Impact of Environmental Factors on Bacteriocin Promoter Activity in Gut-Derived Lactobacillus salivarius

ABSTRACTBacteriocin production is regarded as a desirable probiotic trait that aids in colonization and persistence in the gastrointestinal tract (GIT). Strains ofLactobacillus salivarius, a species associated with the GIT, are regarded as promising probiotic candidates and have a number of associated bacteriocins documented to date. These include multiple class IIb bacteriocins (salivaricin T, salivaricin P, and ABP-118) and the class IId bacteriocin bactofencin A, which show activity against medically important pathogens. However, the production of a bacteriocin in laboratory media does not ensure production under stressful environmental conditions, such as those encountered within the GIT. To allow this issue to be addressed, the promoter regions located upstream of the structural genes encoding theL. salivariusbacteriocins mentioned above were fused to a number of reporter proteins (green fluorescent protein [GFP], red fluorescent protein [RFP], and luciferase [Lux]). Of these, only transcriptional fusions to GFP generated signals of sufficient strength to enable the study of promoter activity inL. salivarius. While analysis of the class IIb bacteriocin promoter regions indicated relatively weak GFP expression, assessment of the promoter of the antistaphylococcal bacteriocin bactofencin A revealed a strong promoter that is most active in the absence of the antimicrobial peptide and is positively induced in the presence of mild environmental stresses, including simulated gastric fluid. Taken together, these data provide information on factors that influence bacteriocin production, which will assist in the development of strategies to optimizein vivoandin vitroproduction of these antimicrobials.

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A Conserved PapB Family Member, TosR, Regulates Expression of the Uropathogenic Escherichia coli RTX Nonfimbrial Adhesin TosA while Conserved LuxR Family Members TosE and TosF Suppress Motility

Infection and Immunity ◽

10.1128/iai.01608-14 ◽

2014 ◽

Vol 82 (9) ◽

pp. 3644-3656 ◽

Cited By ~ 6

Author(s):

Michael D. Engstrom ◽

Christopher J. Alteri ◽

Harry L. T. Mobley

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Virulence Factors ◽

Upper Urinary Tract ◽

Host Cells ◽

Promoter Regions ◽

Content Type ◽

Tract Infections

ABSTRACTA heterogeneous subset of extraintestinal pathogenicEscherichia coli(ExPEC) strains, referred to as uropathogenicE. coli(UPEC), causes most uncomplicated urinary tract infections. However, no core set of virulence factors exists among UPEC strains. Instead, the focus of the analysis of urovirulence has shifted to studying broad classes of virulence factors and the interactions between them. For example, the RTX nonfimbrial adhesin TosA mediates adherence to host cells derived from the upper urinary tract. The associatedtosoperon is well expressedin vivobut poorly expressedin vitroand encodes TosCBD, a predicted type 1 secretion system. TosR and TosEF are PapB and LuxR family transcription factors, respectively; however, no role has been assigned to these potential regulators. Thus, the focus of this study was to determine how TosR and TosEF regulatetosAand affect the reciprocal expression of adhesins and flagella. Among a collection of sequenced UPEC strains, 32% (101/317) were found to encode TosA, and nearly all strains (91% [92/101]) simultaneously carried the putative regulatory genes. Deletion oftosRalleviatestosArepression. Thetospromoter was localized upstream oftosRusing transcriptional fusions of putative promoter regions withlacZ. TosR binds to this region, affecting a gel shift. A 100-bp fragment 220 to 319 bp upstream oftosRinhibits binding, suggesting localization of the TosR binding site. TosEF, on the other hand, downmodulate motility when overexpressed by preventing the expression offliC, encoding flagellin. Deletion oftosEFincreased motility. Thus, we present an additional example of the reciprocal control of adherence and motility.

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Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

Journal of Bacteriology ◽

10.1128/jb.00864-15 ◽

2016 ◽

Vol 198 (7) ◽

pp. 1035-1043 ◽

Cited By ~ 35

Author(s):

Na Ke ◽

Dirk Landgraf ◽

Johan Paulsson ◽

Mehmet Berkmen

Keyword(s):

Escherichia Coli ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Living Cells ◽

Functional Protein ◽

Content Type ◽

Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

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Superior Pyronaridine Single-Dose Pharmacodynamics Compared to Artesunate, Chloroquine, and Amodiaquine in a Murine Malaria Luciferase Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00394-18 ◽

2018 ◽

Vol 62 (9) ◽

Cited By ~ 1

Author(s):

Winter A. Okoth ◽

Elijah J. Dukes ◽

David J. Sullivan

Keyword(s):

Single Dose ◽

Fluorescent Protein ◽

Lag Phase ◽

Content Type ◽

Log Reduction ◽

Drug Inhibition ◽

Drug Studies ◽

Dose Dependent

ABSTRACTMany previousin vitroandin vivopreclinical malaria drug studies have relied on low-parasite-number drug inhibition numerically compared to the untreated controls. In contrast, human malaria drug studies measure the high-parasite-density killing near 100 million/ml. Here we compared thein vivosingle-dose pharmacodynamic properties of artesunate and the 4-aminoquinolines pyronaridine, chloroquine, and amodiaquine in aPlasmodium bergheiANKA-green fluorescent protein GFP-luciferase-based murine malaria blood-stage model. Pyronaridine exhibited dose-dependent killing, achieving parasite reductions near 5 to 6 logs at 48 h, with complete cure at 10 mg/kg of body weight compared to artesunate, which exhibited a 48-h dose-dependent killing with a 2-log drop at the noncurative 250-mg/kg dose. Chloroquine, which was noncurative, and amodiaquine, which was partially curative, had nearly the same initial dose-independent killing, with a lag phase of minimal parasite reduction at all doses between 6 and 24 h, followed by a 2.5-log reduction at 48 h. In experiments with drug-treated, washed infected blood transfer to naive mice, chloroquine and amodiaquine showed fewer viable parasites at the 24-h transfer than at the 8-h transfer, measured by a prolonged return to parasitemia, despite a similar parasite log reduction at these time points, in contrast to the correlation of the parasite log reduction to viable parasites with artesunate and pyronaridine. Artesunate in combination with pyronaridine exhibited an initial parasite reduction similar to that achieved with pyronaridine, while with chloroquine or amodiaquine, the reduction was similar to that achieved with artesunate. Single-oral-dose pyronaridine was much more potentin vivothan artesunate, chloroquine, and amodiaquine during the initial decline in parasites and cure.

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Role of Mouse Peptidoglycan Recognition Protein PGLYRP2 in the Innate Immune Response to Salmonella enterica Serovar Typhimurium InfectionIn Vivo

Infection and Immunity ◽

10.1128/iai.00168-12 ◽

2012 ◽

Vol 80 (8) ◽

pp. 2645-2654 ◽

Cited By ~ 19

Author(s):

Jooeun Lee ◽

Kaoru Geddes ◽

Catherine Streutker ◽

Dana J. Philpott ◽

Stephen E. Girardin

Keyword(s):

Salmonella Enterica ◽

Fluorescent Protein ◽

Intraepithelial Lymphocytes ◽

Innate Immune ◽

Th17 Response ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTPeptidoglycan recognition proteins (PGRPs) are a family of innate pattern recognition molecules that bind bacterial peptidoglycan. While the role of PGRPs inDrosophilainnate immunity has been extensively studied, how the four mammalian PGRP proteins (PGLYRP1 to PGLYRP4) contribute to host defense against bacterial pathogensin vivoremains poorly understood. PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidalin vitro, whereas PGLYRP2 is anN-acetylmuramyl-l-alanine amidase that cleaves peptidoglycan between the sugar backbone and the peptide stem. Because PGLYRP2 cleaves muramyl peptides detected by host peptidoglycan sensors Nod1 and Nod2, we speculated that PGLYRP2 may act as a modifier of Nod1/Nod2-dependent innate immune responses. We investigated the role of PGLYRP2 inSalmonella entericaserovar Typhimurium-induced colitis, which is regulated by Nod1/Nod2 through the induction of an early Th17 response. PGLYRP2 did not contribute to expression of Th17-associated cytokines, interleukin-22 (IL-22)-dependent antimicrobial proteins, or inflammatory cytokines. However, we found thatPglyrp2-deficient mice displayed significantly enhanced inflammation in the cecum at 72 h postinfection, reflected by increased polymorphonuclear leukocyte (PMN) infiltration and goblet cell depletion.Pglyrp2expression was also induced in the cecum ofSalmonella-infected mice, and expression of green fluorescent protein under control of thePglyrp2promoter was increased in discrete populations of intraepithelial lymphocytes. Lastly,Nod2−/−Pglyrp2−/−mice displayed increased susceptibility to infection at 24 h postinfection compared toPglyrp2−/−mice, which correlated with increased PMN infiltration and submucosal edema. Thus, PGLYRP2 plays a protective rolein vivoin the control ofS. Typhimurium infection through a Nod1/Nod2-independent mechanism.

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Elizabethkingia anophelis: Molecular Manipulation and Interactions with Mosquito Hosts

Applied and Environmental Microbiology ◽

10.1128/aem.03733-14 ◽

2015 ◽

Vol 81 (6) ◽

pp. 2233-2243 ◽

Cited By ~ 32

Author(s):

Shicheng Chen ◽

Michael Bagdasarian ◽

Edward D. Walker

Keyword(s):

Fluorescent Protein ◽

Developmental Stages ◽

Genetic Manipulation ◽

Life Stage ◽

Expression System ◽

Aedes Triseriatus ◽

Content Type ◽

Mosquito Midgut

ABSTRACTFlavobacteria (members of the familyFlavobacteriaceae) dominate the bacterial community in theAnophelesmosquito midgut. One such commensal,Elizabethkingia anophelis, is closely associated withAnophelesmosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements ofE. anophelishave not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation ofE. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function inE. anophelis. A flavobacterial expression system based on the promoter PompAwas integrated into theE. anophelischromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-taggedE. anophelisassociated with mosquitoes at successive developmental stages and propagated inAnopheles gambiaeandAnopheles stephensibut not inAedes triseriatusmosquitoes. Feeding NanoLuc-tagged cells toA. gambiaeandA. stephensiin the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected inAedes triseriatusmosquitoes under the same conditions. Of the initialE. anopheliscells provided to larvae, 23%, 71%, and 85% were digested inA. stephensi,A. gambiae, andAedes triseriatus, respectively, demonstrating thatE. anophelisadapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells inA. stephensisignificantly increased when arginine was added to a sugar diet, showing it to be an important amino acid forE. anophelis. Animal erythrocytes promotedE. anophelisgrowthin vivoandin vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.

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To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB

Applied and Environmental Microbiology ◽

10.1128/aem.01056-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 4936-4946 ◽

Cited By ~ 20

Author(s):

Anna Sznajder ◽

Dieter Jendrossek

Keyword(s):

Fluorescent Protein ◽

Ralstonia Eutropha ◽

Yellow Fluorescent Protein ◽

High Capacity ◽

Constitutive Expression ◽

Enhanced Yellow Fluorescent Protein ◽

Content Type ◽

Phb Depolymerase

ABSTRACTThe putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, ofRalstonia eutrophaH16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granulesin vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1or ΔphaP1-phaP5mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditionsin vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion ofphaZd1,phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules norlacZfusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The truein vivofunctions of PhaZd1 and PhaZd2 remain obscure.

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Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

Journal of Bacteriology ◽

10.1128/jb.00249-15 ◽

2015 ◽

Vol 197 (14) ◽

pp. 2383-2391 ◽

Cited By ~ 7

Author(s):

Semen A. Leyn ◽

Irina A. Rodionova ◽

Xiaoqing Li ◽

Dmitry A. Rodionov

Keyword(s):

Carbon Dioxide ◽

Transcriptional Regulation ◽

Comparative Genomics ◽

Dna Binding ◽

Transcriptional Control ◽

Binding Assays ◽

Promoter Regions ◽

Content Type ◽

Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

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Activation of MAT2A-RIP1 signaling axis reprograms monocytes in gastric cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001364 ◽

2021 ◽

Vol 9 (2) ◽

pp. e001364

Author(s):

Yan Zhang ◽

Hui Yang ◽

Jun Zhao ◽

Ping Wan ◽

Ye Hu ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Metabolism ◽

Vital Role ◽

Methionine Metabolism ◽

Promoter Regions ◽

Normal Tissues ◽

Methionine Cycle

BackgroundThe activation of tumor-associated macrophages (TAMs) facilitates the progression of gastric cancer (GC). Cell metabolism reprogramming has been shown to play a vital role in the polarization of TAMs. However, the role of methionine metabolism in function of TAMs remains to be explored.MethodsMonocytes/macrophages were isolated from peripheral blood, tumor tissues or normal tissues from healthy donors or patients with GC. The role of methionine metabolism in the activation of TAMs was evaluated with both in vivo analyses and in vitro experiments. Pharmacological inhibition of the methionine cycle and modulation of key metabolic genes was employed, where molecular and biological analyses were performed.ResultsTAMs have increased methionine cycle activity that are mainly attributed to elevated methionine adenosyltransferase II alpha (MAT2A) levels. MAT2A modulates the activation and maintenance of the phenotype of TAMs and mediates the upregulation of RIP1 by increasing the histone H3K4 methylation (H3K4me3) at its promoter regions.ConclusionsOur data cast light on a novel mechanism by which methionine metabolism regulates the anti-inflammatory functions of monocytes in GC. MAT2A might be a potential therapeutic target for cancer cells as well as TAMs in GC.

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Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

Microorganisms ◽

10.3390/microorganisms9020379 ◽

2021 ◽

Vol 9 (2) ◽

pp. 379

Author(s):

Breanne M. Head ◽

Christopher I. Graham ◽

Teassa MacMartin ◽

Yoav Keynan ◽

Ann Karen C. Brassinga

Keyword(s):

Growth Kinetics ◽

Legionella Pneumophila ◽

Fluorescent Protein ◽

Disease Incidence ◽

Acanthamoeba Castellanii ◽

Infection Model ◽

Intracellular Infection ◽

Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

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Prrx1 promotes stemness and angiogenesis via activating TGF-β/smad pathway and upregulating proangiogenic factors in glioma

Cell Death and Disease ◽

10.1038/s41419-021-03882-7 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Zetao Chen ◽

Yihong Chen ◽

Yan Li ◽

Weidong Lian ◽

Kehong Zheng ◽

...

Keyword(s):

Stem Cells ◽

Therapeutic Strategy ◽

Critical Role ◽

Glioma Stem Cells ◽

Promoter Regions ◽

Aberrant Expression ◽

Smad Pathway ◽

Tgf Β1

AbstractGlioma is one of the most lethal cancers with highly vascularized networks and growing evidences have identified glioma stem cells (GSCs) to account for excessive angiogenesis in glioma. Aberrant expression of paired-related homeobox1 (Prrx1) has been functionally associated with cancer stem cells including GSCs. In this study, Prrx1 was found to be markedly upregulated in glioma specimens and elevated Prrx1 expression was inversely correlated with prognosis of glioma patients. Prrx1 potentiated stemness acquisition in non-stem tumor cells (NSTCs) and stemness maintenance in GSCs, accompanied with increased expression of stemness markers such as SOX2. Prrx1 also promoted glioma angiogenesis by upregulating proangiogenic factors such as VEGF. Consistently, silencing Prrx1 markedly inhibited glioma proliferation, stemness, and angiogenesis in vivo. Using a combination of subcellular proteomics and in vitro analyses, we revealed that Prrx1 directly bound to the promoter regions of TGF-β1 gene, upregulated TGF-β1 expression, and ultimately activated the TGF-β/smad pathway. Silencing TGF-β1 mitigated the malignant behaviors induced by Prrx1. Activation of this pathway cooperates with Prrx1 to upregulate the expression of stemness-related genes and proangiogenic factors. In summary, our findings revealed that Prrx1/TGF-β/smad signal axis exerted a critical role in glioma stemness and angiogeneis. Disrupting the function of this signal axis might represent a new therapeutic strategy in glioma patients.

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