at providing a summary of the current knowledge on the response of L. monocytogenes toward these stresses and the mechanisms of stress resistance employed by this important food-borne bacterium. Circumstances when L. monocytogenes cells become more sensitive or more resistant are mentioned and existence of a cross-resistance when multiple stresses are present is pointed out.

Tumor necrosis factor α (TNFα) drives the pathophysiology of human autoimmune diseases and consequently, neutralizing antibodies (Abs) or Ab-derived molecules directed against TNFα are essential therapeutics. As treatment with several TNFα blockers has been reported to entail a higher risk of infectious diseases such as leishmaniasis, we established an in vitro model based on Leishmania -infected human macrophages, co-cultured with autologous T-cells, for the analysis and comparison of anti-TNFα therapeutics. We demonstrate that neutralization of soluble TNFα (sTNFα) by the anti-TNFα Abs Humira ® , Remicade ® , and its biosimilar Remsima ® negatively affects infection as treatment with these agents significantly reduces Leishmania -induced T-cell proliferation and increases the number of infected macrophages. By contrast, we show that blockade of sTNFα by Cimzia ® does not affect T-cell proliferation and infection rates. Moreover, compared to Remicade ® , treatment with Cimzia ® does not impair the expression of cytolytic effector proteins in proliferating T-cells. Our data demonstrate that Cimzia ® supports parasite control through its conjugated polyethylene glycol (PEG) moiety as PEGylation of Remicade ® improves the clearance of intracellular Leishmania . This effect can be linked to complement activation, with levels of complement component C5a being increased upon treatment with Cimzia ® or a PEGylated form of Remicade ® . Taken together, we provide an in vitro model of human leishmaniasis that allows direct comparison of different anti-TNFα agents. Our results enhance the understanding of the efficacy and adverse effects of TNFα blockers and they contribute to evaluate anti-TNFα therapy for patients living in countries with a high prevalence of leishmaniasis.

Functional impairment of T-cells and a concomitant augmented expression of programmed death-1 (PD-1) have been observed in visceral leishmaniasis patients, as well as in experimental models for visceral and cutaneous leishmaniasis. The PD-1/PD-1-ligand (PD-1/PD-L) interaction negatively regulates T-cell effector functions, which are required for parasite control during leishmaniasis. The aim of this study was to elucidate the impact of the PD-1/PD-L axis in a human primary in vitro infection model of Leishmania major ( Lm ). Blocking the PD-1/PD-L interaction with nivolumab increased T-cell proliferation and release of the proinflammatory cytokines TNFα and IFNγ during the cocultivation of Lm- infected human monocyte-derived macrophages (hMDMs) or dendritic cells (hMDDC) with autologous PD-1 + -lymphocytes. As a consequence Lm infection decreased, being the most pronounced in hMDDC, compared to proinflammatory hMDM1 and anti-inflammatory hMDM2. Focusing on hMDDC, we could partially reverse effects mediated by PD-1 blockade by neutralizing TNFα but not by neutralizing IFNγ. Furthermore, PD-1 blockade increased intracellular expression of perforin, granulysin, and granzymes in proliferating CD4 + -T-cells, which might be implicated in reduction of Lm -infected cells. In all, our data describe an important role for the PD-1/PD-L axis upon Lm infection using a human primary cell system. These data contribute to a better understanding of the PD-1-induced T-cell impairment during disease and its influence on immune effector mechanisms to combat Lm infection.

Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed--in contrast to viable parasites--that apoptotic-like parasites enter an LC3(+), autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4(+) T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells' autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

Any outbreak of an animal disease classified as a List A disease by the Office International des Epizooties, such as classical swine fever (CSF), has severe consequences for animal welfare, livestock production, exports of animals and animal products and the environment. Experience shows that early detection and response to a suspected disease outbreak will maximise the effectiveness of the emergency response actions and minimise the social, economic and environmental costs associated with the outbreak. The development and implementation of measures designed to minimise the risk of diseases entering a country or region has been the predominant animal health management strategy in most countries. However, even the strongest preventive management systems do not guarantee that outbreaks of animal diseases will not occur. Tracing, a procedure that begins with a known infected individual, herd or flock, and which traces all possible locational and interactive exposures in both directions, back towards the source and forward to contacts, is the backbone of disease emergency management. The authors provide an introduction to, and general overview of, tracking and tracing systems used during a recent epidemic of CSF in the Netherlands from 1997 to 1998.